Ex Parte Bentwich

Patent Trial and Appeal Board

Dec 11, 2012

10536560 (P.T.A.B. Dec. 11, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte ITZHAK BENTWICH
__________

Appeal 2012-001111
Application 10/536,560
Technology Center 1600
__________

Before TONI R. SCHEINER, JEFFREY N. FREDMAN, and
STEPHEN WALSH, Administrative Patent Judges.

WALSH, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134(a) from the rejection of
claims directed to an isolated viral nucleic acid, a probe, and a vector. The
Patent Examiner rejected the claims for indefiniteness, anticipation, and
obviousness. We have jurisdiction under 35 U.S.C. § 6(b). We reverse the
pending rejections but enter a new ground of rejection.
Appeal 2012-001111
Application 10/536,560

2
STATEMENT OF THE CASE
Claims 21-34, 50, 52, and 53 are on appeal. Claim 21 is
representative and reads as follows:
21. An isolated first viral nucleic acid or complement thereof, wherein
(a) the first viral nucleic acid consists of 15-24 nucleotides;
(b) a second viral nucleic acid consisting of 50 to 131 nucleotides
comprises the first viral nucleic acid;
(c) the second viral nucleic acid is capable of forming a hairpin,
wherein
(i) the hairpin comprises two stem segments and an
intervening loop segment;
(ii) the two stem segments each consists of 14-71
nucleotides;
(iii) the loop segment consists of 3 to 19 nucleotides;
(iv) the first and second stem segments are at least 30.8%
complementary; and
(v) one of the stem segments of the hairpin comprises the
first viral nucleic acid,
(d) the first viral nucleic acid is capable of binding to a binding site
of a mRNA; and
(e) the first viral nucleic acid is capable of inhibiting expression of
a protein encoded by a mRNA, wherein the mRNA comprises
the binding site;
wherein a viral genome comprises the sequence of the first and second
viral nucleic acids.

The Examiner rejected the claims as follows:
I. claims 21-34, 50, 52, and 53 under 35 U.S.C. § 112, second
paragraph, as indefinite (Ans. 8);
II. claims 21, 22, 33, 34, 50, 52, and 53 under 35 U.S.C. § 102(b) as
anticipated by each of Khvorova (Ans. 9-10), Usman (id. at 10-11),
Stacey (id. at 11-12), Berlin (id. at 12-13), Baker (id. at 13-14), and
Lieven (id. at 14);
Appeal 2012-001111
Application 10/536,560

3
III. claims 21 and 52 under 35 U.S.C. § 102(b) as anticipated by each of
Zhu (Ans. 14-15), Ghiringhelli (id. at 15), Baumstark (id. at 15-16),
Ozdarendeli (id. at 16), and Davison (id.); and
IV. claims 21-34, 50, 52, and 53 under 35 U.S.C. § 103(a) as unpatentable
over Lai, Zhu, Ghiringhelli, Baumstark, Ozdarendeli, Davison, and
Perry (Ans. 17-20).

I
The Examiner’s position is that “the claims as currently written do not
particularly point out why the various structural limitations specifically set
forth for and only relevant to the ‘second’ viral nucleic acid are necessary to
claim the ‘first’ viral nucleic acid, and therefore, it is concluded that the
subject matter regarded as applicant's invention is not distinctly claimed.”
(Ans. 8.)
Appellant contends “it is permissible for claimed subject matter to be
limited by reference to unclaimed subject matter.” (App. Br. 6.)
The rejection is reversed because the reasoning given in the rejection
is insufficient to show the claims are indefinite. First, the statute requires the
Applicant to claim “the subject matter which the applicant regards as his
invention.” 35 U.S.C. § 112, second paragraph (emphasis added).
Appellant contends that claimed subject matter may be limited by reference
to unclaimed subject matter, and the rejection concedes the point. Second,
“[t]he test for definiteness is whether one skilled in the art would understand
the bounds of the claim when read in light of the specification.” Miles
Laboratories Inc. v. Shandon Inc., 997 F.2d 870, 875 (Fed. Cir. 1993). The
rejection did not address the proper test.
Appeal 2012-001111
Application 10/536,560

4
We appreciate the Examiner’s implied concern that the claim scope is
very broad, but breadth is not identical to indefiniteness.

II
The rejections over Khvorova, Usman, Stacey, Berlin, Baker, and Lieven.
Claim 21 is drawn to a viral nucleic acid consisting of 15-24
nucleotides, further defined by (c)(v) that when embedded in a second viral
nucleic acid that can form a hairpin loop, it will be a stem segment in the
hairpin, by (d) that it is capable of binding a mRNA, by (e) that it is capable
of inhibiting expression of a protein encoded by a mRNA having the binding
site mention in (d), and by the final wherein clause requiring that its
sequence must be present in a viral genome.
Notwithstanding claim 21’s final wherein clause, the rejections over
Khvorova, Usman, Stacey, Berlin, Baker, and Lieven dismiss the “viral”
descriptor and give it no weight. (Ans. 9.) The rejections find claim 21
anticipated by nucleic acids said to be similar to SEQ ID NO:2079, but not
derived from a viral source. (We recognize that Appellants elected SEQ ID
NO:2079 for examination in response to a requirement by the Examiner.)
Appellant groups Khvorova, Usman, Stacey, Berlin, Baker, and
Lieven as the “Non-Viral References,” and we will do the same. (App. Br.
11.) Appellant contends that none of the non-viral references anticipates the
claims because none discloses all the limitations of the claims. (Id.)
Specifically, Appellant contends:
in order to anticipate the instant claims the sequence of the prior
art nucleic acid must be present in the genome of a virus.
That is, the prior art nucleic acid must be 100% identical to a
sequence found within a virus genome. The Examiner fails to
Appeal 2012-001111
Application 10/536,560

5
establish that anyone of the cited nucleic acids from the Non-
Viral References is 100% identical to a sequence from a viral
genome. The Examiner is unable to make this showing because
not a single one of the cited nucleic acids from the Non-Viral
References is found in any known viral genome.

(App. Br. 11-12.)
In response, the Examiner acknowledges that none of the references
described a nucleic acid with a sequence that is in a viral genome.
Nevertheless, the Examiner found Appellant’s point unpersuasive in the
absence of “objective evidence showing that the cited prior art nucleic acids
will not be found in a later sequenced virus genome.” (Ans. 30.) In other
words, there is no evidence that the sequence is inherent in a viral genome,
but it might be. As there is no evidence of record that one of the cited prior
art sequences necessarily exists in a viral genome, none of the rejections are
sustainable. “The mere fact that a certain thing may result from a given set
of circumstances is not sufficient.” In re Robertson, 169 F.3d 743, 745 (Fed.
Cir. 1999).

III
The rejections over Zhu, Ghiringhelli, Baumstark, Ozdarendeli, and
Davison.
Claim 21 is drawn to a viral nucleic acid consisting of 15-24
nucleotides, and further defined by additional claim limitations.
The Examiner’s position is that each of the cited references discloses
a viral nucleic acid that contains 15-24 nucleotides embedded in a longer
sequence illustrated as part of a hairpin loop. The rejections find the
drawings sufficient to show (i) the structural requirements of claim 21 are
Appeal 2012-001111
Application 10/536,560

6
met, and (ii) the functional properties recited in claim 21 are inherent in the
prior art nucleic acids.
The rejections cannot be sustained because claim 21 defines a nucleic
acid “consisting of” no more than 24 nucleotides, and none of the references
described a nucleic acid that short.

IV
The Issue
The Examiner’s position is that Lai taught “a computational,
bioinformatics-based approach based the structural features of known
miRNAs combined with comparative genomics allows one to discover
unknown miRNAs ‘in a given sequenced genome.’” (Ans. 17.) The
Examiner concluded:
It would have been obvious to one of ordinary skill in the art at
the time the invention was made to utilize a computational
algorithm-based bioinformatics methodology to identify
potential miRNAs located within the non-coding regions of
viral genomes such as the noncoding regions of the HSV-1 of
Perry et al. and isolate miRNAs from the HSV-1 genome.

(Id. at 19.)
Appellant contends:
The issue here is not simply whether one of ordinary skill in the
art would reasonably expect the genome of a virus to contain
hairpin structure in general, but rather whether one of
ordinary skill in the art would reasonably have expected a
virus genome to contain the very specific structure of a
miRNA hairpin precursor. Applicant submits that the
Examiner has failed to establish that such a reasonable
expectation existed at the time of filing.

Appeal 2012-001111
Application 10/536,560

7
(App. Br. 14.)
We agree with Appellant that the rejection is insufficient to establish a
reasonable expectation of success in finding the claimed 15-24 nucleotide
viral molecule. The rejection established that it may have been obvious to
apply Lee’s discovery software to the viral sequences disclosed in the
references, such as Perry’s HSV-1, because it was a promising field of
experimentation. That is not enough to support a reasonable expectation of
success in finding 15-24 mers having the structural/functional properties
defined in claim 21. See In re O’ Farrell, 853 F.2d 894, 903 (Fed. Cir.
1988). The obviousness rejection is reversed.

V
Pursuant to 37 C.F.R. § 41.50(b), we enter the following new ground
of rejection: Claim 21 is anticipated under 35 U.S.C. 102(b) by Wang1
(attached,) as evidenced by Klump2 (attached).
As elected species SEQ ID NO:2079 was not found in the prior art,
the search has been expanded. Wang is available under § 102 (a) and 102(b)
because Appellant’s priority applications with filing dates prior to April
2001 do not describe the subject matter of claim 21.
Wang teaches a first “viral” nucleic acid, AS-3, which is 20
nucleotides in length (Wang 1045, Table 1).

1 Aikun Wang et al., Specific Inhibition of Coxsackievirus B3 Translation
and Replication by Phosphorothioate Antisense Oligodeoxunucleotides, 45
ANTIMICROB. AGENTS CHEMOTHER. 1043-1052 (2001).
2 Wolfgang M. Klump et al., Complete Nucleotide Sequence of Infectious
Coxsackievirus B3 cDNA: Two Initial 5' Uridine Residues Are Regained
during Plus-Strand RNA Synthesis, 64 J. VIROLOGY 1573-1583 (1990).
Appeal 2012-001111
Application 10/536,560

8
Wang teaches a second viral nucleic acid, loop H of the Cocksackie
virus IRES, which comprises AS-3, where the second viral nucleic acid is
capable of forming a hairpin (see Wang 1045, figure 1, loop H). The loop H
hairpin comprises two stem segments and an intervening loop segment from
nucleotides 581 to 624 of the Cocksackie B3 virus sequence, where the 20-
nucleotide AS-3 oligonucleotide is shown as hybridizing to the stem (Wang
1045, Figure 1).
Klump evidences that the sequence of loop H, nucleotides 581 to 624
of the Cocksackie B3 virus sequence, is
ctggctgcttatggtgacaattgagagattgttaccatatagct (Klump 1576, Figure 2) which
can be folded into a stem loop as shown below where the stem portion is
more than 30% complementary.

Wang teaches that AS-3 binds to the loop H IRES sequence (Wang
1045, Table 1) and inhibits translation of the Cocksackie VP-1 protein
(Wang 1046, figure 2).
Klump evidences that the Cocksackie B3 virus sequence comprises
the sequence of both the first viral nucleic acid, the oligonucleotide AS-3,
and the second viral nucleic acid capable of forming a hairpin, loop H or the
larger complete Cocksackie B3 virus sequence IRES sequence (Klump
1576, figure 2).
Appeal 2012-001111
Application 10/536,560

9
Therefore, the AS-3 oligonucleotide of Wang reasonably anticipates
claim 21. We leave it to the Examiner to determine the applicability of this
reference to the remaining claims.

37 C.F.R. § 41.50(b)
This decision contains a new ground of rejection pursuant to
37 C.F.R. § 41.50(b) (effective September 13, 2004, 69 Fed. Reg. 49960
(August 12, 2004), 1286 Off. Gaz. Pat. Office 21 (September 7, 2004)).
37 C.F.R. § 41.50(b) provides “[a] new ground of rejection pursuant to this
paragraph shall not be considered final for judicial review.”
37 C.F.R. § 41.50(b) also provides that the Appellant, WITHIN TWO
MONTHS FROM THE DATE OF THE DECISION, must exercise one of
the following two options with respect to the new ground of rejection to
avoid termination of the appeal as to the rejected claims:
(1) Reopen prosecution. Submit an appropriate
amendment of the claims so rejected or new evidence relating
to the claims so rejected, or both, and have the matter
reconsidered by the Examiner, in which event the proceeding
will be remanded to the Examiner. . . .

(2) Request rehearing. Request that the proceeding be
reheard under § 41.52 by the Board upon the same record. . . .

SUMMARY
We reverse the rejection of claims 21-34, 50, 52, and 53 under
35 U.S.C. § 112, second paragraph.
We reverse the rejections of claims 21, 22, 33, 34, 50, 52, and 53
under 35 U.S.C. § 102(b) as anticipated by each of Khvorova, Usman,
Stacey, Berlin, Baker, and Lieven.
Appeal 2012-001111
Application 10/536,560

10
We reverse the rejections of claims 21 and 52 under 35 U.S.C.
§ 102(b) as anticipated by each of Zhu, Ghiringhelli, Baumstark,
Ozdarendeli, and Davison.
We reverse the rejection of claims 21-34, 50, 52, and 53 under
35 U.S.C. § 103(a) as unpatentable over Lai, Zhu, Ghiringhelli, Baumstark,
Ozdarendeli, Davison, and Perry.
Claim 21 is rejected under U.S.C. § 102(b) as anticipated by Wang as
evidenced by Klump.

REVERSED; 37 C.F.R. § 41.50(b)

Attachments: Form 892
Wang et al.
Klump et al.

lp

Notice of References Cited

Application/Control No.

10/536,560
Applicant(s)/Patent Under
Reexamination
Itzhak Bentwich
Examiner

Dana Shin
Art Unit

1600

Page 1 of 1

U.S. PATENT DOCUMENTS

* DOCUMENT NO. DATE NAME CLASS SUBCLASS
DOCUMENT
SOURCE **
APS OTHER

A

B

C

D

E

F

G

H

I

J

K

L

M

FOREIGN PATENT DOCUMENTS

* DOCUMENT NO. DATE COUNTRY NAME CLASS SUBCLASS
DOCUMENT
SOURCE **
APS OTHER

N

O

P

Q

R

S

T
NON-PATENT DOCUMENTS

* DOCUMENT (Including Author, Title Date, Source, and Pertinent Pages)
DOCUMENT
SOURCE **
APS OTHER

U

Aikun Wang et al., Specific Inhibition of Coxsackievirus B3 Translation and Replication
by Phosphorothioate Antisense Oligodeoxunucleotides, 45 ANTIMICROB. AGENTS
CHEMOTHER. 1043-1052 (2001).

V

Wolfgang M. Klump et al., Complete Nucleotide Sequence of Infectious Coxsackievirus
B3 cDNA: Two Initial 5' Uridine Residues Are Regained
during Plus-Strand RNA Synthesis, 64 J. VIROLOGY 1573-1583 (1990).

