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Journal of Virology, October 2000, p. 9802-9807, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Replicon Vectors Derived from Sindbis Virus and
Semliki Forest Virus That Establish Persistent Replication in
Host Cells
Silvia
Perri,*
David
A.
Driver,
Jason P.
Gardner,
Scott
Sherrill,
Barbara A.
Belli,
Thomas W.
Dubensky Jr., and
John M.
Polo
Vaccines and Gene Therapy, Chiron
Corporation, Emeryville, California 94608
Received 24 April 2000/Accepted 26 July 2000
 |
ABSTRACT |
Alphavirus replicon vectors are well suited for applications where
transient, high-level expression of a heterologous gene is required.
Replicon vector expression in cells leads to inhibition of host
macromolecular synthesis, culminating in eventual cell death by an
apoptotic mechanism. For many applications, including gene expression
studies in cultured cells, a longer duration of transgene expression
without resulting cytopathic effects is useful. Recently, noncytopathic
Sindbis virus (SIN) variants were isolated in BHK cells, and the
mutations responsible were mapped to the protease domain of
nonstructural protein 2 (nsP2). We report here the isolation of
additional variants of both SIN and Semliki Forest virus (SFV)
replicons encoding the neomycin resistance gene that can establish
persistent replication in BHK cells. The SIN and SFV variant replicons
resulted from previously undescribed mutations within one of three
discrete regions of the nsP2 gene. Differences among the panel of
variants were observed in processing of the nonstructural polyprotein
and in the ratios of subgenomic to genomic RNAs. Importantly,
high-level expression of a heterologous gene was retained with most
replicons. Finally, in contrast to previous studies, efficient
packaging was obtained with several of the variant replicons. This work
expands the utility of noncytopathic replicons and the understanding of
how alphavirus replicons establish persistent replication in cultured cells.
| |
TEXT |
Alphavirus vectors, derived
principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and
Venezuelan equine encephalitis virus, are widely used for gene
expression studies in vitro and are being developed for both vaccine
and gene therapy applications (25). Typically, these vectors
are constructed in a format known as a replicon, due to the
self-amplifying nature of the vector RNA (30). Replicons
contain both the cis and trans alphavirus genetic
elements required for RNA replication, as well as heterologous gene
expression via the native subgenomic promoter. Upon introduction into
cells, replicon RNA is translated to produce four nonstructural proteins (nsPs), which together comprise the alphaviral replicase. Replication proceeds through a minus-strand RNA intermediate and subsequently generates two distinct positive-strand RNA species, corresponding to a genomic-length vector RNA and an abundant subgenomic RNA encoding the heterologous gene (27). The replicon RNA
can be packaged into virion-like particles by providing the structural proteins in trans, from in vitro-transcribed defective
helper RNA (4, 15-17) or using packaging cell lines
(16). Alternatively, the replicon RNA can be introduced
directly into cells as plasmid DNA (2, 6, 8, 13).
In most mammalian cells, host macromolecular synthesis is inhibited
following the introduction of alphavirus replicons, leading to eventual
cell death by an apoptotic mechanism (11, 25). Thus,
application of these vectors for some gene therapy applications and
extended gene expression studies in cultured cells is limited. Given
the many other attractive features of the alphavirus replicon system,
it would be useful to extend the utility of these vectors to include
long-term expression and reduced cytopathogenicity options.
Under appropriate conditions, alphaviruses and alphavirus-derived
vectors can establish persistence in cultured cells (14, 26,
29) or exhibit delayed onset of cytopathic effects
(9). The establishment of SIN replicon persistence in BHK
cells has been associated with mutation of the protease domain of nsP2
(7, 10), and studies have suggested that the use of such
mutants for long-term expression may be possible (1, 3). It
remains to be determined whether mutation of other alphavirus nsPs or nsP2 domains can provide a noncytopathic phenotype by a similar or
alternative mechanism.
To expand the utility of the noncytopathic replicon and further explore
how persistence is established, we isolated additional SIN replicons
with this phenotype, as well as SFV replicons with a similar phenotype.
