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Journal of Virology, September 2004, p. 9645-9651, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9645-9651.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Mutant of Sindbis Virus Which Is Able To Replicate in Cells with Reduced CTP Makes a Replicase/Transcriptase with a Decreased K_m for CTP

Mei-Ling Li,¹ Yen-Huei Lin,^1, H. Anne Simmonds,² and Victor Stollar^1*

Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey,¹ Purine Research Unit, Guy's Hospital, London Bridge, Great Britain²

Received 30 December 2003/ Accepted 13 April 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
We reported earlier the isolation and characterization of a Sindbis virus mutant, SV_PZF, that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SV_CPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SV_PZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SV_CPC was selected for its ability to grow in cells in which only the level of CTP was reduced. Although SV_PZF was cross-resistant to CPC, SV_CPC was not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SV_CPC-infected mosquito cells had a lower K_m for CTP (but not for UTP) than did the enzyme made in SV_STD-infected mosquito cells. SV_PZF and SV_CPC represent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sindbis virus (SV), an enveloped positive-strand RNA virus, is the prototype virus of the family Togaviridae, genus Alphavirus (see references 27 and 31 for reviews). The 11,703-nucleotide (nt)-long single-stranded genome has a 5' cap and a 3' poly(A) tail. The four nonstructural (ns) proteins are encoded by the 5' two-thirds of the viral genome, whereas the three structural proteins (the capsid protein, C, and the two envelope proteins, E1 and E2) are encoded by a subgenomic (SG) RNA whose sequence is colinear with the 3' one-third of the viral genome.

The four ns proteins, nsP1, nsP2, nsP3, and nsP4, are derived from the proteolytic cleavage of two large polyproteins, P123 and P1234. The latter is made by low-efficiency readthrough of an opal codon located near the 3' end of the nsP3 coding sequence.

Specific biochemical functions have been associated with three of the four ns proteins: nsP1 has both RNA methyltransferase and RNA guanylyltransferase activities and is thus involved in the modification of the 5' termini of the positive-strand genomic and SG RNAs (1, 21). The N-terminal domain of nsP2 has an RNA helicase activity (12), while the C-terminal domain is a protease (13) that serves to process the P123 and P1234 polyproteins. nsP2 also has an RNA-triphosphatase activity (32), thus associating it with the modification of the 5' termini of the viral positive-strand RNAs. Studies with a viral mutant, ts6 (2), and the presence of the consensus GDD motif have pointed to nsP4 as the viral RNA-dependent RNA polymerase (RDRP). The biochemical function of nsP3 is not known.

Synthesis of the positive-strand genomic RNA, and of the genomic size negative-strand RNA, and the transcription of the SG positive-strand RNA are regulated temporally by the processing of the ns polyproteins (17, 29). According to the model proposed by Lemm et al. (17), nsP4 and uncleaved P123 serve as the minus-strand RNA replicase. Cleavage of P123 results in the cessation of minus-strand synthesis and the more efficient synthesis of both genomic and SG plus-strand RNAs. More recently, Fata et al. (10) have suggested that the switch from minus-strand synthesis to the synthesis of SG RNA is associated with the cleavage of P23. The mechanism whereby the processing of P123 and P1234 regulates the synthesis of viral RNA is not known.

With the goal of identifying amino acid residues in nsP4 that influence the binding of ribonucleoside triphosphate (rNTP) substrates to the SV replicase/transcriptase, we had earlier isolated a mutant of SV, SV_PZF, that is able to grow in Aedes albopictus cells treated with pyrazofurin (PZF), a pyrimidine analog that inhibits orotic acid decarboxylase, an enzyme in the pyrimidine biosynthetic pathway (20). Treatment of cells with PZF results in a reduction in the size of the UTP and CTP pools. Thus, SV_PZF, in contrast to the parental virus, SV_STD, is able to grow in cells with lowered levels of UTP and CTP. We identified three amino acid changes in the SV_PZF-encoded nsP4 protein, all of which are essential for the PZF resistance phenotype: Met 287 to Leu, Lys 592 to Ile, and Pro 609 to Thr. Since the three-dimensional structure of the alphavirus nsP4 protein had not been described, we derived a tentative structure for SV nsP4 by molecular modeling with the human immunodeficiency virus reverse transcriptase (14, 22) as the template. In this structure, residue 287 was localized to the finger domain of nsP4 and residues 592 and 609 were localized to the thumb domain.