W

X

*A copy of this reference is not being furnished with this Office action. (See Manual of Patent Examining Procedure, Section 707.05(a).)
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
0066-4804/01/$04.0010 DOI: 10.1128/AAC.45.4.1043–1052.2001
Apr. 2001, p. 1043–1052 Vol. 45, No. 4
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Specific Inhibition of Coxsackievirus B3 Translation and Replication by
Phosphorothioate Antisense Oligodeoxynucleotides
AIKUN WANG, PAUL K. M. CHEUNG, HUIFANG ZHANG, CHRISTOPHER M. CARTHY, LUBOS BOHUNEK,
JANET E. WILSON, BRUCE M. MCMANUS, AND DECHENG YANG*
Department of Pathology and Laboratory Medicine, University of British Columbia-St. Paul’s Hospital,
Vancouver, British Columbia V6Z 1Y6, Canada
Received 2 June 2000/Returned for modification 30 August 2000/Accepted 24 January 2001
The 5* and 3* untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary
structures that have been confirmed to play important regulatory roles in viral cap-independent internal
translation initiation and RNA replication. We previously demonstrated that deletions in different regions of
the 5* UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly
that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides
(AS-ODNs) were applied to target crucial sites within the 5* and 3* UTRs. In this study, seven phosphoro-
thioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa
cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR,
and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3
were strongly inhibited by the AS-ODNs complementary to different regions of the 5* and 3* UTRs. The most
effective sites are located at the proximate terminus of the 5* UTR (AS-1), the proximate terminus of the 3* UTR
(AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region
(AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral
protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with
AS-1 and AS-7 targeting the termini of the 5* and 3* UTRs. The percent inhibition values of AS-1 and AS-7 for
CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7,
respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.
Coxsackievirus B3 (CVB3) is a member of the genus En-
terovirus of the family Picornaviridae (3). This virus is the most
important viral myocarditis pathogen in humans and animals
(34). Such importance is reflected in data from the World
Health Organization global surveillance of viral diseases,
where the coxsackie B viruses were ranked the number one
cause of clinically evident cardiovascular diseases (14). In ad-
dition, there is considerable clinical and experimental evidence
indicating that dilated cardiomyopathy, another common heart
disease, may be a late consequence of viral myocarditis (19, 23,
34).
CVB3 is a single-stranded positive-polarity RNA virus. Like
other picornaviruses, the 59 untranslated region (UTR) of the
CVB3 genome is unusually long (741 nucleotides [nt]) but, unlike
eukaryotic mRNAs, is not capped with a 7-methylguanosine
triphosphate group. Instead, it is covalently linked to a virus-
encoded oligopeptide (VPg) (31). The viral genome is approx-
imately 7.4 kb long with a polyadenyl tail at the 39 end. The
primary sequence of the genomic RNA serves as mRNA to
direct synthesis of viral proteins using host protein transla-
tional machinery. Picornavirus RNA encodes a single long
polyprotein, which is processed initially into three precursor
polyproteins (P1, P2, and P3). Further processing of these
precursors by three virus-encoded proteases, 2A, 3C, and 3CD,
gives rise to mature structural and nonstructural proteins in-
cluding four capsid proteins and the RNA-dependent RNA
polymerase essential for viral replication (27).
It has long been known that the majority of cellular mRNAs
in eukaryotic organisms initiate translation via a cap-depen-
dent ribosomal scanning mechanism (18, 26). However, the
initiation of protein translation in picornaviruses occurs by an
unusual mechanism involving direct internal binding of the
ribosome to a sequence element of the 59 UTR of viral RNA
(20), termed an internal ribosomal entry site (IRES) (9, 21,
22). The IRES directs binding of the small ribosomal subunit
to viral RNA near the 39 border of the IRES, independent of
a cap structure at the 59 terminus of the RNA. Recently, our
work has confirmed the presence of an IRES within the 59
UTR of CVB3 RNA by mutational analysis using both bicis-
tronic plasmids and full-length CVB3 mutants (33, 54). Fur-
ther mapping of various mutations demonstrated that the cru-
cial sequence of the IRES of CVB3 is located roughly at
stem-loops G, H, and I, spanning nt 439 to 639. This critical
sequence was further analyzed by site-directed mutagenesis
and demonstrated that the critical nucleotides of the IRES
span the pyrimidine-rich tract between stem-loops G and H. A
46-nt deletion in this region abolished viral translation and
infectivity (33). Therefore, the IRES plays an important role in
the translation initiation of viral proteins. In addition, our
recent work also found that the 59 proximate terminus of 59
UTR is critical for translation initiation of CVB3. Deletion of
nt 1 to 63 of 59 UTR greatly inhibited CVB3 translation (54).
Similar findings have been reported in other picornaviruses,
suggesting that the 59 cloverleaf structure of the 59 UTR may
be responsible for viral replication (16, 53). In addition, recent
* Corresponding author. Mailing address: Cardiovascular Research
Laboratory, University of British Columbia, St. Paul’s Hospital, 1081
Burrard St., Vancouver, British Columbia, Canada V6Z 1Y6. Phone:
(604) 806-8200. Fax: (604) 806-8208. E-mail: dyang@mrl.ubc.ca.
1043
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

reports have suggested that the 39 UTRs of several picornavi-
ruses are involved in viral RNA replication (35, 37, 41). Thus,
by blocking crucial sites within the 59 and 39 UTRs of CVB3
through sequence-specific hybridization, viral protein transla-
tion and RNA replication will be inhibited.
Antisense (AS) RNA or DNA oligonucleotides have been
considered promising agents for inhibiting viral replication due
in part to their high specificity for viral RNA sequences. These
agents have been successfully employed to inhibit human im-
munodeficiency virus (32), hepatitis B and C virus (5, 38, 39,
42), influenzavirus (2, 17), coronavirus (1), and respiratory
syncytial virus (40). Recently, the Food and Drug Administra-
tion approved the first AS-based therapeutic product for the
treatment of retinitis caused by cytomegalovirus infections in
patients with AIDS (11). Although viral replication could be
inhibited by unmodified oligonucleotides, their vulnerability to
nuclease attack, combined with their intracellular distribution
and uptake properties, have limited their therapeutic potential
(4, 24). To avoid this problem and to improve the cellular
uptake of oligonucleotides, phosphorothioate oligodeoxynu-
cleotides (ODNs) encapsulated in liposomes were used in this
study. Because the mode of AS action is highly specific, it is
essential to carefully select appropriate viral target sequences.
Based on our previous mutational mapping and other reports
on CVB3 and other picornaviruses (15, 33, 35, 37, 50, 54). The
59 and 39 UTRs of CVB3 RNA were chosen as the major
targets for designing As ODNs. In this report, seven AS-ODNs
were synthesized and evaluated in HeLa cells infected with
CVB3. Four of the seven showed specific and dose-dependent
inhibition of CVB3 gene expression. Two of the four targeting
the 59 and 39 proximate termini of CVB3 genomic RNA dem-
onstrated the strongest antiviral activity.
MATERIALS AND METHODS
Design and synthesis of AS-ODNs. ODNs targeting the 59 UTR were designed
based on our previous mutational mapping of the cis- and trans-acting transla-
tional sequence elements (33, 54), such as the putative translation initiation
factor binding site, the IRES, and the surrounding sequence of the initiation
codon. The ODNs located at the 39 UTR were designed according to the tertiary
structure of the 39 UTR of CVB3 RNA (35, 50). These three ODNs targeting the
39 UTR were designed to disrupt the kissing interaction of two predominant
hairpin loops. To avoid using too many control oligomers in the first round of the
evaluation, an ODN (AS-S) with the same length and an average GC content but
with a random sequence was designed as a general control of all seven ODNs.
After the highest inhibitory activities were obtained with AS-1 and AS-7, these
two AS-ODNs were chosen for further evaluation of their specificity using two
new controls for each, which were designed with scrambled and reverse se-
quences, respectively. The controls were analyzed with DNA Strider software to
avoid any sequence complementation with CVB3 genomic RNA.
The AS-ODNs were synthesized by the standard phosphoramidite chemistry
method using an Applied Biosystems DNA/RNA synthesizer on a 1-mM scale at
the Biotechnology Laboratory, University of British Columbia. In order to re-
place the phosphodiester bonds within the oligonucleotides with phosphorothio-
ates, the oxidation step was substituted with a sulfurization procedure using
Beaucage’s reagents. The oligonucleotide derivatives were purified by reverse-
phase high-pressure liquid chromatography and lyophilized, and the powder was
dissolved in distilled water. All oligonucleotides were 20-mers. Figure 1 shows
the location of each AS-ODN within the 59 and 39 UTRs of CVB3 RNA (25)
used in this investigation. The AS-ODN sequences are shown in Table 1.
Virus, cell culture, and transfection. Stock CVB3 was generously provided by
Reinhard Kandolf and was stored at 280°C. Virus was grown in HeLa cells
(American Type Culture Collection), and titers were routinely redetermined at
the beginning of all individual experiments.
Transfection of HeLa cells with ODNs was conducted in 24-well plates. HeLa
cells were seeded in plates (1.2 3 105 cells/well) and grown in minimum essential
medium (MEM) containing 10% fetal calf serum at 37°C. After incubation for
20 h, the cells reached about 85% confluency and were washed with phosphate-
buffered saline (PBS). A transfection mixture containing AS-ODN (final con-
centration of 1.0 or 10 mM) and 4 mg of Lipofectin (GIBCO-BRL) in 200 ml of
Opti-medium (GIBCO-BRL) was added to each well. The transfection mixture
was prepared as follows. In a sterile tube, 16 ml of Lipofectin was added to 384
ml of Opti-medium, mixed well gently, and kept at room temperature for 60 min.
In another sterile tube, 4 ml of the oligonucleotide at appropriate concentration
was added to 400 ml of Opti-medium, and the sample was mixed well gently and
kept at room temperature for 60 min. The two tubes were combined, mixed well,
and kept at room temperature for another 30 min. HeLa cells were overlaid with
this final transfection mixture of Lipofectin-ODN (200 ml/well) and incubated for
6 h at 37°C. Then the cells were washed with PBS and infected with 200 ml of
CVB3 supernatant at a multiplicity of infection (MOI) of 0.01 for 60 min. After
infection, the cells were washed with PBS, overlaid with 200 ml of complete
MEM, and incubated for 24 h at 37°C in a humidified 5% CO2 incubator. Finally,
the supernatants from each treatment were collected by centrifugation at 4,000 3
g for 5 min. The resulting supernatant was aliquoted and kept at 280°C until use.
Western blot detection of CVB3 structural protein VP1. Thirty microliters of
the resulting supernatant from each ODN treatment was denatured at 95°C for
3 min and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis. The sample was then transferred to a nitrocellulose membrane at 75 V for 60
min. The membrane was blocked with PBS containing Tween 20 and 5% milk at
room temperature for 2 h and then incubated with primary antibody (rabbit
immunoglobulin G to CVB3 VP1) (Denka Seiken Co., Ltd.) diluted 1:2,000 at
4°C overnight. The membrane was washed by PBS containing Tween 20 for 30
min and incubated with secondary antibody (anti-rabbit immunoglobulin G con-
jugated to horseradish peroxidase) (Transduction Ltd.). The membrane was
washed again for 30 min. The signal detection was conducted by the enhanced
chemiluminescence method per the manufacturer’s instructions (Amersham
Pharmacia Biotech, Inc.). Films were analyzed by densitometric scanning of the
bands, and the density values of CVB3 VP1 were represented as means 6
standard deviations (SD). The means 6 SD of controls were normalized to a
value of 100. Mean 6 SD values of the treatment groups were calculated with
respect to the control values. All experiments were performed four times.
Detection of CVB3 RNA by RT-PCR. One hundred microliters of supernatant
containing CVB3 particles from each treatment was added to 1 ml of TRIZOL
Reagent (GIBCO-BRL). Viral RNA was extracted following the procedure
described in the instructional manual. At the final step, the pellets were dissolved
in 30 ml of diethyl pyrocarbonate-treated water and than kept at 280°C. Reverse
transcription (RT) was conducted according to the manufacturer’s instructions
(GIBCO-BRL) using 5 ml of extracted CVB3 RNA and 1.2 ml of 20 mM RT
primer (GCATTCAGCCTGGTCTCA, nt 780 to 797 of CVB3 RNA genome).
After incubation at 42°C for 60 min, the samples were heated at 99°C for 5 min
to stop the reaction. In order to amplify CVB3 cDNA, a PCR was carried out by
following the standard method in a volume of 100 ml containing 5 ml of RT
product and 2 ml of 20 mM upstream primer (AGCCTGTGGGTTGATCCCAC,
nt 8 to 27) and 2 ml of 20 mM downstream primer (AATTGTCACCATAAGC
AGCCA, nt 581 to 601). The reaction was run for 20 cycles with the following
parameters: denaturation at 94°C for 30 s, annealing at 58°C for 40 s, and
extension at 72°C for 45 s. For the negative control, water was substituted for
cDNA. Twenty microliters of PCR product from each sample was analyzed by
0.8% agarose gel electrophoresis. The bands were scanned using a densitometer,
and the mean density for each band was calculated as described above. Each
experiment was repeated three times.
Plaque assay. Viral plaque assay was carried out using supernatants collected
from the HeLa cell monolayers treated with AS-ODN at a final concentration of
10 mM. HeLa cells were seeded into 6-well plates (8 3 105 cells/well) and
incubated at 37°C for 20 h. When cell confluency reached approximately 90%,
cells were washed with PBS to remove fetal bovine serum and then overlaid with
1 ml of supernatant diluted 1:10. The cells were incubated at 37°C for 60 min, the
supernatants were removed, and the cell were washed with PBS. Finally, cells
were overlaid with 2 ml of sterilized soft Bacto Agar-MEM (1.5% Bacto
Agar–23 MEM [1:1]). The cells were incubated at 37°C for 72 h, fixed with
Carnoy’s fixative (75% ethanol–25% acetic acid) for 30 min and then stained
with 1% crystal violet. The plaques were counted, and the viral PFU per milliliter
was calculated. Supernatants from HeLa cell monolayer treated with control
AS-ODNs were used as controls. Each experiment was repeated three times. The
inhibitory activity of each AS-ODN was calculated with respect to the value for
the corresponding control.
Statistical analysis. All values are expressed as means 6 SD. Statistical sig-
nificance was evaluated using the Student t test for paired comparison. A P value
of ,0.01 was considered statistically significant.
1044 WANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

RESULTS
Establishment of in vitro cell system. To establish an opti-
mal in vitro evaluation system using HeLa cells, three param-
eters were tested. The first was cell confluency. The optimal
cell confluency for transfection should usually be 30 to 50%.
However, at this cell confluency, it is hard to determine the
effects of AS-ODNs on viral multiplication because most trans-
fected cells died quickly after only 12 h with CVB3 infection.
This observation was attributed to the high transfection effi-
ciency leading to nonspecific toxicity to the cells, especially at
a high oligonucleotide concentration. Through a series of ex-
periments, we found that the optimal cell confluency for AS-
ODN transfection followed by CVB3 infection is 85%, even
though the transfection efficiency would be somewhat affected.
The second consideration was the MOI. Unlike some viruses,
FIG. 1. Targets of the AS-ODNs within the proposed secondary structures of the 59 and 39 UTRs of CVB3 RNA (50, 54). The 59 UTR (nt 1
to 741) contains three AS-ODN blocking sites. AS-1 blocks the proximal terminus of the 59 UTR at nt 1 to 20. AS-2 (nt 557 to 576) and AS-3 (nt
583 to 602) target the IRES region. AS-2 is complementary to the polypyrimidine tract of the IRES core sequence. AS-3 targets the downstream
region of the AS-2 near the 39 boundary of the IRES. AS-4 (nt 733 to 752) blocks the translation initiation codon AUG region including 9 nt of
the 59 UTR. Within the 39 UTR (nt 7300 to 7399), three AS-ODN targets were selected. AS-5 (nt 7301 to 7320) and AS-6 (nt 7340 to 7359) target
stem-loops L and M, respectively. AS-7 (nt 7380 to 7399) blocks the 39 proximal terminus of the 39 UTR. The general scrambled oligomer control,
AS-S, was synthesized at the same length and random sequence with no annealing target in the CVB3 genomic RNA. Additional oligomer controls
for AS-1 and AS-7 are listed with all other oligomers together in Table 1.
TABLE 1. AS phosphorothioate ODNs used in anti-CVB3 evaluation
ODNa Sequence (59 ➝ 39) Target in CVB3 RNAb
AS-1 CAACCCACAGGCTGTTTTAA nt 1–20, 59 end of the 59 UTR
AS-1-s CGTAGTACACTTACTAACGC Scrambled sequence of AS-1
AS-1-r AATTTTGTCGGACACCCAAC Reverse sequence of AS-1
AS-2 AGGAATAAAATGAAACACGG nt 557–576, IRES core sequence
AS-3 CAATTGTCACCATAAGCAGC nt 583–602, IRES, downstream of AS-2
AS-4 TGAGCTCCCATTTTGCTGTA nt 733–752, AUG start codon region
AS-5 TTATTTCAAATTGTCTCTAA nt 7301–7320, 39 UTR, stem-loop L
AS-6 TATCTGGTTCGGTTAGCACA nt 7340–7359, 39 UTR, stem-loop M
AS-7 CCGCACCGAATGCGGAGAAT nt 7380–7399, 39 end of the 39 UTR
AS-7-s ACGACGTCGATCGAACGACG Scrambled sequence of AS-7
AS-7-r TAAGAGGCGTAAGCCACGCC Reverse sequence of AS-7
AS-S ACGTTGCAACGTCGTATCAT General scrambled sequence
a All oligomers are 20 nt long. AS-S is scrambled AS as a general control.
b The nucleotide positions within CVB3 genomic RNA are shown (25).
VOL. 45, 2001 INHIBITION OF CVB3 GENE EXPRESSION BY AS-ODNs 1045
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