Mutations that conferred the establishment of persistent replication
were mapped to several regions of nsP2 for both SIN and SFV replicons,
in addition to the same residue 726 mutation identified previously
(7, 10). These mutations had various effects on the levels
of genomic and subgenomic replicon RNA and, in some cases, processing
of the nonstructural polyprotein.
Selection of replicons that establish persistent replication.
To select alphavirus replicon variants capable of establishing
persistent replication, the neomycin phosphotransferase gene (neo) was placed under the control of the subgenomic
promoter in both SIN- and SFV-derived replicons. These plasmids,
designated pSINBV-neo and pSFV-neo, were derived from pRSIN
(8) and pSFV1 (15) (GIBCO-BRL), respectively.
neo-containing replicons were transcribed in vitro from
linearized plasmid template; in some experiments, the DNA templates
were subjected to prior random mutagenesis using the bacterial strain
XL-1 Red Mutator (Stratagene). Replicon RNAs were transfected into BHK
cells, and the cells were subjected to G418 selection (Geneticin; 0.5 mg/ml; GIBCO-BRL) at 24 h posttransfection. Drug-resistant
colonies were obtained from both nonmutagenized and mutagenized
replicons. In addition, colonies were obtained after infection of BHK
cells with packaged vector particles containing nonmutagenized
neo replicon, generated as previously described
(16). These data indicated that drug resistance was
associated with the replicon RNA and that adaptive replicon mutations
could occur within the cell.
Within each selection, the drug-resistant BHK cells were pooled and
expanded. To confirm that neo expression was associated with
alphavirus replicon RNA species, poly(A)-selected mRNA was extracted
from the pools (Triazol [GIBCO-BRL] followed by Oligotex [Qiagen])
and analyzed by Northern blot hybridization with a
32P-labeled DNA fragment derived from the neo
gene (Fig. 1A). The neo
sequence indeed was found within both genomic- and subgenomic-length RNA species for all pools. This analysis also indicated variations in
the RNA profiles among SIN and SFV pools, particularly with respect to
the relative ratios between subgenomic and genomic RNA and the
appearance of new RNA species migrating faster than the genomic RNA
(Fig. 1A, lanes S5, S8, S9, SF1, and SF2). Such variation suggested
possible phenotypic differences among the selected variants.
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FIG. 1.
Analysis of Neo-resistant BHK pools derived from SIN and
SFV replicons. The SIN-derived pools are designated S1 to S10; the
SFV-derived pools are named SF1 and SF2. (A) Northern blot analysis of
poly(A)-selected RNA extracted from BHK cells either transfected (lanes
S1, S2, S4 to S10, SF1, and SF2) or infected (lane S3) with replicon
RNA and selected with G418. Pools were obtained using nonmutagenized
replicon (lanes S1 to S3 and SF1) or replicon transcribed from
templates that had been subjected to one round (lanes S4 and S7), two
rounds (lanes S8 and SF2), three rounds (lanes S5 and S9), or four
rounds (S6 and S10) of mutagenesis. In vitro-transcribed genomic RNA
from the two parental replicons (lanes SIN and SFV) was used as a
marker for the full-length replicon, and poly(A)-selected mRNA from
naive BHK cells was used as a mock control (lanes mock). The blot was
hybridized with a probe specific for the neo gene. Expected
sizes for vector subgenomic RNAs are 1.2 kb for SIN and 1.65 kb for
SFV. (B) Complementation analysis. Expression was measured after
introduction of nsP-deleted defective  -Gal replicons into SIN- and
SFV-derived Neo-resistant BHK pools. Detection of -Gal expression
was performed using a luminescent -Gal assay kit (Clontech), and the
signal was measured in relative light units (RLU). Data are the means
of two independent electroporations; the background reading obtained
with naive BHK cells was subtracted from all samples. Images were
processed with Adobe Photoshop software.
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To confirm that Neo resistance was conferred by replicon RNA, naive BHK
cells were electroporated with 5 to 10 µg of poly(A)-selected
mRNA
extracted from either SIN- or SFV- derived Neo-resistant
pools or from
naive BHK cells as control. Approximately 48 h
postelectroporation,
the transfected cells were subjected to G418
selection. Transfection
with mRNA from both SIN- and SFV-derived pools
rapidly generated
high numbers of Neo-resistant cells. In contrast,
transfection
of control mRNA gave no colonies over an extended period
of
time.