CVB3 replicates and assembles very fast in HeLa cells. There-
fore, it is important to choose a suitable MOI for CVB3 to
evaluate the antiviral effects of AS-ODNs effectively. MOIs of
0.1, 0.01, and 0.001 were tested with the AS-ODNs at a final
concentration of 10 mM. We found that an MOI of 0.01 was
the best choice for this experiment because the most meaning-
ful inhibitory effects were obtained under this condition. The
third parameter was the dosage of AS-ODNs. ODNs at two
different final concentrations (1 and 10 mM) were evaluated
under the optimal conditions mentioned above. Ten micromo-
lar appeared to be the best dosage for this in vitro evaluation
system (see Fig. 2). Under these conditions, additional exper-
iments were conducted by transfection of control ODN (AS-S)
to determine the toxic effects on HeLa cell growth with the
treatment using Lipofectin only as AS-S’s negative control.
The results demonstrated no significant differences between
the AS-S and Lipofectin groups in terms of cell growth, CVB3
VP1 synthesis, and RNA replication, indicating that AS-S is a
reliable control AS-ODN (data not shown). Last, the active
AS1, AS-7, and their corresponding scrambled and reverse
ODN controls (10 mM) were simply added to cell culture
without transfection for 6 h, and no cellular toxicity on HeLa
cells in terms of morphology and cell count was found.
Inhibitory effects of AS-ODNs on CVB3 VP1 protein synthe-
sis. To evaluate the effects of AS-ODNs on CVB3 translation,
viral structural protein VP1 was detected by Western blotting
after transfection. Since CVB3 RNA encodes a single long
polyprotein including structural and nonstructural proteins,
the synthesis of structural protein VP1 fully represents CVB3
translation efficiency. In order to compare the antiviral action
of each AS-ODN with the negative-control oligomer AS-S, the
statistically analyzed means and SD were normalized by a
value which converted the VP1 mean of control group AS-S to
100. Two different doses of ODNs were used to evaluate their
antiviral activities. When a 10 mM concentration ODNs was
used (Fig. 2), several oligomers showed potent antiviral activ-
ity. Of these oligomers, AS1 and AS7 blocking the termini of 59
and 39 UTRs showed the strongest inhibitory effects on VP1
synthesis. Compared with the negative controls (AS-S), the
percent inhibition values of CVB3 VP1 synthesis were 84.6,
75.2, 40.6, 56.3, and 88.5 for AS-1, AS-2, AS-3, AS-4, and AS-7,
respectively. However, no marked inhibitory activities were
observed in groups AS-5 and AS-6. On the contrary, ODN
AS-5 slightly stimulated the synthesis of CVB3 VP1, which may
be due to experimental variation, since similar results were not
obtained from other evaluations of the same ODN.
The evaluation was also performed at a final AS-ODN con-
centration of 1 mM. Similar inhibitory patterns were observed
but at a lower degree of inhibition (Fig. 3) compared with
treatment at the 10 mM dosage. The percentages of inhibition
for CVB3 VP1 synthesis were 67.9, 55.7, 33.1, 55.3, and 72.8 for
AS-1, AS-2, AS-3, AS-4, and AS-7, respectively. Again, no
significant inhibitory activities for AS-5 and AS-6 were ob-
served.
Based on the first round of evaluation, the two most active
oligomers, AS-1 and AS-7, were chosen for further evaluation.
The specificities of these two oligomers were confirmed by
using additional corresponding scrambled and reverse se-
quences as negative controls. At 10 mM dosage, the percent
inhibition values of CVB3 VP1 synthesis were 70.6 for AS-1
and 73.7 for AS-7 compared with the scrambled control value
(AS-1-s or AS-7-s). There were significant differences between
AS and scrambled oligomers. Similar inhibitory results were
obtained with reverse control groups, namely, 66.5 for AS-1
and 76.7 for AS-7 (Fig. 4).
Inhibitory effects of AS-ODNs on CVB3 RNA replication. To
evaluate the effects of AS-ODNs on CVB3 replication, viral
genomic RNA was amplified and quantitatively analyzed by
FIG. 2. Inhibitory effects of AS-ODNs, at a final concentration of 10 mM, on CVB3 structural protein VP1 synthesis. (A) Western blot analysis.
HeLa cells were transfected with 10 mM AS-ODNs by a Lipofectin method for 6 h, infected with CVB3 at an MOI of 0.01 for 1 h, and then
incubated in complete MEM for 24 h. The cell-free supernatants containing viral proteins were fractionated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. The viral protein VP1 was detected by Western blotting and enhanced chemiluminescence analysis using rabbit
antiserum to VP1. Seven AS-ODNs and a control oligomer (AS-S) are indicated above the lanes and correspond to the bars in panel B. (B)
Quantitation of VP1 protein in the inhibition assay using the AS-ODNs indicated in panel A. The VP1 bands were measured by scanning X-ray
film using a densitometer. The mean density of each product was calculated with respect to the control values as described in Materials and
Methods. Each experiment was repeated four times. The SD values of data are shown as error bars in the figures. The differences between the
values for AS-S and ODNs AS-1, AS-2, AS-3, AS-4, and AS-7 were statistically significant (P , 0.01).
1046 WANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

RT-PCR and densitometry. We performed PCR with different
numbers of cycles and found that 20 cycles was optimal for
demonstrating the most significant differences among samples
treated with different AS-ODNs. In order to compare the
antiviral action of each AS-ODN with the negative-control
oligomer, the statistically analyzed means and SD were nor-
malized by a value, which converted the CVB3 cDNA mean of
the control group AS-S to 100. Two different doses of AS-
ODNs were used to evaluate their antiviral activities. When 10
mM ODN was applied (Fig. 5), the inhibitory trend on CVB3
RNA synthesis was quite similar to that seen for VP1 synthesis.
Of the seven oligomers, AS-1 and AS-7 blocking the termini of
59 and 39 UTRs, respectively, again showed the strongest an-
tiviral activity. Compared with the control oligomer AS-S, the
percentages of inhibition were 88.2, 63.3, 45.5, 59.9, and 87.9
for AS-1, AS-2, AS-3, AS-4, and AS-7, respectively. There
were no remarkable inhibitory effects on CVB3 RNA synthesis
for AS-5 and AS-6. When 1 mM AS-ODN was employed,
similar data were obtained (Fig. 6). The percentages of inhi-
bition for CVB3 RNA synthesis were 81.2, 58.1, 26.1, 50.9, and
79.1 for AS-1, AS-2, AS-3, AS-4, and AS-7, respectively. No
significant inhibitory effects were observed in groups AS-5 and
AS-6. Again, the evaluation was further conducted to confirm
the specificity of the most potent oligomers AS-1 and AS-7 by
FIG. 3. Inhibitory effects of AS-ODNs, at a final concentration of 1 mM, on CVB3 structural protein VP1 synthesis. HeLa cells were transfected
with 1 mM AS-ODN by a Lipofectin method for 6 h, infected with CVB3 at an MOI of 0.01 for 1 h, and then incubated in complete MEM for
24 h. The cell-free supernatants were collected by centrifugation. The viral protein VP1 in supernatants was detected and quantitated by the
methods described in the legend to Fig. 2. Each experiment was repeated four times. The differences between the values for AS-S and ODNs AS-1,
AS-2, AS-3, AS-4, and AS-7 were statistically significant (P , 0.01).
FIG. 4. Further controlled evaluation of specific inhibition of AS-1 and AS-7, at a final concentration of 10 mM, on CVB3 structural protein
VP1 synthesis. HeLa cells were transfected with 10 mM AS-1, AS-1-s, AS-1-r, AS-7, AS-7-s, or AS-7-r by a Lipofectin method and then infected
with CVB3 at an MOI of 0.01. After incubation, the cell-free supernatants were collected, and the viral protein VP1 for each sample was detected
(A) and quantitated by densitometry (B). The means 6 SD of the controls were normalized to a value of 100. Mean 6 SD values of ODN
treatments were calculated with respect to the control values. The data from four independent experiments were analyzed. The difference between
each control and its respective ODN was statistically significant (P , 0.01).
VOL. 45, 2001 INHIBITION OF CVB3 GENE EXPRESSION BY AS-ODNs 1047
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

using their corresponding scrambled and reverse sequence as
negative controls. At 10 mM dosage, compared with the scram-
bled control AS-1-s and AS-7-s, the percentages of inhibition
of CVB3 RNA synthesis were 79.6 for AS-1 and 79.7 for AS-7.
There were significant differences of antiviral activity between
AS and scrambled oligomers. Similar data were obtained with
reverse control oligomers (80.3 for AS-1 and 78.7 for AS-7)
(Fig. 7).
Inhibitory effect of AS-ODN on CVB3 infectivity. A viral
plaque assay was employed to measure the antiviral activity of
AS-ODNs at the optimal dosage of 10 mM determined above.
The viral plaques were counted 3 days after overlaying soft
agar on a HeLa cell monolayer (Fig. 8), and the numbers of
PFU per milliliter were calculated. By normalizing the data
with that of the negative-control AS-S, we found that AS-
ODNs AS-1 and AS-7 showed the strongest antiviral activities
FIG. 5. Inhibitory effects of AS-ODNs, at a final concentration of 10 mM, on CVB3 RNA replication. HeLa cells were transfected with 10 mM
AS-ODN by a Lipofectin method for 6 h, infected with CVB3 at an MOI of 0.01 for 1 h, and then incubated in complete MEM for 24 h. The
cell-free supernatants were collected by centrifugation. Viral RNA was prepared from 100 ml of the resulting supernatant of each treatment.
RT-PCR was conducted for 20 cycles under standard conditions. (A) Agarose gel electrophoresis of RT-PCR products. Twenty microliters of PCR
product from each treatment was run on a 0.8% agarose gel. The AS-ODNs and the control (AS-S) are marked above the lanes. (B) Quantitation
of the viral RNA in the inhibition assays using AS-ODNs corresponding to those in panel A. The bands of RT-PCR products were scanned using
a densitometer. The mean density of each band was calculated with respect to that of the control as described in Materials and Methods. Each
experiment was repeated three times. The differences between the values for AS-S and ODNs AS-1, AS-2, AS-3, AS-4, and AS-7 were statistically
significant (P , 0.01).
FIG. 6. Inhibitory effects of AS-ODNs, at a final concentration of 1 mM, on CVB3 RNA replication. HeLa cells were transfected with 1 mM
AS-ODN by a Lipofectin method for 6 h, infected with CVB3 at an MOI of 0.01 for 1 h, and then incubated in complete MEM for 24 h. Viral
RNA was detected by RT-PCR (A) and quantitated by densitometry (B) as described in the legend to Fig. 5. Each experiment was repeated three
times. The differences between the values for AS-S and ODNs AS-1, AS-2, AS-3, AS-4, and AS-7 were statistically significant (P , 0.01).
1048 WANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

(78.4 and 77.4%), followed by AS-2 (50.7%) and AS-4 (45%).
AS-3 showed only slight inhibition (18%) of plaque-forming
ability. However, AS-5 and AS-6 did not demonstrate notice-
able inhibition of CVB3 infectivity. These results correlated
very well with those obtained by measuring inhibitory effects
on CVB3 VP1 production and RNA replication. These obser-
vations were further confirmed by plaque assays with AS-1 and
AS-7 and their corresponding controls mentioned above. The
FIG. 7. Further controlled evaluation of specific inhibition of AS-1 and AS-7, at a final concentration of 10 mM, on CVB3 RNA replication.
HeLa cells were transfected with 10 mM AS-1, AS-1-s, AS-1-r, AS-7, AS-7-s, or AS-7-r by a Lipofectin method and followed by infection with CVB3
at an MOI of 0.01. After incubation, the cell-free supernatants were collected and the viral RNA was detected by RT-PCR (A) and quantitated
by densitometry (B) as described in the legend to Fig. 4. Each experiment was repeated three times. The difference between each control and its
respective ODN was statistically significant (P , 0.01).
FIG. 8. Inhibition of plaque formation by AS-ODNs at a final concentration of 10 mM. (A) Viral plaque assay. HeLa cell monolayers at ;90%
confluency were infected for 1 h with supernatants containing viral particles and diluted 1:10, and then were overlaid with 2 ml of 0.75% Bacto
Agar–MEM. Seventy-two hours postinfection, cells were fixed with Carnoy’s fixative and stained with 1% crystal violet. The plaques were counted,
and the viral titer (PFU per milliliter) was calculated. The bars in panel B represent the values (PFU per milliliter) obtained from four experiments.
The differences in values (PFU per milliliter) between AS-S and ODNs AS-1, AS-2, AS-3, AS-4, and AS-7 were statistically significant (P , 0.01).
VOL. 45, 2001 INHIBITION OF CVB3 GENE EXPRESSION BY AS-ODNs 1049
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

percent inhibition values of CVB3 infectivity normalized
against scrambled and reverse controls are 80.5 and 76.7 for
AS-1 and 79.9 and 80.1 for AS-7, respectively (Fig. 9).
DISCUSSION
The most important factor in determining effectiveness and
specificity of AS-ODNs is the selection of target sites within
CVB3 RNA. The unusually long 59 UTR of CVB3 RNA forms
a highly ordered secondary structure and plays an important
role in controlling viral translation, replication, and cardioviru-
lence (10, 33, 54). Our recent mutational mapping of the IRES
has confirmed that ribosomes initiate translation in CVB3 by
binding to the polypyrimidine stretch in the IRES of 59 UTR
and then migrating to the AUG initiation codon (54). In this
situation, AS-ODNs presumably block translation through the
inhibition of binding of ribosomes and/or other RNA-binding
proteins that are essential in forming the ribosomal complex
and in the migration of ribosomal subunits along the mRNA.
Therefore, it is possible that the AS-ODN (AS-2) functions in
translation arrest by blocking the landing of the ribosome
and/or RNA-binding proteins at the IRES (Fig. 1). In addition,
it should be noted that the polypyrimidine tract located be-
tween stem-loops G and H (59UUCAUUUU39, nt 562 to 569)
appears as an open single-stranded structure based on the
computer-predicted secondary structure model (54). This im-
plies that AS-ODN can easily form stable hybridization at this
region. From this point of view, it is obvious why this AS-ODN
showed such potent inhibitory effects, while another AS-ODN
(AS-3) also binding to IRES region did not show the same
potency in inhibiting the synthesis of CVB3 VP1. Because the
target site of AS-3 is in a hairpin-like secondary structure, it is
difficult for AS-3 to form a stable duplex with the viral se-
quence. Similar situations occurred for AS-5 and AS-6, target-
ing two hairpin-like structures of 39 UTR. As for AS-4 covering
the translation start codon, the strong inhibitory effect is ex-
pected, since AS-ODNs blocking the translation initiation
codon should theoretically show a certain degree of inhibitory
effect on a mRNA’s translation initiation. Similar observations
have been reported by other investigators (2, 5, 39, 49).
Interestingly, two of the AS-ODNs with the strongest inhib-
itory effects are located at both ends of CVB3 RNA. It is
known that the 59 and 39 UTRs of the CVB3 RNA form highly
ordered tertiary structures serving as recognition features for
the RNA-binding domain of host proteins and have important
cis-acting functions in the replication and translation of the
FIG. 9. Further controlled evaluation of specific inhibition of plaque formation by AS-1 and AS-7 at a final concentration of 10 mM. All the
procedures are the same as described in the legend to Fig. 8 except that two more controlled oligomers, scrambled and reverse sequences, for each
AS-ODN were used. (A) Viral plaque assay. (B) Values (PFU per milliliter) for the control and treatment in each group. Each experiment was
repeated three times. The means 6 SD of controls were normalized to 100, and the values of treatments were calculated accordingly. The
difference between each control and its respective ODN treatment was statistically significant (P , 0.01).
1050 WANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

RNAs (8, 35, 37, 41, 53, 54). Evidence to date suggests that a
cloverleaf structure of poliovirus formed by the 59-terminal 88
nt of the RNA binds viral proteins 3AB and 3CD and a host
protein of 36 kDa (16). A cellular factor that specifically binds
to the 39 UTR of poliovirus, coxsackievirus, and rhinovirus was
detected. Mutations within the 39 UTR, which decrease the
affinity of the RNA for the cellular factor, decrease RNA
replication and virus viability (36). Furthermore, our concur-
rent UV cross-linking experiments suggest that certain host
proteins are able to interact with both the 59 UTR (nt 1 to 249)
and 39 UTR (nt 7299 to 7399) terminal regions of CVB3
genome (P. K. M. Cheung, C. M. Carthy, L. Bohunek, A. K.
Wang, J. E. Wilson, B. M. McManus, and D. C. Yang, unpub-
lished data). The extensive stem-loops and secondary struc-
tures within the 59 and 39 UTRs are likely the recognition sites
of certain common binding proteins connecting both ends of
the viral genome (28–30). The interaction between the 59 and
39 ends of the viral genome via common binding protein(s) has
been suggested to play a crucial role in viral replication and
possibly in translation initiation (28–30, 52). In addition, there
has been evidence of the viral 59 and 39 UTRs interacting to
enhance viral translation and/or transcription (42, 52); hence,
a closed-loop translation model of mRNA has been proposed
(6, 12, 46, 51). Thus, blocking the terminal region of CVB3
RNA either in the 59 UTR or 39 UTR can possibly inhibit
binding of cellular proteins which serve as translation or rep-
lication initiation factors and in turn, block the formation of a
circular replication unit of viral RNA. Such a mechanism may
explain why two AS-ODNs (AS-1 and AS-7) blocking termini
of CVB3 RNA showed such powerful inhibitory effects. In
addition, CVB3 translation and replication may affect each
other and thereby result in a negative-feedback effect. When
translation is inhibited, the synthesis of structural proteins
(such as VP1) and the nonstructural proteins (including RNA-
dependent RNA polymerase) essential for virus replication is
reduced, which will make it difficult for viral RNA transcrip-
tion and particle assembly to occur. Decreased viral RNA
synthesis will in turn reduce translation efficiency. More im-
portantly, some of the AS-ODNs may block not only the land-
ing of translational machinery but also the binding of the
protein factors required within viral RNA for gene expression.
Therefore, the AS-ODNs can inhibit CVB3 gene expression at
both the translational and transcriptional levels.
The patterns of inhibition observed in this study were con-
sidered sequence specific regarding AS-1 and AS-7 because
strict ODN controls (scrambled and reverse) for AS-1 and
AS-7 were used. However, it still can be argued that the anti-
viral effects may be due in part to the so-called sequence-
dependent but nonantisense effect in which sequence-specific
interactions between ODNs and cellular proteins occur (7, 45,
47, 48). Since our AS-ODNs did not produce notable negative
effects on HeLa cell growth in the tests of toxicity, this possi-
bility is unlikely. Additionally, several reports indicated the
nonsequence-specific effects of ODNs, particularly with the
chemically modified molecules. For example, ODNs can bind
in a sequence-independent manner the gp120 protein of hu-
man immunodeficiency virus type 1, viral polymerases, RNase
H, bovine serum albumin, the receptor for platelet-derived
growth factor, and other cellular proteins (7, 13, 43–45).
Whether this similar event can happen in CVB3 needs to be
explored in the future to determine the detailed mechanisms of
antiviral action of our AS oligomers.
In conclusion, AS phosphorothioate ODNs targeting the
termini of 59 and 39 UTRs, the core sequence of the IRES, and
the translation initiation codon region possess specific and
strong anti-CVB3 activity. This is the first report to demon-
strate that translation and replication of CVB3 in tissue culture
cells can be specifically inhibited by AS-ODNs. Our observa-
tions suggest that these oligomers have great potential for
further development as effective, ameliorative therapeutic
agents for the control of coxsackievirus-induced heart, pan-
creas, brain, liver, and muscle diseases.
ACKNOWLEDGMENTS
We thank Reinhard Kandolf, University of Tubingen, Germany for
providing us with CVB3.
This work was supported in part by a grant from the Medical Re-
search Council of Canada (D. C. Yang and B. M. McManus), student-
ships from the Heart and Stroke Foundation of British Columbia and
Yukon (P. Cheung and C. Carthy).
REFERENCES
1. Abdou, S., J. Collomb, F. Sallas, A. Marsura, and C. Finance. 1997. Beta-
cyclodextrin derivatives as carriers to enhance the antiviral activity of an
antisense oligonucleotide directed toward a coronavirus intergenic consen-
sus sequence. Arch. Virol. 142:1585–1602.
2. Abe, T., T. Hatta, K. Takai, H. Nakashima, T. Yokota, and H. Takaku. 1998.
Inhibition of influenza virus replication by phosphorothioate and liposomally
encapsulated oligonucleotides. Nucleosides Nucleotides 17:472–478.
3. Abelmann, W. H. 1973. Viral myocarditis and its sequelae. N. Engl. J. Med.
24:145–152.
4. Agrawal, S. 1999. Importance of nucleotide sequence and chemical modifi-
cations of antisense oligonucleotides. Biochim. Biophys. Acta 1489:53–68.
5. Alt, M., R. Renz, P. H. Hofschneider, G. Paumgartner, and W. H. Casel-
mann. 1995. Specific inhibition of hepatitis C viral gene expression by anti-
sense phosphorothioate oligodeoxynucleotides. Hepatology 22:707–717.
6. Borman, A. M., F. G. Deliat, and K. M. Kean. 1994. Sequences within the
poliovirus internal ribosome entry segment control viral RNA synthesis.
EMBO J. 13:3149–3157.
7. Crooke, S. T., and C. F. Bennet. 1996. Progress in antisense oligonucleotide
therapeutics. Annu. Rev. Pharmacol. Toxicol. 36:107–129.
8. Currey, K. M., and B. A. Shapiro. 1997. Higher structures of coxsackievirus
B3 59 nontranslated region RNA. Curr. Top. Microbiol. Immunol. 223:169–
190.
9. Dorner, A. I., B. L. Semler, R. J. Jackson, R. Hanecak, E. Duprey, and E.
Wimmer. 1984. In vitro translation of poliovirus RNA: utilization of internal
initiation sites in reticulocyte lysate. J. Virol. 50:507–514.
10. Dunn, J. J., N. M. Chapman, S. Tracy, and J. R. Romero. 2000. Genomic
determinants of cardiovirulence in coxsackievirus B3 clinical isolates: loca-
tion to the 59 nontranslated region. J. Virol. 74:4787–4797.
11. Fox, J. L. 1998. FDA approves first antisense drug for CMV retinitis. ASM
News 64:678–679.
12. Gallie, D. R. 1991. The cap and poly (A) tail function synergistically to
regulate messenger RNA translational efficiency. Genes Dev. 5:2108–2116.
13. Gao, W. Y., F. S. Han, C. Storm, W. Egan, and Y. C. Cheng. 1992. Phos-
phorothioate oligonucleotides are inhibitors of human DNA polymerases
and RNase H: implications for antisense technology. Mol. Pharmacol. 41:
223–229.
14. Grist, N. R., and D. Reid. 1993. Epidemiology of viral infections of the heart,
p. 23–31. In J. E. Banatvala (ed.), Viral infections of the heart. Edward
Arnold, London, England.
15. Haller, A. A., J. C. Nguyen, and B. L. Semler. 1995. Minimum internal
ribosomal entry site required for poliovirus infectivity. J. Virol. 67:7461–
7471.
16. Harris, K. S., W. Xiang, L. Alexander, W. S. Lane, A. V. Paul, and E.
Wimmer. 1994. Interaction of poliovirus polypeptide 3CDpro with the 59 and
39 termini of the poliovirus genome. Identification of viral and cellular
cofactors needed for efficient binding. J. Biol. Chem. 269:27004–27014.
17. Hatta, T., Y. Nakagawa, K. Takai, S. Nakada, T. Yokota, and H. Takaku.
1996. Inhibition of influenza virus RNA polymerase and nucleoprotein gene
expression by unmodified, phosphorothioated and liposomally encapsulated
oligonucleotides. Biochem. Biophys. Res. Commun. 223:341–346.
18. Hershey, J. W. B. 1991. Translation control in mammalian cells. Annu. Rev.
Biochem. 60:717–755.
19. Hosenpud, J. D., R. J. Novick, T. J. Breen, and O. P. Daily. 1994. The registry
VOL. 45, 2001 INHIBITION OF CVB3 GENE EXPRESSION BY AS-ODNs 1051
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