To determine whether vector RNA was actively replicating in
Neo-resistant cells, various cell pools were transfected with
defective
SIN and SFV replicon RNAs that encoded a
-galactosidase
(
-Gal)
reporter but had the nsP genes deleted. Amplification
and subgenomic
transcription of the
-Gal mRNA could occur with
these defective
vectors only if functional nsPs were provided
in
trans by
replicons already present in the drug-resistant pools.
The
defective
-Gal replicons were transcribed from plasmids
pSINBVdlnsP-
gal
(derived from pSINBV-
gal [
8] by
deleting the
BspEI fragments)
and pSFV3dlnsP-
gal (derived
from pSFV3-
gal [
15] [GIBCO-BRL]
by deleting the
PstI fragments). After transfection of the defective
replicons,
-Gal expression was observed in all but one pool (Fig.
1B). This result clearly demonstrated that the variant replicons
were
actively replicating in cells and provided
trans
complementation.
Pool SF1 did not show demonstrable
-Gal expression,
indicating
a defect reducing either replication of the variant SFV
vector
or the vector's ability to initiate subgenomic transcription in
trans. Since low levels of subgenomic RNA were observed for
SF1
in the Northern analysis (Fig.
1A, lane SF1), the lack of
-Gal
expression may be a consequence of reduced subgenomic
transcription.
Mapping the adaptive genetic determinants of persistence.
To
identify the causal mutations, representative pools S1, S2, SF1, and
SF2 were chosen for mapping based on their unique RNA profiles in the
Northern analysis. The complete nsP genes of SIN and SFV variant
replicons present in these pools were cloned by reverse transcription
(RT)-PCR in three and four fragments, respectively (Fig. 2A and
B). Each amplified fragment then was substituted for the corresponding fragment in wild-type pSINBV-neo or
pSFV-neo, and three independent replicon clones were generated for each
nsP fragment substitution. Replicon RNA was transcribed in vitro from
the constructs and transfected into naive BHK cells. Following G418
selection, the number of colonies obtained for each construct was
compared to the number of colonies obtained with the parental wild-type
replicon. For all but one pool, a single specific fragment substitution
resulted in the establishment of persistent Neo resistance (Fig. 2A and
B). For the SF2 pool, which was derived from vector that had undergone
two rounds of mutagenesis, two fragments, SF2A and SF2C, independently
conferred the phenotype. Thus, both SIN and SFV replicons that
established persistent replication could result from substitution with
a defined fragment.
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FIG. 2.
Mapping of causal mutations in the variant replicons. (A
and B) Schematic illustrations of the cloning strategy used to map the
replicon variants. Depicted are the neo-containing SIN and
SFV replicons, with corresponding RT-PCR-amplified fragments generated
from the Neo-resistant BHK pools. Also shown are nsP coding regions and
the restriction sites used in fragment substitutions. nt., nucleotide.
The ability of each fragment substitution to efficiently confer Neo
resistance (+) compared to the parental vectors ( ) is indicated;
"nt" denotes fragments not tested. (C) Sequence alignments of the
nsP2 regions in which the mutations were located are shown for several
alphaviruses. Bold characters indicate amino acid residues where
mutations were found; the change is indicated above the alignment for
the SIN-derived variants and below the alignment for the SFV-derived
variants. In variant SF1B,  indicates the deletion of amino acid
D469. Since the length of nsP2 varies between SIN and SFV,
codon numbering is indicated for each virus. White boxes highlight
identical residues among all of the alphaviruses aligned; gray boxes
highlight conservative changes. Images were processed with Adobe
Photoshop software.
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The defined fragments were sequenced entirely and compared to the
parental replicon sequence. Each SIN and SFV variant contained
only a
single amino acid substitution within the nsP2 protein
(Fig.
2C).