of the International Society for Heart and Lung Transplantation: eleventh
official report—1994. J. Heart Lung Transplant. 13:561–570.
20. Jackson, R. J., M. T. Howell, and A. Kaminski. 1990. The novel mechanism
of initiation of picornavirus RNA translation. Trends Biochem. Sci. 15:477–
483.
21. Jang, S. K., M. V. Davies, R. J. Kaufman, and E. Wimmer. 1989. Initiation
of protein synthesis by internal entry of ribosomes into the 59 nontranslated
region of encephalomyocarditis virus RNA in vivo. J. Virol. 63:1651–1660.
22. Jang, S. K., H.-G. Kräusslich, M. J. H. Nicklin, G. M. Duke, A. C. Palmen-
berg, and E. Wimmer. 1988. A segment of the 59 nontranslated region of
encephalomyocarditis virus RNA directs internal entry of ribosomes during
in vitro translation. J. Virol. 62:2636–2643.
23. Johnson, R. A., and I. Palacios. 1982. Dilated cardiomyopathy in the adult.
N. Engl. J. Med. 307:119–126.
24. Juliano, R. L., and S. Akhtar. 1992. Liposomes as a drug delivery system for
antisense oligonucleotides. Antisense Res. Dev. 2:165–176.
25. Klump, W. M., I. Bergmann, B. C. Muller, D. Ameis, and R. Kandolf. 1990.
Complete nucleotide sequence of infectious coxsackievirus B3 cDNA: two
initial 59 uridine residues are regained during plus-strand RNA synthesis.
J. Virol. 64:1573–1583.
26. Kozak, M. 1989. The scanning model for translation: an update. J. Cell Biol.
108:229–241.
27. Krausslich, H. G., and E. Wimmer. 1988. Viral proteinases. Annu. Rev.
Biochem. 57:701–754.
28. Lahser, F. C., L. E. Marsh, and T. C. Hall. 1993. Contributions of the brome
mosaic virus RNA-3 39-nontranslated region to replication and translation.
J. Virol. 67:3295–3303.
29. Lai, M. M. C. 1998. Cellular factors in the transcription and replication of
viral RNA genomes: a parallel to DNA dependent RNA transcription. Vi-
rology 244:1–12.
30. Leathers, V., R. Tanguay, M. Kobayashi, and D. R. Galli. 1993. A phyloge-
netically conserved sequence within viral 39 untranslated RNA pseudoknots
regulates translation. Mol. Cell. Biol. 13:5331–5347.
31. Lee, Y. F., A. Nomoto, B. M. Detjen, and E. Wimmer. 1977. The genome
linked protein of picornaviruses: a protein covalently linked to poliovirus
genome RNA. Proc. Natl. Acad. Sci. USA 74:59–63.
32. Lisziewicz, J., D. Sun, M. Klotman, S. Agrawal, P. Zamecnik, and R. Gallo.
1992. Specific inhibition of human immunodeficiency virus type 1 replication
by antisense oligonucleotides: an in vitro model for treatment. Proc. Natl.
Acad. Sci. USA 89:11209–11213.
33. Liu, Z. W., C. M. Carthy, P. K. Cheung, L. Bohunek, J. E. Wilson, B. M.
McManus, and D. C. Yang. 1999. Structural and functional analysis of the 59
untranslated region of coxsackievirus B3 RNA: in vivo translational and
infectivity studies of full-length mutants. Virology 265:206–217.
34. McManus, B. M., and R. Kandolf. 1991. Myocarditis: evolving concepts of
cause, consequence, and control. Curr. Opin. Cardiol. 6:418–427.
35. Melchers, W. J. G., J. G. J. Hoenderop, H. J. Bruinsslot, C. W. A. Pleij, E. V.
Pilipenko, V. I. Agol, and J. M. D. Galama. 1997. Kissing of the two pre-
dominant hairpin loops in the coxsackie B virus 39 untranslated region is the
essential structural feature of the origin of replication required for negative-
strand RNA synthesis. J. Virol. 71:686–696.
36. Mellits, K. H., J. M. Meredith, J. B. Rohll, D. J. Evans, and J. W. Almond.
1998. Binding of a cellular factor to the 39 untranslated region of the RNA
genomes of entero- and rhinoviruses plays a role in virus replication. J. Gen.
Virol. 79:1715–1723.
37. Mirmomeni, M. H., P. J. Hughes, and G. Stanway. 1997. An RNA tertiary
structure in the 39 untranslated region of enteroviruses is necessary for
efficient replication. J. Virol. 71:2363–2370.
38. Moriya, K., M. Matsukura, K. Kurokawa, and K. Koike. 1996. In vivo
inhibition of hepatitis B virus gene expression by antisense phosphorothioate
oligonucleotides. Biochem. Biophys. Res. Commun. 218:217–223.
39. Offensperger, W. B., S. Offensperger, E. Walter, K. Teubner, G. Igloi, H. E.
Blum, and W. Gerok. 1993. In vivo inhibition of duck hepatitis B virus
replication and gene expression by phosphorothioate modified antisense
oligodeoxynucleotides. EMBO J. 12:1257–1262.
40. Player, M. R., L. Barnard, and P. F. Torrence. 1998. Potent inhibition of
respiratory syncytial virus replication using a 2-5A-antisense chimera tar-
geted to signals within the virus genomic RNA. Proc. Natl. Acad. Sci. USA
95:8874–8879.
41. Rohll, J. B., D. H. Moon, D. J. Evans, and J. W. Almond. 1995. The 39
untranslated region of picornavirus RNA: features required for efficient
genome replication. J. Virol. 69:7835–7844.
42. Schultz, D. E., C. C. Hardins, and S. M. Lemon. 1996. Specific interaction of
glyceraldehyde 3-phosphate dehydrogenase with the 59 nontranslated RNA
of hepatitis A virus. J. Biol. Chem. 271:14134–14142.
43. Stein, C. A. 1995. Dose antisense exist? Nat. Med. 1:119–121.
44. Stein, C. A., and Y. C. Cheng. 1993. Antisense oligonucleotides as therapeu-
tic agents—is the bullet really magical? Science 261:1004–1012.
45. Stein, C. A., and A. M. Krieg. 1994. Problems in interpretation of data
derived from in vitro and in vivo use of antisense oligodeoxyribonucleotides.
Antisense Res. Dev. 4:67–69.
46. Tarun, S. Z., and A. B. Sachs. 1997. Association of the yeast poly (A) tail
binding protein with translation initiation factor eIF4G. EMBO J. 15:7168–
7177.
47. Vaerman, J. L., C. Lammineur, P. Lewalle, F. Deldime, M. Blumenfeld, and
P. Martiat. 1995. BCR-ABL antisense oligodeoxyribonucleotides suppress
the growth of leukemic and normal hematopoietic cells by a sequence spe-
cific but nonantisense mechanism. Blood 86:3891–3896.
48. Wagner, R. W. 1995. The state of art in antisense research. Nat. Med.
1:1116–1118.
49. Wakita, T., and J. R. Wands. 1994. Specific inhibition of hepatitis C virus
expression by antisense oligodeoxynucleotides. J. Biol. Chem. 269:14205–
14210.
50. Wang, J., J. M. Bakkers, J. M. Galama, H. J. Bruins Slot, E. V. Pilipenko,
V. I. Agol, and W. J. Melchers. 1999. Structural requirements of the higher
order RNA kissing element in the enteroviral 39 UTR. Nucleic Acids Res.
27:485–490.
51. Wells, S. E., P. W. Hillner, R. D. Vale, and A. B. Sachs. 1998. Circulization
of mRNA by eukaryotic translational initiation factors. Mol. Cell 2:135–140.
52. Witherell, G. W., and E. Wimmer. 1994. Encephalomyocarditis virus internal
ribosomal entry site RNA-protein interactions. J. Virol. 68:3183–3192.
53. Xiang, W., K. S. Harris, L. Alexander, and E. Wimmer. 1995. Interaction
between the 59-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential
for RNA replication. J. Virol. 69:3658–3667.
54. Yang, D. C., J. E. Wilson, D. R. Anderson, L. Bohunek, C. Cordeiro, R.
Kandolf, and B. M. McManus. 1997. In vitro mutational and inhibitory
analysis of the cis-acting translational elements within the 59 untranslated
region of coxsackievirus B3: potential targets for antiviral action of antisense
oligomers. Virology 228:63–73.
1052 WANG ET AL. ANTIMICROB. AGENTS CHEMOTHER.
on D
ecem
ber 4, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://aac.asm
.org/
D
ow
nloaded from

JOURNAL OF VIROLOGY, Apr. 1990, p. 1573-1583 Vol. 64, No. 4
0022-538X/90/041573-11$02.00/0
Copyright C) 1990, American Society for Microbiology
Complete Nucleotide Sequence of Infectious Coxsackievirus B3
cDNA: Two Initial 5' Uridine Residues Are Regained
during Plus-Strand RNA Synthesis
WOLFGANG M. KLUMP,t INGRID BERGMANN, BARBARA C. MULLER, DETLEV AMEIS,
AND REINHARD KANDOLF*
Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Federal Republic of Germany
Received 23 June 1989/Accepted 20 December 1989
A full-length reverse-transcribed, infectious cDNA copy of coxsackievirus B3 (CVB3) was used to determine
the nucleotide sequence of this cardiotropic enterovirus. Comparison of the nucleotide sequence and the
deduced amino acid sequence of the viral precursor polyprotein with the sequences of other group B
coxsackieviruses (CVB1 and CVB4) demonstrates a high degree of genetic identity. They share about 80%
homology at the nucleotide level and about 90% when the amino acid sequences of the polyproteins are
compared. The potential processing sites of the coxsackievirus polyproteins, as deduced from alignment with
the poliovirus sequence, are conserved among these enteroviruses with the exception of the cleavage sites
between VP1 and 2APro and between polypeptides 2B and 2C. Comparison of the 5' termini of the enteroviral
genomes reveals a high degree of identity, including the initial 5' consensus UUAAAACAGC, suggesting
essential functions in virus replication. An important finding concerning the molecular basis of infectivity was
that both recombinant CVB3 cDNA and in vitro-synthesized CVB3 RNA transcripts are infectious, although
two initial 5' uridine residues found on the authentic CVB3 RNA were missing. Here, we report that
cDNA-generated CVB3, as well as CVB3 generated by in vitro-synthesized RNA transcripts, regains the
authentic initial 5' uridine residues during replication in transfected cells, indicating that the picornaviral
primer molecule VPg-pUpU may be uridylylated in a template-independent fashion. The generation of virus or
virus mutants with infectious recombinant CVB3 cDNA and in vitro-synthesized infectious CVB3 transcripts
should provide a valuable means for studying the molecular basis of the pathogenicity of this cardiotropic
enterovirus.
Enteroviruses of the human Picornaviridae, such as the
group B coxsackieviruses (types 1 to 5), are the most
common agents known to cause viral myocarditis (1, 12, 24,
34, 53). Other human enteroviruses, comprising at present
over 70 serotypes (e.g., various group A coxsackieviruses
and echoviruses), have also been associated with human
heart disease. These agents are capable of producing dilated
cardiomyopathy of acute onset or leading to life-threatening
arrhythmias. Particularly intriguing is the concept of entero-
virus persistence in chronic dilated cardiomyopathy evolv-
ing from acute or subacute infections of the human heart (13,
17).
The structure and molecular genetics of the enteroviruses
are well understood, chiefly by analogy with the extensively
studied poliovirus (for a review, see reference 52). The
coxsackieviruses possess a single-stranded RNA genome of
about 7,500 nucleotides (nts) of positive polarity, which is
covalently linked at the 5' end to a small virus-encoded
protein, VPg (3B, according to the systematic nomenclature
of picornavirus proteins [36]). The viral RNA, which is
infectious (22, 39), is polyadenylylated at the 3' end and
functions as an mRNA in the cytoplasm of infected cells. It
is translated into a large precursor polyprotein, which is
processed by virus-encoded proteinases to yield the mature
structural and nonstructural proteins (19). Replication of
picornaviral RNA has been studied extensively (52, and
references therein), although the individual steps in RNA
* Corresponding author.
t Present address: Department of Pharmacology M-036, School of
Medicine, University of California, San Diego, La Jolla, CA 92093.
replication, e.g., initiation of plus-strand RNA synthesis,
have not yet been elucidated unambiguously. Distinct pro-
teins may be involved in the formation of a replication
complex, including the virus-encoded RNA-dependent RNA
polymerase (3DPOI), VPg (3B) or its precursor molecule 3AB,
and possibly other virus- or host cell-encoded factors (2, 27,
52, 55).
In order to study the molecular basis of pathogenicity and
to introduce in situ hybridization as a diagnostic tool in
patients with a clinical suspicion of enteroviral heart disease,
we have previously cloned the genome of the cardiotropic
coxsackievirus B3 (CVB3), which had been propagated in
cultured human myocardial cells (14-16). Full-length re-
verse-transcribed recombinant cDNA generated infectious
virus antigenically identical to CVB3 upon transfection into
mammalian cells, demonstrating the biological intactness of
the cloned cDNA copy. The isolation of cDNA fragments
representing the genomic RNA of CVB3 has also been
described by others (20, 48), and a nucleotide sequence of
CVB3 based on subgenomic cDNA fragments has been
reported by Lindberg et al. (20). We now present the entire
nucleotide sequence of infectious, full-length reverse-tran-
scribed recombinant CVB3 cDNA and have compared this
with that of other human enteroviral genomes.
In this context, we report on transfection experiments
with circular recombinant CVB3 cDNA and in vitro-synthe-
sized CVB3 RNA transcripts, demonstrating that inherent
infectivity of these CVB3 constructs is independent of the
presence of two initial 5' uridine residues found on the
authentic viral RNA. Remarkably, the authentic viral 5'
terminus is restored in the RNA genome of both cDNA-
1573
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

1574 KLUMP ET AL.
derived CVB3, as well as in vitro-synthesized RNA-derived
virus progeny.
MATERIALS AND METHODS
Sequence determination. Full-length reverse-transcribed,
infectious CVB3 cDNA (16) was used to determine the
nucleotide sequence of CVB3. For this purpose, subgenomic
CVB3 cDNA fragments were inserted into the SmaI site of
pUC19 (54) and sequenced by the chain termination method
(3, 37). Fragments from the cloned CVB3 cDNA were
generated by digestion with BglI or PvuII and subsequent
incubation with nuclease Bal 31 or by digestion with one of
the frequently cleaving restriction endonucleases, HaeIII,
AluI, and RsaI. A total of 93% of the CVB3 nucleotide
sequence was obtained from both strands, and the remainder
was obtained from multiple determinations on one strand of
at least two independent subclones.
To determine the sequence of the very 5' end of the viral
genome, 1 ,ug of purified CVB3 RNA (16) was primed with
an 18-base-long synthetic oligonucleotide, complementary to
a region of viral RNA comprising nucleotide positions 62 to
78. Single-stranded cDNA was synthesized with reverse
transcriptase as previously described (16). The primer-ex-
tended cDNA was separated on an 8% denaturating poly-
acrylamide gel and sequenced by the method of Maxam and
Gilbert (23). Analogously, the 5' sequence of cDNA-gener-
ated CVB3, as well as the 5' sequence of viruses generated
by in vitro-synthesized CVB3 RNA, was obtained.
Computer analysis. The program library of the University
of Wisconsin Genetics Computer Group (6) was used for
sequence analysis on a Microa VAX II computer system.
Alignments were performed by the GAP program at a gap
weight penalty of 5 and a length weight penalty of 0.3.
Secondary structures and free energies for the 5' noncoding
(5' nc) region (bases 1 to 741) were calculated by the folding
program of Zuker and Stiegler (56).
Plasmid construction. A 7.5-kilobase EcoRI fragment ob-
tained by partial digestion of the infectious CVB3 cDNA
plasmid pCB3-M1 (16) was inserted in both orientations into
the dual-promoter plasmid pSPT18 (constructed at Pharma-
cia) containing both the SP6 and T7 promoter sequences,
diametrically opposed and separated by the multiple cloning
site of pUC-18 (54). The resulting recombinant plasmids
were designated pCB3/T7 and pCB3/SP6 (Fig. 1) and al-
lowed the in vitro synthesis of plus-strand CVB3 RNA under
the control of the T7 and SP6 promoter, respectively,
whereas minus-strand transcripts are synthesized under the
control of the opposite promoter. The nucleotide sequences
at the ligation junctions were determined directly from
plasmid DNA (3). The T7 promoter of pCB3/T7 directs
synthesis of plus-strand transcripts with 34 nonviral nts at
the 5' end. pCB3/SP6-derived SP6 transcripts contain 82
nonviral nucleotides at the 5' end (see legend to Fig. 1). Both
plasmids, pCB3/T7 and pCB3/SP6, were found to be infec-
tious upon transfection into HeLa cells.
In vitro transcription. Plasmid pCB3/T7 was digested with
Sall [cuts 65 nts after the 3' poly(A) tail], and the resulting
linear DNA was isolated by phenol extraction and ethanol
precipitation. RNA transcription was carried out essentially
as described previously (4, 50). Briefly, 1 ,ug of Sall-digested
pCB3/T7 DNA served as a template for plus-strand RNA
synthesis with T7 polymerase (400 U/ml) for 60 min at 37°C
in 50 RI that contained 40 mM Tris hydrochloride (pH 7.5),
10 mM NaCl, 6 mM MgCl2, 2 mM spermidine hydrochloride,
10 mM dithiothreitol, 500 ,uM each of ATP, CTP, UTP, and
T7 Eco RI T7 Eco RI
Pvu I (iPvuI(
SPS pCB3/T7 SPS pCB3/SP6
Sal I 10.6 kb Sal I s, 10.6 kb
Eco RI Eco RI
FIG. 1. Plasmids containing infectious CVB3 cDNA (16) under
the control of the T7 and SP6 promoters. Plasmids pCB3/T7 and
pCB3/SP6 were constructed by insertion of infectious CVB3 cDNA
(open bars) in both orientations into the dual-promoter plasmid
pSPT18 as described in Materials and Methods. Infectious plus-
strand CVB3 RNA was synthesized in vitro from plasmid pCB3/T7
following linearization with Sall or from plasmid pCB3/SP6 follow-
ing linearization with PvuI. Plus-strand CVB3 RNA transcripts
synthesized in vitro from pCB3/T7 under the control of the T7
promoter contain 34 nonviral nts at the 5' end: 5'GGGAGACCC
GAAUUCUCCAAGACAUCCCCCCCCCAAAACAGC.... Plus-
strand CVB3 RNA transcripts synthesized from pCB3/SP6 under
the control of the SP6 promoter contain 82 nonviral nts at the 5' end:
5'GAAUACAAGCUUGCAUGCCUGCAGGUCGACUCUAGAG
GAUCCCCGGGUACCGAGCUCGAAUUCUCCAAGACAUCCC
CCCCCCAAAACAGC.... Nucleotides underlined represent the 5'
terminal region of in vitro-synthesized CVB3 RNA. Note that two
initial 5' uridine residues of the authentic CVB3 RNA are missing
that are not contained in the infectious CVB3 cDNA (16). kb,
Kilobases; amp, ampicillin resistance.
GTP, bovine serum albumin (100 ,ug/ml), and RNasin (500
U/mI). After the DNA template was digested with DNase I
(20 U/ml) for 15 min at 37°C, RNA transcripts were isolated
by phenol extraction and ethanol precipitation. Analogously,
plus-strand RNA was synthesized from plasmid pCB3/SP6
after digestion with PvuI [cuts 1,652 nts after the poly(A)
tail], except that transcription was performed with SP6
polymerase.
Synthesis of minus-strand RNA was identical to that
described above, except that plasmid pCB3/T7 was linear-
ized with PvuI while plasmid pCB3/SP6 was linearized with
SalI and transcription was performed under the control of
the opposite promoter.
Transfection of cells and detection of CVB3. At 18 h before
being transfected, HeLa cells were plated onto 60-mm petri
dishes at about 5 x 105 cells per dish. RNA transfections
were performed with DEAE-dextran as a facilitator (49). A
total of 10 to 15 ,ug of in vitro-synthesized CVB3 RNA in 500
,ul of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
(HEPES)-buffered saline (137 mM NaCl, 5 mM KCI, 0.7 mM
Na2HPO4, 6 mM glucose, 20 mM HEPES adjusted to a pH
of 6.98 with 0.5 M NaOH) was mixed with 0.5 ml of
DEAE-dextran (1 mg/ml in HEPES-buffered saline). The
RNA-DEAE-dextran mixture was added to HeLa cells
(about 80% confluent) and was incubated at room tempera-
ture for 30 min; then, 4 ml of Dulbecco modified Eagle
minimal minimal medium was added and incubation was
continued for 2.5 h at 37°C. At the end of the incubation, the
cells were washed twice with Dulbecco modified Eagle
minimal medium and maintained in medium supplemented
with 10% fetal bovine serum and kanamycin at 50 pkg/ml.
Complete lysis of cell cultures transfected with plus-strand
RNA transcripts was observed at 48 to 96 h posttransfection.
The supernatant medium of lysed cultures was taken and
submitted to a specific antibody neutralization assay (16) to
examine the identity of transfection-derived viruses. In
addition, plaque assays (15) were carried out to determine
J. VIROL.
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

INFECTIOUS COXSACKIEVIRUS B3 cDNA 1575
specific infectivities in dilution experiments and to assess the
plaque morphology of transfection-derived first-passage vi-
rus.
DNA transfections of circular recombinant CVB3 plas-
mids were performed with HeLa cells as described previ-
ously (16).
Materials. Sources were as follows: plasmid pSPT18,
Pharmacia; avian myeloblastosis virus reverse transcriptase,
J. W. Beard, Life Sciences (St. Petersburg, Fla.); Bal 31,
Bethesda Research Laboratories, Inc.; RNasin and all other
enzymes, Boehringer Mannheim Biochemicals; dideoxynu-
cleotide sequencing kit, New England BioLabs, Inc.; chem-
ical sequencing kit, Dupont, NEN Research Products; [a-
32P]dCTP and [_y-32P]ATP, Amersham Corp.
RESULTS
Nucleotide sequence of the CVB3 genome. The nucleotide
sequence of the CVB3 genome was determined from sub-
cloned fragments of infectious recombinant CVB3 cDNA.
The sequence of the two initial 5' uridine residues of the
genome that was not contained in the infectious CVB3
cDNA was obtained from primer-extended cDNA with a
synthetic oligodeoxynucleotide corresponding to positions
62 to 78 of the authentic viral RNA. The complete nucleotide
sequence of CVB3 is shown in Fig. 2, including an alignment
with the genomes of CVB1 (10) and CVB4 (11). The CVB3
genome consists of 7,399 nts in addition to a 3' terminal
poly(A) stretch. The 5' nc region comprises 741 nts, followed
by an open reading frame that encodes the viral polyprotein
consisting of 2,186 codons with a start codon (AUG) at
position 742 and a stop codon (UAG) at position 7297. The 3'
nc region consists of 100 nts. Results from independently
obtained CVB3 cDNA clones revealed that up to 59 adenine
residues constitute the 3' poly(A) stretch. The base compo-
sition of the CVB3 genome excluding the poly(A) stretch is
A (28.8%), C (23.3%), G (24.5%), and U (23.4%).
Comparison of the sequence of the infectious CVB3
cDNA with the CVB3 nucleotide sequence reported by
Lindberg et al. (20) showed 65 nucleotide changes through-
out the genome that result in 41 amino acid changes.
Regarding the partial CVB3 sequence reported by Tracy et
al. (48) comprising the 5' part of the genome (nts 1 through
3822), 41 nts were found to be changed corresponding to 24
amino acid changes. Some of these are likely to be changes
which occurred when the virus was propagated in different
cell systems, reflecting a selective adaption process to myo-
cardial cells. At two positions within the gene region coding
for the 2A proteinase (2APro), the infectious CVB3 cDNA
exhibits differences which cause significant alterations in the
amino acid sequence. One nucleotide insertion after position
3299 together with 1 nt deletion at position 3333 leads to a
frameshift within the previously reported CVB3 sequences
(20, 48) (Fig. 3). This frameshift results in an altered peptide
sequence of 12 amino acids. The majority of amino acids in
the immediate vicinity of the cleavage site between the
structural protein VP1 and 2APro are changed in a noncon-
servative fashion. In addition, insertions after positions 3486
(1 nt) and 3502 (2 nts) within the partial sequence reported by
Tracy et al. (48) lead to three amino acid changes and the
appearance of an additional amino acid (leucine) within
2APrO. Alignment of these two regions of the infectious
CVB3 cDNA sequence with other enterovirus sequences
reveals a high degree of sequence identity with CVB1 (100%)
(10), CVB4 (83%) (11), and, less pronounced, with poliovirus
type 1 (PV1) (50%) (18, 32), whereas the previously reported
CVB3 sequences (20, 48) clearly deviate (Fig. 3).
Sequence comparison with other group B coxsackieviruses.
Comparison of the nucleotide sequence of the CVB3 genome
with the sequences of the potentially cardiotropic CVB1 and
CVB4 (compare the sequences in Fig. 2) revealed high
degrees of overall identity, 80.7 and 78.3%, respectively.
The nc regions are highly conserved among the coxsackie-
viruses: CVB3-to-CVB1 identity, 94.1% (5' nc) and 89.4%
(3' nc); CVB3-to-CVB4 identity, 83.4% (5' nc) and 92.3% (3'
nc). One insertion (CVB4, 1 nt after position 32) and three
deletions (CVB3, 1 nt after position 115; CVB1, 1 nt each at
positions 118 and 7399) were observed within the 5' nc and 3'
nc regions, respectively.
The majority of nucleotide differences among group B
coxsackieviruses appear to be almost evenly distributed
over the entire open reading frame encoding the viral poly-
protein and occur frequently (71.3%) at third-nucleotide
positions. As expected, the regions coding for the structural
proteins VP1, VP2, and VP3 are less conserved than the
regions coding for VP4 and the nonstructural proteins.
Notable deletions and insertions are observed in the coding
regions of VP1 and VP2. A total of 15 nts are inserted within
the gene region of VP1 of CVB4 (after nt 2703), 6 nts are
deleted within VP1 (nt 2673 and nts 2680 through 2684) and
VP2 of CVB4 (nt 1404 and nts 1414 through 1418), and 9 nts
are inserted within VP1 of CVB3 (nts 3283 through 3291).
Alignment of the deduced CVB3 polyprotein sequence
with those of CVB1, CVB4, and PV1 (Fig. 4) allows the
identification of putative cleavage sites. In comparison to
those of PV1, cleavage sites are conserved, with the excep-
tion of the cleavage sites between polypeptides 2B and 2C
(2B/2C) and between VP1 and 2APrO (VP1/2APrO). In PV1,
2B/2C is a target of the 3C proteinase (3CPrO) (8). However,
a Q-N pair is found to be present in CVB3, CVB1, and CVB4
at a position corresponding to the Q-G cleavage site of PV1.
Since no alternative Q-G site is found at a proximal position,
we suggest that the Q-N pair is cleaved by the coxsackievi-
rus 3CPro, generating termini of polypeptides 2B and 2C at
equivalent positions with respect to PV1.
In the case of poliovirus, the cleavage between VP1 and
2APrO is mediated by 2APro, leading to the proper release of
the structural precursor protein P1 (46). In comparison to the
Y-G cleavage site of PV1 comprising the cleavage between
VP1 and 2APrO, the corresponding amino acids are not
conserved among the group B coxsackieviruses (Fig. 4).
Assuming cleavage sites with properties similar to the polio-
virus Y-G pair (aromatic amino acid-G), the F (or Y)-G pairs
located two residues downstream with respect to PV1 would
represent suitable cleavage sites in the group B coxsackie-
viruses. Alternatively, the T-G pair (two residues upstream)
has been suggested (10, 30). To verify the proposed cleavage
site for CVB3, protein sequence studies are required.
The alignment of the polypeptides of CVB1, CVB3,
CVB4, and PV1 (Fig. 4) indicates that identity among the
coxsackieviral polyproteins is clearly higher compared with
that of PV1. Taking into consideration the deduced cleavage
sites of the polyproteins, the degree of identity among the
viral proteins has been determined and is summarized in
Table 1. This comparison reveals that the structural protein
VP4 and all nonstructural polypeptides are highly conserved
among the coxsackieviruses (90.9 to 99.0% identity) and that
polypeptides 2B and 2C with yet unknown functions are
almost identical (98 to 99%). In contrast, the structural
proteins VP2, VP3, and VP1 exhibit a pronounced diversity
(69.4 to 82.9% identity) which is restricted to clusters of
heterologous sequences alternating with highly conserved
regions (compare sequences in Fig. 4). Highly homologous
VOL. 64, 1990
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