Although the precise location of these amino acid changes
differed
among the SIN and SFV variants, the amino terminus (amino
acid [aa] 1 in variant S1 and aa 10 in variant SF2A) and a small
region of the
carboxy terminus (aa 726 in variant S2 and aa 713
in variant SF2C)
seemed to be targeted preferentially. The latter
region is within the
putative protease domain of nsP2 (
12),
where mutation of
P
726 previously was found to reduce cytopathogenicity
of
both SIN (
7) and a SIN-based replicon (
10).
Interestingly,
the S1 mutation mapped within the nsP1-nsP2 cleavage
recognition
site (
27). Furthermore, the SF1B variant
contained an in-frame
deletion of aa 469, within a different region of
nsP2.
Properties of the cloned variants.
To characterize the cloned
replicon variants, we examined the impact of each mutation on RNA
replication and heterologous gene expression. The ratios of subgenomic
and genomic RNA were evaluated in drug-resistant cell lines obtained
using the cloned SIN and SFV replicon variants, as well as naive BHK
cells electroporated 2 h earlier with parental replicon RNAs.
Cells were labeled with [3H]uridine (100 µCi/ml) in the
presence of dactinomycin (1 µg/ml) for 7 h. Total RNA was
extracted from the cells and separated by gel electrophoresis
(19), the gels were treated and exposed to film
(10), and the genomic and subgenomic RNA bands were excised
and subjected to scintillation counting (Fig.
3A). Although a direct comparison could
not be made with the transiently transfected parental vectors, the
individual variant replicons clearly showed differences in molar ratios
of subgenomic to genomic RNA among each other. This result suggested
that the nsP2 mutations affected the levels of genomic replication
and/or subgenomic transcription and that variants S2 and SF2C had
smaller amounts of genomic RNA than other variants. A longer exposure
confirmed the presence of both genomic and subgenomic RNAs in S2 (data
not shown).
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FIG. 3.
Analysis of RNA replication and heterologous gene
expression. (A) Stable BHK cell lines derived using the cloned variant
vectors (lanes S1, S2, SF2A, SF1B, and SF2C) and naive BHK cells
electroporated with parental vector RNAs (lanes SINBV and SFV) were
labeled with [3H]uridine. Total RNA was extracted and
quantitated, and equivalent amounts were run in an agarose-formaldehyde
gel. Molar ratios of subgenomic to genomic RNA were measured by
scintillation counting of excised gel fragments. Size differences
between SIN and SFV vectors are reflected in the subgenomic RNA size.
Exposure time for the transfected SINBV and SFV control lanes was
shorter than for the drug-resistant cell lanes. (B) BHK cells were
electroporated with parental and variant vector RNA encoding E-GFP. At
24 h posttransfection, the cells were analyzed by flow cytometry,
and mean fluorescence intensity (MFI) of the GFP-positive cell
population was plotted. Data are the means of four independent
electroporations, with the standard deviations shown (n = 4). The background MFI reading from mock-transfected BHK cells was
subtracted from all the samples. Images were processed with Adobe
Photoshop software.
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The level of heterologous gene expression among variants and parental
replicons was next compared by using replicons expressing
the E-GFP
(enhanced green fluorescent protein) reporter gene (Clontech).
BHK
cells were electroporated with in vitro-transcribed replicon
RNA, and
the level of GFP expression was assayed 24 h later by
flow
cytometry (Fig.
3B). Although transfection efficiency varied
among
replicons, GFP expression levels within individual transfected
cells
were similar to or slightly lower than levels in the parental
replicons
for most variants. In contrast to all other variants,
SFV1B showed
inefficient expression of GFP. Since the subgenomic-to-genomic
RNA
ratio was lower for SF1B than for other variants (Fig.
3A),
the
observed defect may be a consequence of low subgenomic transcription
levels.
We then analyzed whether the mutations differentially affected
plus-strand or minus-strand RNA synthesis. To differentiate
the levels
of each RNA species, semiquantitative RT-PCR was performed
on
equivalent amounts of total RNA extracted from either Neo-resistant
BHK
cell lines containing the cloned SIN and SFV variant replicons
or naive
BHK cells electroporated 24 h earlier with the parental
replicons.