1576 KLUMP ET AL. J. VIROL.
CVBI U C U A U U U. UUl CA
CVB4 UA G A A UC A C AGUUC C CA GA AUG UUAUGA CA,A
148 CACAGUUACACCKGUCCCGCGGACAAAUCCCCGUMGGAACUIGUtCGCACAUXAAACUGACCGGAGUCGGGUCCCG
U C A A C A G
C UGU GC U GU AG UG C C C G G G A AC AG
U C G A A
U C U G U UG C GCA G U C
U A G
GUU G C AUtG G C U CCU
598 AAUUGAGAGAJJCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUWGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAULMACA NCAJUUAAGUUGAAUACAGCAJGA
C GU U C A U C UIC CA AC A A I G
A U A U A A C UOG U CG UCUGGA ACG AU X UCUUU AU GGAC C GAUl
A A G A G C U C AC C UA U CA A A UA
A GG A A C U UU GCC UA C GU U C C AC UU A UG A A U A UiAU A
898 UUCACAGAACCAGUGMAAGAUAUCAUGAUUAAAUCAUACCAGCUCCA CCCCACAGUAGAGGAGUGCGGAUACAGUGACAGGGCAGAUXAAUCACAUUAGGUAACtUCCAACCAAACGACUCAGGAAUGCGCCAACGUGGUGGUG
U UG G CU C GA G AU G U UCC A U UU UUC A CCCG U C AA GU U UU U
G AG G G A G G G G G U U C AUtU A CC G G U AA G UA C
G U G A UG CA AC GC A A C G C C tUUA GC G AU GAU G U UA A
G CC C U GCCGAA G A G C UU G AG U CGUGA U AA G UGCAGGGG UC A
118 VAUGC A AA U UU C AA CUUAGGCGACAU CGGGACMUA CAAG CGCA AUAtGG GUGUACGAGUGAACGG
UACAUGAC AGACGGUAG UUAAAG AAACUGAcCMGUCA U G CtCU GC GU U G U
1648 UGCGCACCUAGACCACGACGUCMUCGCGUGCCMUUCGGCACCVMACUCGGGGLAGCUGUCAA VUAGCAUGACGCGACUGACtUUCCMAA
AUGGAUKU AAU C MAAU CAAMC CCUAACGUAUMMUGCGUAA ACGCAUACG U VUVU
tiACGACGUA U U GU AG A A ....G AAAU G AAGG G AGU GCU AUA GC AGG U G V UU
G UA A G GA G CACG A UCGUCUC UG GCACCUGACGACCGUGCUA CA ACU
A~U AG U U UGCA ACUACCA GGC U VGCA UUGAAAUCUGAACUUCGUGA GCU GGG
CCMMA AUAGACAAAGC C G C C UGA A CA ACC A CVGGUU CCCCUACGUU U C U
GA GUAGCGUUCAGAUG CGAGAGV CU AGCU CAGACAAAAGUAUCCUAACAG G CCG UAAGU
G AG GGC UGA CAGU AAC AAGACA UG A AC CA ACACUG OG VAU G
A U A C UAGCA A UAAGCA GAG CAUAGAC UAGCCCA AUCUGAGC UCCU GG GU GGG
C GA UAG AGUGM AG G CUAGA GCAA C C UAC A U U CUl ACGccc G UC AGc U
CG GG AU A UGAU CG A U CCGUAUG GCU C UUGCMAAAAG AA UGMGC A GCU CCVAA
239 tVGtUGUCAGGCCAUGAGGCACAGGACCUtUCCUUUCGCGCAUACUACU CCA~CCAGAGt GGAAGA CGCGZAAPUAACCC AUtAGGUtGGGUGCGUCVGUACAWWM4GtGGGCACACCAAGUAUACCAIGGtGCAWCUCUG
GCA GAc GUC UVAG A CCUA U AVGGAGACGGUCGUU GCACUAUGU UCCCC
ACCAG G GMU ACAMC Al AACA AGACGGGCAAGAAC UAG GC ACGAUCCGC AUG UGCMU C VGAG
228AAGCC UCCAUACGUUUUCAtCAGAGGAACC AGGGUAUAC tG U C GAA UUUGGIC AUCUCAGCtOCAUUUK" MWUGAC AGCACC ACUUM
U G UAUUCGGGAGA A G AAGAUGCU c CCAUU AAUVA CAUU AUA.GUAUC
UCAGG A AUGCCAG ACACGGAcUVGUC UGUGMCUGAGCUUM U AllG A G GCG UG
A39tUGAAUCAG ACUAU CUG GUCAAAGGUGUUXAACAGAACAUGGAGCUAGU GGAACUGGAA UGCCAC GAUCAGACUAACAGGCACUGCAC
GCUACCG A UC CUGAAAGC AGA GUU CGG AUGGACCG ACAGAC CCGU G CAC C C A GU AAGc
GACUUCA UUC MGAC A UCG UCC AUCGUCGAC GACAG CGAUCCCG CAC UG U CAACUAAMGA
258GUGUCAGGCACACUCACAAUAGUCCGUGACCUAUVAGAACCACGCUAA UGCC UUCGGCW Z"f CU GCAVGCA CAUAGCGCWMAACti-
CAU U U UCVACACU UAAVGUMC AG GAA AA UU UA UU G CCA GCC UUACG CUC C AGUM
UGC U A AUCG U CA GGAAGG GGGACCV AAU CAGIUCACA C AGCC..UCtAU C
268GGCGCC CC....... ACGGA CGA CGtUA4CCAAAUGCACCACAALLS AACUGU AGA AUtCt 4GGGUCGACUGUGAGCGCGUU GGUCUAGCAAGt CACAUGCCtAC
. CG C AG UACCACAC AG GG C GCAACC CCAG GCGGCM U AGA GUUC GV A GU
.GAUCAACAACIG U C ACU AA GC AGC AGC CU t UUC GGU GU IC CC UGAGC
G ACGGU C CGAA CAGA GU CGGCGAGGU CUG ACGCCCGGG AUG U G UAGU A AG
CGCACUCCUU CAGGA AU CC GG ACG CCGAGC UGGA CAC UVAAM MGU
3583 CAtGUGCUUUUAGCAGCtGAAUUUGCMCGMCCUAGUGACUGUGWCGUACCUAA UGM XAGCGG UUCAANGCAGCGACCGU
3733 GCMUAMAAGCACCAUGGGAAtCUAGCUUCAACGAMGCAUCGGCUCCVGGCUA~VCCUAMACCUAGGACtCAAGUCACCCUAGAGMAMAGAACGUGGGCUCAAACUCCAGCGGAGAC MACUACUAAAAGCALCACUAC
GIGCU CAG A U CCACVG AA G CAG AG GCGC CAUti A GC
I A UCU GA V UU ACAC GGC AGU A AGU A G GACCA
VP4
VP2
VP3
VP1
2A
FIG. 2. Nucleotide sequence of infectious CVB3 cDNA (top) in comparison with the sequences of CVB1 (middle) (lizuka et al. [10]) and
CVB4 (bottom) (Jenkins et al. [11]). The CVB3 sequence is shown in its entirety together with the corresponding nucleotide changes of CVB1
and CVB4. The gene regions of the distinct viral proteins are defined by vertical lines by analogy to the poliovirus genome (18, 32). The
interrupted line after nucleotide position 3303 indicates the proposed border between the CVB3 gene regions of VP1 and 2APro. Dots represent
nucleotide deletions.
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

VOL. 64, 1990 INFECTIOUS COXSACKIEVIRUS B3 cDNA
C: CWIIUGUUAUAtGIG6U GAGGAUACCCAGCUCCGGCGCCtCLClAGCUUUtC66UUGUACCUCG UC1_166MUAACGAGLAXCNWGAtCCCUAU6GGCU
A A UGG G G A C C UUA A A A U C U CC C A A AA U G A C G C U C U A
C C U CG G A A U U AU A U C U C C U A U G C C A C
GAC GAAAlLUACAAtlWUU CUGCAGUUGGAlUGJGUUCAAUUCAGAAUUCUUGAUGGU6AUAAUUCCAG AGGUCAGAA6ACCAUUCCUG
G G U C G C C C C A G AU C U A U CC A A U A U U A
61CG G A A G U C A AG G C G U A G U G A AG G U A A
AACAAAACAtCCCAUUAUGAAUCAG6CGCACAACGACAGACGCCCAICAAUGACCAGlACAAUMLEUAUGLAJCWLCCACllU KCGAGUXGMCUAUCGCUAGtG
U G CG G U C U AU A U U G U A G G U U G A U C G A A G U G C
GU C G AC C GG U C U A A U A C U GC G C A G C U A G U A
6C ^UCCUI UCAAGtCCMN)GCCGJWW6ACCUGlAtUGU^UtUCUCCUGCACGGGAGCCCt6GUt6CCGCA GUGGCAACAAACUGAU6GMQIGWCU
A A U C C U U AG A U C U CG GC C A C G
C U AAA C W U U A A G G A U U C GC AU A
GGAACL6CACUCAGUG UACAUACCGCCAGACCGACUCGCGGUACAACAGAGCCGUGGSUCAAGGCGASCUCUCUNWAAL CCUGAGG UGUCUCCtUG UUtICCMAtGUUtCAG6UA
G U U U U A C UU U A U C U U C A U G U U U U U C G
A GUA U CU A C U U C A A C A A C G CG G U G U
GAUUUUGUCACCCAUG CUGCCC U tUGUfiCUCACCGUUUGt UUGGCA GACUGCAGGAW ACUCC AACCGUGUANACGGC6GMG" CCUAAGA
C G A A G U U C C U A C C C A U C U G AC U G U
C A A G G A U CU C U C C A U G C C C G G A C C CGUC A G C U U
C C G U GG G UA A C G A CUA A C U A G
U A C C C G U A A C G GA A A C G UC AG U
CAGGtCAGAUAUAUACAUGCUAGUCACCGAGALGUUUAGGGAGUAUC CAUAGCGUGGGGACCACGCUUGAGGCACCCACCA A
A C U G A U C A A U C U U G C GA U CG A U U G C UA G G
A G 6 C U G A C C G C U C GG C GA C G G A U U CA U A C
CCGCCC UCUCAAAUCGGCCI-YCI W C KC = C( I U tl
A U A U A C A G6G A C A C U C U GA
A AUA C A U U G G A G CC AU A U G G G C A GUUUW G AA A U C C C A C
ACCACAUtGIGU6ALCGAUCI_AUAt4AAUUC UCUGAGGllUCUCCMC6MWCAAGCAAAAGCUUtW tC6CC
U U C A A C C A C AC A C G A U AG A C U CC U A A UA U
A U C U C U U CU C A G A G A G GA U A A UA A A G U UC U U
G6UCCAA UGAG AGAUCAAGCACGGUGAAAMCUG hAUGCGAGUUUACCAtUGCAUCUU6CllAGUGCGUCCGUUGCCACGCAGCAC 6GCACCAUCUAGAGCAUCAAGGUCUGGGCUA
A C G U G A C U C U U U U U U A AA
C G C U A G C UA U CC A U C G U UC U C G C G
C U G U A G U GC A A U U G A G U
AU A GA U U A UC GG G C C A G UUc A UC C U
_ew =A C~ ~~ ~~ ~~~~ ~~~ ~ULMXACAUIXC
C U U C G A U G U G U G U G G U C U A U
A A C C CAM C U U U A C U GU A C U U U G
_U W CCUCAMCA~~~~~~~~CAACCC_ c
U A C 6 U AC G CU C A C G C A AC C U C U A
A UC U U UCCG U AUU CU U C UA A UCC U C U
A C U A C U G C A C CCA C A C A C C U A
CA A A C C U C A A A C AC C C AC U AU C G U A CC U C
_ U S 6~~~~~~AGCACUGAACG UAACCAAUAAA U
UA U U U U U GAU C C GU A U A U U AC U U G C A U C
A U U U G UG U U C UA G A U CG A U U U A G G C
CWUMiCCISUCCC CUUCCU^CG CG U GUCG AACG AU A C_ L =11- GAU
U A A C GAC C C CG C A A C G A A C6C G G A
U A C A C A G C AG U C G U A AC C G A A
UUAC AUG C GGQCC1GGUGAGCUUACA8CUtUCACGC C GUCUUGAUG
A U UC C A C C A U A G A U C UC
C A A C C C U U G A U GG A U G A A G C A C C U 6 U C U A U
UUIUCU666 1 1 W CV6UCU66~~~~~~UAAAUG UCUA M CUACUUGAUAUUGUCACCCAtA
G C G U U A U C G U C A C U GC ACG A U U G C U C
C C c u c c U CC U C C U GUC G UC G A A C G A U A C
C A A C C A C C A U C C U UGC U A
A A C C AC U C U A G C C C C U C U C CU U G G U U
U U C A U G C 6 CC 6 U G A GU U C A G U C U C G U U U A U
U U A U U C C C C G A AC A G A U C U U U C U
C _ CC U W ;GGCAUC
W A C C C U U C C GU CU G C C
A U U U C A G C G U C G A U U C CU 6 C
_6@C CCCUCC_~~~~~~~~~~~~~~~~~~~~~~~~~--AUUGAUAUtGUGC.. .. . .. .. . . ...~~~~~~~~~uc
c
AA G CU A
U GC C U A U
U C C U G
AUCCMAUCWUA
C CA U G G
CA U G
G C U A U A
1577
2B
2C
3A
3B
3C
3D
c UA
c A
7399
G
FIG. 2-Continued.
peptide sequences are found corresponding to the antiparal-
lel oriented beta strands and short alpha helices which
determine the core structure of PV1 capsid proteins (9).
Diverging sequences correlate to distinct loops connecting
the conserved core elements of VP2, VP3, and VPI. CVB3
peptide sequences at positions 75 through 86 of VP1 (site 1),
197 through 210 of VP1 and 135 through 174 of VP2 (site 2),
and 54 through 66, 75 through 80 of VP3, 72 through 76 of
VP2, and the C terminus of VP1 (site 3) correspond to PV
sequences containing the main immunodominant antigenic
sites of the three poliovirus strains PV1, PV2, and PV3 (25,
29). These regions are part of the highly divergent sequences
CVB3 3883
CVB1
CVB4
4033
4183
4333
4483
4633
4783
4933
5083
5233
5383
5533
5683
5833
5983
6133
6283
6433
6583
6733
6883
7033
7183
7333
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

1578 KLUMP ET AL.
CVB3
CVB3*
CVB3+
CVB3
CVB3*
CVB3+
3300 3333
.UUUGGACAACAAUCAGGGGCAGUGUAUGUGGGGAAC
A
A
I W T T I R G S V C . NI WT TI RG6SV C GD
CVB1 F Q-S6AVYVGN
CV84 Y G H S . .........X. V...... ....~~~~~~~~~~~~~~~~~~.
PVl F H QN K vYT A G
FIG. 3. Alignment of two regions withi
CVB3 cDNA with various enterovirus seq
amino acid sequence of infectious CVB3 c
(CVB3) reveals a high degree of identity to
(11) but lower homology to PV1 (18, 32).
determined by Lindberg et al. (20) (CVB3*)
(CVB3+) clearly deviate from the CVB3 sequ
a consequence of the frameshift between nt
cated). Note that the majority of amino ac
nonconservatively changed. A second frame
and 3502 was only found to be present in
reported by Tracy et al. (48) (CVB3+), leadin
sequence with conserved amino acid changer
identical amino acids.
among the coxsackieviruses, suggestin
major role in the formation of the antige
The 5' terminus of the CVB3 genome
features conserved among the picornaviru
sequence is highly conserved among diff
Enteroviruses have the first 10 nts, 5' L
common (Fig. 5). In addition, two pairs
are found to be present within the first 9
ing positions (nts 2 through 9 and 79 thri
18 and 26 through 35, in the case of CV
are common to other human pathogenic
as rhinoviruses, and to a lesser extent, he
5). Computer-calculated secondary st
shown) indicate that inverted repeats fac
of unique secondary structures presumal
functions in virus replication. In addition
all picornaviral genomes sequenced so fa
5' uridine residues. However, their rele
are still obscure.
Two initial 5' uridine residues missing
binant CVB3 cDNA are present in cDNA
quencing of the 5' terminus of infectious
cDNA revealed that two 5' uridine resid
were found to be encoded in the au
Instead, deoxycytidine residues have 1
corresponding to the oligo(dC) tract ger
ing of the CVB3 genome (16). This
question of how the recombinant CVB3
modified 5' terminus is capable of ind
cycle. To examine whether this modifi
during transfection of mammalian cells,
isolated from cDNA-derived CVB3. TI
quence of the genome of cDNA-gen
determined by primer extension and chei
performed for the determination of the
authentic wild-type RNA. Strikingly, th
cDNA-generated CVB3 genome was fou
that of the wild-type RNA containing
dues. These residues must have been I
initiation of an infectious cycle upon tra
binant CVB3 cDNA into permissive cel
3487 3502 Restoration of the authentic CVB3 5' end takes place in the
11 .U6U6C6UCCMAAAAMC. cytoplasm of transfected cells. To prove whether the authen-
// u AGC tic 5' terminus is restored through a cytoplasmic event,
transfection experiments were performed with in vitro-
/ c A $ K-N synthesized RNA transcripts, which should initiate an infec-
// c A^ S KN tious cycle independent of a nuclear event. For this purpose,
II L Ce v Q K s the recombinant CVB3 cDNA was set under the control of
// c A S R N both the T7 and SP6 promoters and was used for the
// c A S K s synthesis of full-length CVB3 RNA transcripts of plus-strand
/1 c E S R R polarity (compare structures shown in Fig. 1). These RNA
n 2APro of infectious molecules also lacked the two 5' uridine residues and con-
uences. The deduced tained additional nonviral nucleotides at both ends. A spe-
;DNA presented here cific infectivity of 14 to 30 PFU/,ug of T7- and SP6-derived
CVB1 (10) and CVB4 transcripts was observed in transfected HeLa cells (n = 3
The CVB3 sequences different experiments each), whereas virion RNA showed an
and Tracy et al. (48) infectivity of 2 x 105 PFU/[Lg. Total lysis of cells overlaidence presented here as with fluid medium was observed within 2 to 4 days upon
ids in this region are transfection of in vitro-synthesized plus-strand RNA. As
shift between nts 3487 expected, no infectious cycle was initiated upon transfection
the partial sequence of minus-strand RNA transcripts. To examine whether the
g to a different peptide authentic CVB3 5' terminus had been restored in plus-strand
s. Shaded areas depict RNA-transfected cells, the 5' nucleotide sequence of the
virus progeny was determined. The analysis revealed the
presence of two initial 5' uridine residues identical to the 5'
sequence 5'UUAAAA. . . in cDNA-derived CVB3 (Fig. 6).
ig that they play a This finding demonstrates that the authentic CVB3 5' termi-
enic determinants. nus has been restored in the cytoplasm upon transfection of
] contains structural in vitro-synthesized RNA transcripts.
ises. The 5' terminal Biological properties of transfection-derived CVB3. The
erent enteroviruses. supernatant medium of transfected cultures was tested after
JUAAAACAGC, in complete lysis of the cells in a specific antibody neutraliza-
of inverted repeats tion assay to examine the identity of transfection-derived
10 nts at correspond- viruses. Neutralization was achieved with CVB3 serotype-
ough 86, 10 through specific antiserum but not with CVB1 or CVB5 antiserum,
'B3). These features indicating that the cDNA-generated viruses, as well as
picornaviruses such viruses generated through in vitro-synthesized RNA tran-
epatitis A virus (Fig. scripts, were authentic CVB3. With regard to tissue tropism,
tructures (data not transfection-derived viruses were found to induce myocardi-
ilitate the formation tis in mice and are also capable of establishing a persistent
bly serving essential carrier state infection in cultured human myocardial fibro-
, it is of interest that blasts, as was previously described for the parental CVB3
tr contain two initial (14, 15). In addition, transfection-derived viruses were found
vance and function to grow to titers of about 2 x 108 to 4 x 108 PFU/ml upon
passage in HeLa cells, which is typically observed for
in infectious recom- wild-type stocks. cDNA-derived virus, as well as virus
A-derived virus. Se- generated through transfection of in vitro-synthesized CVB3
recombinant CVB3 RNA, clearly revealed wild-type virus plaque morphology
lues are missing that (Fig. 7).
thentic viral RNA.
taken their position
nerated during clon-
finding raised the
cDNA containing a
lucing an infectious
cation is conserved
genomic RNA was
he 5' nucleotide se-
erated viruses was
mical sequencing, as
very 5' end of the
e 5' terminus of the
nd to be identical to
two 5' uridine resi-
provided during the
Lnsfection of recom-
lls (Fig. 6).
DISCUSSION
Picornavirus research is in a very dynamic phase following
the demonstration that cloned cDNA copies of poliovirus
(33) and CVB3 (16) and in vitro-synthesized RNA transcripts
of poliovirus (50), human rhinovirus 14 (26), and hepatitis A
virus (4) are infectious. In addition, recombinant DNA
techniques have contributed to an improved diagnosis of
picornavirus infections. We have previously developed an in
situ hybridization assay capable of detecting enterovirus
RNA in myocardial tissue with full-length reverse-tran-
scribed recombinant CVB3 cDNA as an enterovirus group-
specific probe (14). By this approach, the presence of
enterovirus RNA has been assessed in endomyocardial
biopsy samples of a significant number of patients with
myocarditis or dilated cardiomyopathy or both (13, 17).
By comparison of the CVB3 genome to those of other
J. VIROL.
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

VOL. 64, 1990 INFECTIOUS COXSACKIEVIRUS B3 cDNA 1579
CVB3 1 NGAQVSTQKTGAHETRLNASGNSIIHYTNINYYKDAASNSARQDFTQOPGKFTEPVKDIMIKSLPALN SPTVEECGYSV4RSItLGNSTITTQECANVVVGYGVWPOYLKDSEATAEDQPTQV4
CVB1 G N SA V E N G
CVB4 S S S V V S E
PV1 S V NSNR Y G T N T R S A SK S S I VL TA M NI A VLQL A S A R E R NPV E
125 PDVATCRFYTLDSVQWQKTSPGWWWKLPDALSNLGLFGQNMQYHYLGRTGYTVHVQCNASKFHQGCLLVVCVPEAENGCATLDNT. . PSSAELLGGDTAKEFADKPVASGSNKLVQRVVYNAGMG VP2
E NN A QN I SN N KF S N RN T TE GTSND K tA W
N K EMQ A F EN S I TNAE A AYGD C E S EQN.. ATGKTA TA C
A T S T E R ROM Y S A G FA CLAGDSNTT NHT YQNANP EKGGT TGTFTPDNNQTSPA RFCPVDYL
248 VG.... VGNLTIFPHQWINLRTNNSATIVNPYTNSVPMDNNFRHNNVTLMVIPFVPLDYCPGSTTYVPITVTIAPNCAEYNGLR... LAGHQ GLPTMNTPGSCQFLTSODFQSPSNPQYDVTP
I Y L I N SE SP SS V T T F
Y I F I A VT ASS I V S L T F
L NGTLL AFV I C L L V LSI S VK WGIAIL LA NFASE SPEI L C F NIT PRL V N Y A N C L EF
365 EIRIPGEVKNLNEIAEVDSWPVQNVGEKVNSMEAYQIPVRSNEGSGTQVFGFPLQPGYSSVFSRTLLGE ILNYYTHWSGSIKLTFNfCGSANATGKFLLAYSPPGAGAPTKRVDAMLGTHVIWD VP3
Q R N N TDNN GLK Q SDNRR ANN LN V KN R
N Q R IN LKANLNT RVQ TDEN G I A LQ L V DS KN
PIO N L I THI FDLSAT K T N RVRLSDKPHTOWPILCLS S ASDPRL HN A L F L FN L VS A DP K KE
490 VGLQSSCVLCIPWISQTHYRFVASDEYTAGGFITCWYQTNIVVPADAQSSCYINCFVSACNDFSVRLLKDTPFISQQNFFQ G ..... ...... PVEDAITAAIGR ..VADTVGTGPTNSEAIPA
V Y VE A YV I V T D L N R D Y ESVER NV SSK S
V Y VD K S S VI E K N R Q K T Y T ESVERN IAR S Q
I TMVV N T QTID SF E Y SVF R LSTPREND LG R TH E KALA LGQNLESNIDNT RETVG TS DALPN EAS H KE
601 LTMETGHTSQWPGOTNQTRHVKNYHSRSESTIENFLCRSACVYFTEYKNSGA .....KRYAEWVLTPRQAAQLRRKLEFFTYVRFDLELTFVITSTQQPSTTQNQDAQILTHQINYVPPGGPV VP1
S S YAT N NSE G INT V L RKLE L A E ATSV PVQ Q
V D S H S IY..I Y S ESNNL INT V NN I C N H EM AT S VPVQ
V A NPL S V VQHR S S FA G TINTVD PASTTNKD LF V KI YKDTV S N V A..NFTE N GH LNQVY A
721 PDKVDSYVWQTSTNPSVFWTEGNAPPRNSIPFLSIGNAYSNFYDGWSEFS ........ RNGVYGINTLNNNGTLYARHVNAGSTGPIKSTIRIYFKPKHVKAWIPRPPRLCQYEKAKNVNFQPSG
T tDA I C TQ N EAGQ V V Q N T
TS ND I N TN N O I Y S I DS P GLT YV K S DVEA
E W D T S I Y Y T A I V YVG S H F KVPLKOQSAALGDSL MS OF I AV V DHNPTKVT K V L IRV C AVA Y.GPG DYKDGT
IV
838 VTTTRQSITTNTNTG AF:GQQSGAVYVGNYRVVNRHLATSAOWQNCVWESYNRDLLVSTTTAHGCDI IARCQCTTGVYFCASKNKHYPISFEGPGLVEVQESEYYPRRYQSHVLLAMGFSEPGO 2A
SN... RE R D R V K
AEALI... PY' H K NV D T S V K T
L PLSTKDL ... YIG H NK TAG KIC Y QD L A NVNWS TESR Q T S N NA Y E RR Y V V TFQYNE A N IGH AS
961 CGGILRCEHGVIGIVTNGGEGVVGFADIRDLLWLEDOANEQ GVKDYVEQLGNAFGSGFTNQICEQVNLLKESLVGQDSILEKSLKLVK I ISALVIVVRNHOL ITVTATLALIGCTSSPWRWL 2B
V V IV V
L V
H I A L A S YAY EE ITN I S A Q SOKITE TVTS..T T L N I S IT YE TT L L DA Q
1085 KQKVSQYYGIPMAERQ NNSWLKKFTENTNACKGNEWIAVK IQKF IEWLKVK ILPEVREKHEFLNRLKQLPLLESQIATIEQSAPSQSDQEQLFSNVQYFAHYCRKYAPLYAAEAKRVFSLEKKN
SG I K
H G K S
RK ACDVLE YVIK GD AC A L VSN S D E I QA D L VTK R EN N S H C EH I N RWLSIQSKRF V IQK HTI
1209 SNYIQFKSKCRIEPVCLLLHGSPGAGKSVATNLIGRSLAEKLNSSVYSLPPDPDFDGYKQQAVIMDLCQNPDGKDVSLFCQMVSSVDFVPP2CEEKGILFTSPFVLASTNASIAT S 2C
N H V T A Al RE TT S G N A W T E I S NY SSR SP A
1334 OSRALARRFHFDNNIEVISNYSQNGKINNSVKTCDDECCPVNFKKCCPLVCGKAIQfIDRRTQVRYSLNMLVTENREYNHRHSVGTTLEALFQ GPPVYREIKISVAPETPPPPAIADLLKS 3A
E A I I
E R K A T V
H D A D Q ME RD L A ATEN KNCHQ R L KSSR I QIT NIIN R R SNI NC LQ KDL OI.KTS EC N QA
1458 VDSEAVREYCKEKGWLVPE INSTLQIEKHVSRAF ICLQALTTFVSVAGI IYI IYKLFAGFQ GAYTGVPNQKPRVPTLRQAKVQ GPAFEFAVAURNSSTVKTEYGEFTMLGIYORWAVLPRH 3B
K V
I D I I K
QE 0 EK I .N T QV T RNIN NTI V AA VVV H L K N I T G DY A IV AT SK VH NV I T
1581 AKPGPTI3LCVGVLWAKELVOKDGTNLELTLLKLNRNEKFRDIRGfLAKEEVEVNEAVLAINTSKFPNMYIPVGQVTEYGFLNLGGTPTLMYNFPTRACGGVLMSTGKVLGIHVGGN 3C
I R R D
S ES VIDGK El A E QA I IIT K PHIPTQIT T DG IV Y V A Q Y RQ A T ITC I N
1706 GHQGFSMLLKHYFNDEQ GEIEFIESSKtAGFPVINTPSKTKLEPSVFHQVFEGWEPAVLRSGDRLKAFEEAIFSKYIGNNTEYMLEAVDHYATLDISTEPKLEDAVYGTEG
E N R R
SH A KRS TQS QWRP EVY I A A Y V TKN TO V KI E K NS N Q C D
1830 LEALDLTTSAGYPYVALGIKKRDILSKKTKDLTKLKECNDKYGLNLPNVTYVKDELRSIEKVAKGKSRL IEASSLNOSVAMRQTFGNLYKTFHLNPGWVTGSAVGCDPDLFWSKIPVNLDGHLIA 3D
R A I V
A A I V
S N K N Q R TKEQKLL T I L KT EQ MA M K I LNEEK F
1955 FDYSCYDASLSPVWFACLKNLLEKLGYTHKETNYIOYLCNSHHLYROKHYFVRGGNPSGCSGTSIFNSMINNI I IRTLNLKVYKGIDLDQFRNIAYGDDVIASYPWPIDASLLAEAGKGYGLINT
L S ROC
L 0
T A EA V I FGDR.VD NH KN T C K L L T HLK HEV QSD T
2080 PADI(ECFNV NTFLKYFQYPFLVHP IHESIRWTKDPKNTDHSLCLAWNEHEYEEFIRIRWACLTLPASTLRKDF 2185
D AV A
V Q
SAT ET E V F K I E R E LA I A L EY Y R
FIG. 4. Deduced amino acid sequence of infectious CVB3 cDNA in comparison with that of the polyproteins of CVB1 (lizuka et al. [10]),
CVB4 (Jenkins et al. [11]), and PV1 (18, 32). The CVB3 amino acid sequence is shown in its entirety, and deviating amino acids are depicted
for CVB1, CVB4, and PV1. Dots represent amino acid deletions. The proposed cleavage sites of the polyproteins of the coxsackieviruses are
marked by vertical lines by analogy to the biochemically established sites of proteolytic processing of PV1 (18). Note that only the proposed
cleavage sites between polypeptides 2B and 2C and between VP1 and 2APro differ between the coxsackieviruses and poliovirus. To determine
the exact position of the cleavage site of CVB3 between VP1 and 2APrO, direct sequencing of the termini of these polypeptides will be required.
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

1580 KLUMP ET AL.
TABLE 1. Amino acid sequence identity among the proteins of
CVB3 and three other enteroviruses"
Identity between:
Protein CVB3b CVB3 and CVB3 and CVB3 and
CVB1 CVB4 PV1
VP4 69 97.1 94.2 65.2
VP2 263 82.9 79.8 51.0
VP3 238 80.7 79.0 55.5
VP1 281 77.2 69.4 38.8
2APro 150 94.0 92.0 58.0
2B 99 98.0 99.0 49.5
2C 329 98.2 97.9 62.3
3A 89 97.8 93.3 51.7
3B 22 90.9 90.9 77.3
3CPro 183 98.9 97.8 55.2
3DPO' 462 96.3 97.0 79.7
a Sequence identities are expressed as percentages. Details are taken from
lizuka et al. (10) for CVB1, Jenkins et al. (11) for CVB4, and Kitamura et al.
(18) for PV1.
b Number of amino acids of CVB3 proteins based on the cleavage sites
proposed in the legend to Fig. 4.
potentially cardiotropic group B coxsackieviruses, the high
degree of identity among nucleotide and amino acid se-
quences clearly demonstrates their close genetic relation-
ship, which is considerably lower with respect to poliovirus
(compare Table 1 with Fig. 4). Most pronounced is the
degree of identity within the 5' and 3' nc regions and,
regarding the polyprotein sequences, within the nonstruc-
tural proteins and VP4. In the case of the structural proteins
VP1, VP2, and VP3, identity is confined to distinct regions
interspersed between nonhomologous sequences (Fig. 4). As
is evident from the crystallographic structures of various
picornaviruses (9, 21, 35), conserved regions have an impor-
1 2 3 4
AA
A
:........:
4*F a#
.,. 1
C
A
G
C
C
U
G
U
G
G
G
IU
FIG. 6. 5' Terminal sequence of CVB3 generated through trans-
fection of recombinant CVB3 plasmid DNA into HeLa cells. To
obtain the sequence of the 5' terminal region of the genomic RNA of
cDNA-derived CVB3, primer-extended cDNA was synthesized
from purified viral RNA and sequenced chemically as described in
Materials and Methods. Lanes 1 to 4, Labeled DNA fragments
generated from cleavage at G (lane 1), G and A (lane 2), T and C
(lane 3), and C (lane 4). The complementary 5' terminal sequence of
the corresponding viral RNA is shown at the right. Note the
presence of the two initial 5' uridine residues which are not
contained in the recombinant infectious CVB3 cDNA (16). An
identical sequence was obtained by sequencing genomic RNA from
CVB3 generated upon transfection of in vitro-synthesized viral
RNA.
CVB3
CVB1
CVB4
PVl
PV2
PV3
HRV14
HRV2
HAV/LA
HAV/MBB
10 20 30 40 50 60 70 80 90
UUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUAAC
5 -, -
UUAAAACAGCCUGUGGGUUGUUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUACAUC., ~~4-
UUAAAACAGCCUGUGGGUUGUACCCACCCACAGGGCCCAMUGGGCGCUAGCACACUGGUAUUCCGGUACCUUUGUGCGCCUGUUUUAUAC
,.~ ~ ~ ~ ~ -
UUAAAACAGCUCUGGGGUUGUACCCACCCCAGAGGCCCACGUGGCGGCUAGUACUCCGGUAUUGCGGUACCCUUGUACGCCUGUUUUAUA
UUAAAACAGCUCUGGGGUUGUUCCCACCCCAGAGGCCCACGUGGCCAGUACACUGGUAUUGCGGUACCUUUGUACGCCUGUUUUAUACUC
-B 4-
UUAAGCAGCUCUCGGGUUGUUCCCCCCCCAGAGGCCCACGUGGCGGCUAGUACACUGGUAUCACGGUACCUUUGUACGCCUGUUUUAUA
UUCAACAGCGGAUGGGUAUCCCACCAUUCGACCCAUUGGGUGUAGUACUCUGGUACUAUGUACCUUUGUACGCCUGUUUCUCCCCACC
--4 4 -
UUMMACUGGAUCCAGGUUGUUCCCACCUGGAUUUCCCACAGGGAGUGGUACUCUGUUAUUACGGUMACUUUGUACGCCAGUUUUAUCUC
UUCMAG6GGGUCUCCGGAG6UUUCCGGAGCCCCUCUU6GM6AUCCAUGGUGAGGG6ACUUGAUACCUCACC6CCGUUUGCCUAGGCUAU
- G -
UUCMAGAGGGGUCUCCGGGGAUUUCCGGAGUCCCUCUUGGMAGUCCAUGGUGAGGG6ACUUGAUACCUCACCGCCGUUUGCCUAGGCUAU
D 4--4t 4 -
FIG. 5. Common structural features of the 5' terminus of different picornaviruses. The first 90 5' terminal nts of CVB3, CVB1 (10), CVB4
(11), PV1 (18, 32), PV2 (45), PV3 (41), human rhinovirus type 14 (HRV14) (40), human rhinovirus type 2 (HRV2) (38), and hepatitis A viruses
(HAV/LA) (28) and HAV/MBB (31) are presented. Note that the first 10 nts at the 5' end are identical among the enteroviruses. Arrows
indicate perfect repeats of inverted symmetry, 6 to 10 nts in length, which are found at corresponding positions for the human enteroviruses
and rhinoviruses (nts 9 to 20 are complementary to nts 23 to 37, and nts 2 to 11 are complementary to nts 75 to 88). In the case of hepatitis
A virus, the inverted repeat that is complementary to nts 2 to 11 is shifted to nts 31 to 39. Computer-calculated secondary structures (data
not shown) suggest an important function for these inverted repeats during plus- and minus-strand RNA replication.
J. VIROL.
5'
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