Oligonucleotides complementary to either plus- or minus-strand
RNA were
used for detection of strand-specific cDNA. After cDNA
synthesis and
RNase A treatment, a 700-bp fragment corresponding
to a region of
either nsP4 for the SIN variants or nsP3 for the
SFV variants was
amplified by PCR. Each PCR mixture was divided
into multiple aliquots,
and one aliquot was analyzed every five
amplification cycles (Fig.
4). Both plus- and minus-strand RNA
levels were found to be lower with both the S1 and SF2C variants
than
with the parental vectors at 24 h postelectroporation. Similar
results were obtained with the other variants (data not shown).
A
specific fragment of the housekeeping gene BHKp23 (
18) also
was synthesized from each sample as an internal standard, and
similar
amounts of product were obtained in all cases (Fig.
4).
This result
clearly demonstrated that each variant had ongoing
minus-strand
synthesis, which is a requirement for persistent
replication.
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FIG. 4.
Detection of vector minus-strand and plus-strand RNA by
RT-PCR. The cDNA corresponding to either minus strand or plus strand
was amplified using strand-specific primers from total RNA of either
BHK cells lines containing the cloned variants (S1 and SF2C) or naive
BHK cells electroporated 24 h earlier with the parental vectors
(SINBV and SFV). As an internal control, the housekeeping BHKp23 mRNA
was amplified from each sample (p23). Amplification mixtures were
divided into six aliquots, and one aliquot per sample was removed after
5, 10, 15, 20, 25, and 30 amplification cycles, as indicated above the
lanes. Images were processed with Adobe Photoshop software.
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Alphavirus nsPs are translated initially as polyproteins, P1234 and
P123 in SIN and P1234 in SFV. These polyproteins are processed
subsequently into mature monomers by the nsP2 protease (
5,
12), with the processing intermediates playing an important
role
in the early events of RNA replication, including a shift
from
minus-strand to plus-strand synthesis (
23,
27). Since
minus-strand synthesis was maintained with the SIN and SFV variant
replicons, we analyzed the effects of the mutations on polyprotein
processing. Coupled transcription-translation of parental and
variant
replicon RNA was performed with rabbit reticulocyte lysates
(T
NT; Promega) in the presence of
[
35S]methionine. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
(SDS-PAGE) analysis revealed that although all
mutants accumulated
the nsP monomers, mutants S1, SF2A, and SF1B also
accumulated
significant amounts of higher-molecular-weight products
(Fig.
5A and C). Immunoprecipitation of
the in vitro-translated products
from SINBV and S1, with antisera
specific for either nsP1 or nsP3,
indicated that variant S1 accumulated
the P123 and P23 precursors
(Fig.
5B). In contrast to previous results
for mutation of nsP2
residue 726 (
10), we did not observe
any significant processing
difference between variant S2 and its
parental replicon. However,
several amino acid differences exist among
nsPs of the parental
replicons used by each laboratory, and these
differences in genetic
background may affect the processing efficiency.
Nonetheless,
these results suggested that the maintenance of
minus-strand synthesis
may be achieved through altered polyprotein
processing.
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FIG. 5.
In vitro processing of nonstructural polyprotein. (A and
C) Coupled transcription-translation of parental and variant replicon
RNA was performed in the presence of [35S]methionine, in
rabbit reticulocyte lysates, and the products were analyzed by SDS-PAGE
on an 8% gel. (B) The translation products (lanes T) of SINBV and
variant S1 were immunoprecipitated with polyclonal antibodies against
either SIN nsP1 (lanes  1) or SIN nsP3 (lanes 3). Images were
processed with Adobe Photoshop software.