INFECTIOUS COXSACKIEVIRUS B3 cDNA 1581
FIG. 7. Plaque morphology of CVB3 generated through transfec-
tion of wild-type RNA (A), recombinant pCB3-M1 plasmid DNA
(B), recombinant pCB3/T7 plasmid DNA (C), and in vitro-synthe-
sized CVB3 RNA (D). HeLa cell monolayers were infected with
transfection-derived first-passage stocks, as described for plaque
assays in Materials and Methods, indicating that viruses generated
in the absence of two initial 5' uridine residues exhibited wild-type
plaque morphology.
tant function in maintaining polypeptide chains of the struc-
tural proteins in the barrel-like core structure. Structurally
conserved loops and terminal extensions have been sug-
gested to be involved in dynamic viral processes, e.g.,
assembly, receptor binding, and uncoating (7), whereas
highly divergent sequences may participate in the formation
of antigenic determinants. This pattern of conserved and
heterologous peptide sequences is shared by various genera
of picornaviruses, as demonstrated by a comprehensive
alignment of the capsid proteins of 33 picornavirus strains
(30). Peptide sequences, which can be identified by correla-
tion to poliovirus sequences containing antigenic determi-
nants, are highly divergent among the coxsackieviruses and
probably exhibit serotype-specific antigenic properties. In
addition, peptides with sequences common to a subset of
serotypes should provide useful targets for broad-spectrum
clinical diagnosis of potentially cardiotropic enteroviruses.
In this context, we have recently demonstrated that poly-
clonal antibodies generated against bacterially synthesized
fusion proteins of various CVB3 structural proteins clearly
cross-react with a wide variety of group A and B coxsack-
ieviruses, as well as echoviruses (51).
The mechanism by which the recombinant cDNA of a
picornavirus initiates an infectious cycle in transfected cells
is currently poorly understood. Presumably, the recombi-
nant cDNA is transcribed in the nucleus of transfected cells
to produce plus-strand RNA transcripts, which serve as
messengers for the synthesis of virus-directed proteins and
as templates for replication. Regardless of whether these
transcripts are initiated from cellular promoters after inte-
gration or from cryptic promoters present in the vector
DNA, they probably represent oversized transcripts with
extra nonviral sequences at both ends. The in vitro-synthe-
sized CVB3 RNA transcripts also represent oversized tran-
scripts. These RNAs, while about 20 times more infectious
than the plasmid DNAs, are only about 0.01% as infectious
as RNA isolated from the virus. This may be caused by the
presence of oversized nonviral sequences (50) and possibly
also by the absence of two initial 5' uridine residues.
Nonetheless, transfection-derived viruses have biological
properties indistinguishable from those of parental CVB3.
The 5' termini of the transfection-derived viruses and that
of parental CVB3 were found to be identical, demonstrating
that recombinant plasmid DNA, as well as in vitro-synthe-
sized RNA transcripts, has regained two initial 5' uridine
residues during initiation of an infectious cycle. Presumably,
these uridine residues are provided by the picornaviral
nucleotidyl protein VPg-pUpU, which has been proposed as
a component of the initiation complex of poliovirus RNA
synthesis (5, 43, 44, 52). Free VPg-pUpU is known to be
present in poliovirus-infected cells (5) and has been synthe-
sized in vitro in a membrane fraction of poliovirus-infected
cells (43). It was suggested that a precursor of VPg, most
likely polypeptide 3AB, is uridylylated and subsequently
cleaved by the 3C protease (3CPrO) to yield the primer
molecule VPg-pUpU (44, 52). In vitro pulse-chase analyses
have supported a model in which VPg-pUpU can function as
a primer in the elongation reaction (42), and evidence was
obtained by an in vitro genetic approach that implicates
3DPOI in the formation of VPg-pUpU (47). Thus, a very
attractive model for the initiation of picornaviral RNA
synthesis is provided by implying synthesis ofVPg-pUpU by
3D"' without a template, by its hybridization to template
RNA, and by elongation by 3DP01.
Our finding of a correct 5' end of transfection-derived
CVB3 strongly supports uridylylation of VPg in a template-
independent fashion. This implies a unique mechanism al-
lowing the correct initiation of plus-strand RNA synthesis
independent of the presence of two complementary adenines
of the minus strand. Presumably, distinct sequences of the
minus-strand RNA template are involved in this process by
interacting with components of the replication complex. The
inverted repeats identified near the 5' terminus of the viral
genome (compare structures shown in Fig. 5) might play an
important role. It is tempting to assume that these se-
quences, as part of the minus strand, might adopt a distinct
three-dimensional arrangement serving as a signal element
for the interaction with protein entities of the putative
replication complex.
The availability of the nucleotide sequence of an infectious
cDNA clone of a cardiotropic CVB3 should prove to be
useful for further studies on the structure and function of the
viral RNA and provides a basis for a molecular genetic
approach not previously available for the analysis of entero-
virus-induced cardiomyopathy.
ACKNOWLEDGMENTS
We thank C. Schommer and H. Riesemann for excellent technical
assistance. The identity of transfection-derived CVB3 was kindly
established by M. Roggendorf (University of Munich).
This work was supported in part by grant 321-7291-BCT-0370
Grundlagen und Anwendungen der Gentechnologie from the Ger-
man Ministry for Research and Technology. I.B. received support
from a research fellowship from the Alexander von Humboldt-
Stiftung. R.K. is a Hermann and Lilly Schilling Professor of Medical
Research.
VOL. 64, 1990
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

1582 KLUMP ET AL.
LITERATURE CITED
1. Abelmann, W. H. 1973. Viral myocarditis and its sequelae.
Annu. Rev. Med. 24:145-152.
2. Andrews, N. C., and D. Baltimore. 1986. Purification of a
terminal uridylyltransferase that acts as host factor in the in
vitro poliovirus replicase reaction. Proc. Natl. Acad. Sci. USA
83:221-225.
3. Chen, E. Y., and P. H. Seeburg. 1985. Supercoil sequencing: a
fast and simple method for sequencing plasmid DNA. DNA
4:165-170.
4. Cohen, J. I., J. R. Ticehurst, S. M. Feinstone, B. Rosenblum, and
R. H. Purcell. 1987. Hepatitis A virus cDNA and its RNA
transcripts are infectious in cell culture. J. Virol. 61:3035-3039.
5. Crawford, N. M., and D. Baltimore. 1983. Genome-linked
protein VPg of poliovirus is present as free VPg and VPg-pUpU
in poliovirus-infected cells. Proc. Natl. Acad. Sci. USA 80:
7452-7455.
6. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-
sive set of sequence analysis programs for the VAX. Nucleic
Acids Res. 12:387-395.
7. Filman, D. J., R. Syed, M. Chow, A. J. Macadam, P. D. Minor,
and J. Hogle. 1989. Structural factors that control conforma-
tional transitions and serotype specificity in type 3 poliovirus.
EMBO J. 8:1567-1579.
8. Hanecak, R., B. L. Semler, C. W. Anderson, and E. Wimmer.
1982. Proteolytic processing of poliovirus polypeptides: anti-
bodies to polypeptide P3-7c inhibit cleavage at glutamine-
glycine pairs. Proc. Natl. Acad. Sci. USA 79:3973-3977.
9. Hogle, J. M., M. Chow, and D. J. Filman. 1985. Three-dimen-
sional structure of poliovirus at 2.9 A resolution. Science
229:1358-1365.
10. lizuka, N., S. Kuge, and A. Nomoto. 1987. Complete nucleotide
sequence of the genome of coxsackievirus Bi. Virology 156:
64-73.
11. Jenkins, O., J. D. Booth, P. D. Minor, and J. W. Almond. 1987.
The complete nucleotide sequence of coxsackievirus B4 and its
comparison to other members of the Picornaviridae. J. Gen.
Virol. 68:1835-1848.
12. Johnson, R. A., and I. Palacios. 1982. Dilated cardiomyopathies
of the adult. N. Engl. J. Med. 307:119-126.
13. Kandolf, R. 1988. The impact of recombinant DNA technology
on the study of enterovirus heart disease, p. 293-318. In M.
Bendinelli and H. Friedman (ed.), Coxsackieviruses: a general
update. Plenum Publishing Corp., New York.
14. Kandolf, R., D. Ameis, P. Kirschner, A. Canu, and P. H.
Hofschneider. 1987. In situ detection of enteroviral genomes in
myocardial cells by nucleic acid hybridization: an approach to
the diagnosis of viral heart disease. Proc. Natl. Acad. Sci. USA
84:6272-6276.
15. Kandolf, R., A. Canu, and P. H. Hofschneider. 1985. Coxsackie
B3 virus can replicate in cultured human foetal heart cells and is
inhibited by interferon. J. Mol. Cell. Cardiol. 17:167-181.
16. Kandolf, R., and P. H. Hofschneider. 1985. Molecular cloning of
the genome of a cardiotropic coxsackie B3 virus: full-length
reverse-transcribed recombinant cDNA generates infectious
virus in mammalian cells. Proc. Natl. Acad. Sci. USA 82:
4818-4822.
17. Kandolf, R., and P. H. Hofschneider. 1989. Viral heart disease.
Springer Semin. Immunopathol. 11:1-13.
18. Kitamura, N., B. L. Semler, P. G. Rothberg, G. R. Larsen, C. J.
Adler, A. J. Dorner, E. A. Emini, R. Hanecak, J. J. Lee, S. van
der Werf, C. W. Anderson, and E. Wimmer. 1981. Primary
structure, gene organization and polypeptide expression of
poliovirus RNA. Nature (London) 291:547-553.
19. Krausslich, H.-G., M. J. H. Nicklin, C.-K. Lee, and E. Wimmer.
1988. Polyprotein processing in picornavirus replication. Bio-
chimie 70:119-130.
20. Lindberg, A. M., P. 0. K. Stalhandske, and U. Pettersson. 1987.
Genome of coxsackievirus B3. Virology 156:50-63.
21. Luo, M., G. Vriend, G. Kamer, I. Minor, E. Arnold, M. G.
Rossmann, U. Boege, D. G. Scraba, G. M. Duke, and A. C.
Palmenberg. 1987. The atomic structure of Mengo virus at 3.0 A
resolution. Science 235:182-191.
22. Mattern, C. F. T. 1962. Some physical and chemical properties
of coxsackie viruses A9 and A10. Virology 17:520-532.
23. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled
DNA with base-specific chemical cleavages. Methods Enzymol.
65:499-560.
24. Melnick, J. L. 1985. Polioviruses, coxsackieviruses, echovi-
ruses, and newer enteroviruses, p. 739-794. In B. N. Fields
(ed.), Virology. Raven Press, N.Y.
25. Minor, P. D., M. Ferguson, D. M. A. Evans, J. W. Almond, and
J. P. Icenogle. 1986. Antigenic structure of polioviruses of
serotypes 1, 2 and 3. J. Gen. Virol. 67:1283-1291.
26. Mizutani, S., and R. J. Colonno. 1985. In vitro synthesis of an
infectious RNA from cDNA clones of human rhinovirus type
14. J. Virol. 56:628-632.
27. Morrow, C. D., G. F. Gibbons, and A. Dasgupta. 1985. The host
protein required for in vitro replication of poliovirus is a protein
kinase that phosphorylates eukaryotic initiation factor-2. Cell
40:913-921.
28. Najarian, R., D. Caput, W. Gee, S. J. Potter, A. Renard, J.
Merryweather, G. Van Nest, and D. Dina. 1985. Primary struc-
ture and gene organization of human hepatitis A virus. Proc.
Natl. Acad. Sci. USA 82:2627-2631.
29. Page, G. S., A. G. Mosser, J. M. Hogle, D. J. Filman, R. R.
Rueckert, and M. Chow. 1988. Three-dimensional structure of
poliovirus serotype 1 neutralizing determinants. J. Virol. 62:
1781-1794.
30. Palmenberg, A. C. 1989. Sequence alignments of picornaviral
capsid proteins, p. 211-241. In B. L. Semler and E. Ehrenfeld
(ed.), Molecular aspects of picornavirus infection and detection.
American Society for Microbiology, Washington, D.C.
31. Paul, A. V., H. Tada, K. von der Helm, T. Wissel, R. Kiehn, E.
Wimmer, and F. Deinhardt. 1987. The entire nucleotide se-
quence of the genome of human hepatitis A virus (isolate MBB).
Virus Res. 8:153-171.
32. Racaniello, V. R., and D. Baltimore. 1981. Molecular cloning of
poliovirus cDNA and determination of the complete nucleotide
sequence of the viral genome. Proc. Natl. Acad. Sci. USA
78:4887-4891.
33. Racaniello, V. R., and D. Baltimore. 1981. Cloned poliovirus
complementary DNA is infectious in mammalian cells. Science
214:916-919.
34. Reyes, M. P., and A. M. Lerner. 1985. Coxsackievirus myo-
carditis-with special reference to acute and chronic effects.
Prog. Cardiovasc. Dis. 27:373-394.
35. Rossmann, M. G., E. Arnold, J. W. Erickson, E. A. Franken-
berger, J. P. Griffith, H.-J. Hecht, J. E. Johnson, G. Kramer, M.
Luo, A. G. Mosser, R. R. Rueckert, B. Sherry, and G. Vriend.
1985. Structure of a human common cold virus and functional
relationship to other picornaviruses. Nature (London) 317:
145-153.
36. Rueckert, R. R., and E. Wimmer. 1984. Systematic nomencla-
ture of picornavirus proteins. J. Virol. 50:957-959.
37. Sanger, F., S. Nicklen, and A. Coulson. 1977. DNA sequencing
with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA
74:5463-5476.
38. Skern, T., W. Sommergruber, D. Blaas, P. Gruendler, F. Fraun-
dorfer, C. Pieler, I. Fogy, and E. Kuechler. 1985. Human
rhinovirus 2: complete nucleotide sequence and proteolytic
processing signals in the capsid protein region. Nucleic Acids
Res. 13:2111-2126.
39. Sprunt, K., W. M. Redman, and H. E. Alexander. 1959. Infec-
tious ribonucleic acid derived from enteroviruses. Proc. Soc.
Exp. Biol. Med. 101:604-608.
40. Stanway, G., P. J. Hughes, R. C. Mountford, P. D. Minor, and
J. W. Almond. 1984. The complete nucleotide sequence of a
common cold virus: human rhinovirus 14. Nucleic Acids Res.
12:7859-7875.
41. Stanway, G., P. J. Hughes, R. C. Mountford, P. Reeve, P. D.
Minor, G. C. Schild, and J. W. Almond. 1984. Comparison of
the complete nucleotide sequences of the genomes of the
neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin
vaccine derivative P3/Leon 12a1b. Proc. Natl. Acad. Sci. USA
81:1539-1543.
J. VIROL.
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from

INFECTIOUS COXSACKIEVIRUS B3 cDNA 1583
42. Takeda, N., R. J. Kuhn, C.-F. Yang, T. Takegami, and E.
Wimmer. 1986. Initiation of poliovirus plus-strand RNA synthe-
sis in a membrane complex of infected HeLa cells. J. Virol.
60:43-53.
43. Takegami, T., R. J. Kuhn, C. W. Anderson, and E. Wimmer.
1983. Membrane-dependent uridylylation of the genome-linked
protein VPg of poliovirus. Proc. Natl. Acad. Sci. USA 80:
7447-7451.
44. Takegami, T., B. L. Semler, C. W. Anderson, and E. Wimmer.
1983. Membrane fractions active in poliovirus RNA replication
contain VPg precursor polypeptides. Virology 128:33-47.
45. Toyoda, H., M. Kohara, Y. Kataoka, T. Suganuma, T. Omata,
N. Imura, and A. Nomoto. 1984. Complete nucleotide sequences
of all three poliovirus serotype genomes. J. Mol. Biol. 174:
561-585.
46. Toyoda, H., M. J. H. Nicklin, M. G. Murray, C. W. Anderson,
J. J. Dunn, F. W. Studier, and E. Wimmer. 1986. A second
virus-encoded proteinase involved in proteolytic processing of
poliovirus polyprotein. Cell 45:761-770.
47. Toyoda, H., C.-F. Yang, N. Takeda, A. Nomoto, and E. Wim-
mer. 1987. Analysis of RNA synthesis of type 1 poliovirus by
using an in vitro molecular genetic approach. J. Virol. 61:
2816-2822.
48. Tracy, S., H.-L. Liu, and N. M. Chapman. 1985. Coxsackievirus
B3: primary structure of the 5' non-coding and capsid protein-
coding regions of the genome. Virus Res. 3:263-270.
49. Vaheri, A., and J. S. Pagano. 1965. Infectious poliovirus RNA:
a sensitive method of assay. Virology 27:434-436.
50. Van der Werf, S., J. Bradley, E. Wimmer, W. F. Studier, and
J. J. Dunn. 1986. Synthesis of infectious poliovirus RNA by
purified T7 RNA polymerase. Proc. Natl. Acad. Sci. USA
83:2330-2334.
51. Werner, S., W. M. Klump, H. Schonke, P. H. Hofschneider, and
R. Kandolf. 1988. Expression of coxsackievirus B3 capsid
proteins in Escherichia coli and generation of virus-specific
antisera. DNA 7:307-316.
52. Wimmer, E., R. J. Kuhn, S. Pincus, C.-F. Yang, H. Toyoda,
M. J. H. Nicklin, and N. Takeda. 1987. Molecular events leading
to picornavirus genome replication. J. Cell Sci. Suppl. 7:
251-276.
53. Woodruff, J. F. 1980. Viral myocarditis-a review. Am. J.
Pathol. 101:427-479.
54. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved
M13 phage cloning vectors and host strains: nucleotide se-
quences of the M13mpl8 and pUC19 vectors. Gene 33:103-119.
55. Young, D. C., D. M. Tuschall, and J. B. Flanegan. 1985.
Poliovirus RNA-dependent RNA polymerase and host cell
protein synthesize product RNA twice the size of poliovirion
RNA in vitro. J. Virol. 54:256-264.
56. Zuker, M., and P. Stiegler. 1981. Optimal computer folding of
large RNA sequences using thermodynamics and auxiliary
information. Nucleic Acids Res. 9:133-148.
VOL. 64, 1990
on D
ecem
ber 5, 2012 by U
S
P
A
T
E
N
T
&
T
R
A
D
E
M
A
R
K
O
F
F
IC
E
http://jvi.asm
.org/
D
ow
nloaded from