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Finally, we examined the ability of the variant replicons to be
packaged into virion-like particles by supplementing the structural
proteins in
trans from separate capsid- and
glycoprotein-defective
helper RNAs transcribed in vitro
(
16). Interestingly, and in
contrast to previous studies,
some variant replicons could be
packaged as efficiently as the parental
replicon (SINBV-GFP [5
× 10
8 IU/ml] versus S1-GFP
[3.8 × 10
8 IU/ml]) and some with a slightly
decreased efficiency (SFV3LacZ
[3.8 × 10
8 IU/ml]
versus SF2ALacZ [5 × 10
7 IU/ml] and SF2CLacZ
[10
7 IU/ml]). The other replicons were packaged at very
low efficiency
(S2-GFP and SF1BLacZ,
le4 IU/ml).
This report extends previous studies on noncytopathic SIN replicon
variants (
1,
10) with the demonstration that mutations
in
multiple regions of nsP2 can lead to the establishment of persistent
RNA replication. Significantly, similar variants also may be generated
with SFV-derived replicons. For both SIN and SFV, regions of nsP2
encompassing the amino terminus or proximal to the carboxy terminus
seem to be preferential targets for mutations leading to persistent
replication and maintenance of heterologous gene
expression.
Alphavirus replicon vectors have many attractive features, and the
addition of either long-term expression options or decreased
cytopathogenicity should facilitate the expansion of their
applications.
Maintenance of ongoing minus-strand synthesis and
inhibition of
cytopathogenicity are requirements for these vectors to
establish
persistent replication with continued high-level transgene
expression.
In the alphavirus replication cycle, minus-strand RNA
synthesis
occurs only during the first few hours postinfection
(
21,
24).
Extensive work (reviewed in references
23 and
27) supports
proposed
models in which both processing intermediates and mature
nsP monomers
form alphavirus polymerases with different activities.
Final cleavage
of the P23 intermediate converts the polymerase
activity from synthesis
of both minus and plus strands to only
plus-strand genomic and
subgenomic RNA synthesis (
23,
27).
Since nsP2 contains the
protease domain responsible for the nsP
maturation (
12), it
is noteworthy that all noncytopathic variants
characterized to date
(this work and reference
10) result from
mutation of
nsP2. Interestingly, one variant (S1) accumulates
the P123 and P23
processing intermediates, indicating that maintenance
of minus-strand
synthesis may be achieved by deregulating the
switch from minus strand
to plus strand through this pathway.
Similar to previously published
studies where temperature-sensitive
nsP2 mutants did not abolish or
resumed minus-strand synthesis
at the nonpermissive temperature
(
22,
28), one variant in
the present study showed
accumulation of unprocessed nsP and severe
inhibition of
subgenomic RNA synthesis. However, most noncytopathic
variants (this work and reference
10)
displayed high subgenomic
RNA expression, indicating that maintenance
of minus-strand synthesis
through this pathway does not necessarily
result in inhibition
of subgenomic RNA synthesis. Interestingly, this
phenotype is
similar to that of another temperature-sensitive SIN
mutant, 24R1,
in which a mutation in nsP4 permitted continuation of
minus-strand
synthesis without affecting subgenomic RNA synthesis
(
20).
The loss of cytopathogenicity was correlated with a reduction of RNA
replication in a panel of nsP2 Pro
726 mutants
(
10).
Unfortunately, diminished replication also was
associated with
severely decreased replicon packaging efficiency. One
of our variants,
S2, which was equivalent to previously isolated
variants (
10),
could not be packaged efficiently into
virion-like particles.
In contrast, several of our newly identified
variants (S1, SF2A,
and SF2C) both maintained high-level transgene
expression and
also were packaged efficiently, thus increasing the
versatility
of these replicons. For example, the new replicons might be
particularly
useful for extending expression studies in hippocampal
slice cultures
(
9) without perturbation of host cell
metabolism. The degree
of cytopathogenicity and the mechanisms by which
induction of
apoptosis is either inhibited or delayed remain to be
established.
Importantly, this panel of variants provides a basis for
further
studies in both cultured cells and animal models of human
disease.
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FOOTNOTES |
*
Corresponding author. Mailing address: Chiron
Corporation, 4560 Horton St., MS 4.3, Emeryville, CA 94608. Phone:
(510) 923-8144. Fax: (510) 923-2586. E-mail:
silvia_perri{at}cc.chiron.com.
| |
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Journal of Virology, October 2000, p. 9802-9807, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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