SV_PZF was selected on the basis of its ability to replicate in cells with decreased levels of both UTP and CTP. Could we now select a viral mutant on the basis of its ability to replicate in cells in which the level of only one of these rNTPs is decreased? If so, would such a mutant display the same amino acid changes in nsP4 as SV_PZF? To answer these questions, we set out to select a viral mutant resistant to cyclopentenylcytosine (CPC), a compound that inhibits CTP synthase (11) and thus should decrease the level of CTP in treated cells. We expected that UTP levels in these cells would not be decreased, and might even be increased. We report here the properties of the mutant we obtained and identify the mutation responsible for the resistance to CPC.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and viruses. The A. albopictus mosquito (C7-10) cells used have been described previously (7); they were grown at 28°C in Eagle's minimal essential medium (MEM) supplemented with nonessential amino acids and 5% fetal calf serum. BHK cells were grown in Eagle's MEM supplemented with 5% tryptose phosphate and 5% bovine serum. The chicken embryo fibroblasts (CEF) used were either primary cultures (30) or the SL-29 cell line obtained from the American Type Culture Collection. CEF were grown in Eagle's MEM supplemented with 10 mM sodium pyruvate, 5% tryptose phosphate, and 5% bovine serum at 37°C.

Our standard virus (SV_STD) was derived from the HR strain of SV (28). SV_Toto is the virus derived from plasmid Toto1101 (23). Mosquito cells were infected at a multiplicity of infection (MOI) of 2 to 5 PFU per cell and maintained after infection at 34.5°C in medium containing 0.2% bovine serum albumin in place of fetal calf serum. Media from infected cultures were harvested at the indicated times, and virus titers were measured by plaque formation on CEF.

pToto1101 (13,638 bp) is a plasmid that contains a genome length cDNA copy of SV RNA immediately downstream from an SP6 promoter (23). Infectious RNA transcripts generated from pToto were used to transfect C7-10 cells as described previously (19).

Plasmid construction and site-directed mutagenesis. DNA manipulations were performed by standard procedures (24). The chimeric plasmid pToto:SV_CPC5262-7999 was generated as follows. To prepare SV_CPC cDNA, BHK cells were infected with SV_CPC at an MOI of 1 PFU per cell at 34.5°C for 24 h. Total RNA was extracted from cells with Trizol reagent (Invitrogen, Carlsbad, Calif.). SV_CPC cDNA was then synthesized by reverse transcription-PCR with 5 µg of the total RNA extracted from the infected cells, the SuperScript one-step reverse transcription-PCR system (Invitrogen), and the oligonucleotides nt 5161 to 5178 (plus strand) and 8049 to 8028 (minus strand) of the SV RNA sequence as upstream and downstream primers, respectively. The cDNA was digested with SpeI (nt 5262) and AatII (nt 7999) and cloned into pToto1101, replacing the original pToto sequence. The QuikChange kit (Stratagene, La Jolla, Calif.) was used to perform site-directed mutagenesis in accordance with the manufacturer's directions.

Extraction and analysis of ribonucleotides. Mosquito cells were either left untreated or treated with 5 µM CPC for 8 h; ribonucleotides were extracted from cells as described previously (4). Briefly, cells from each 35-mm-diameter plate were lysed with 200 µl of 10% trichloroacetic acid. The precipitate was removed by centrifugation for 1 min at 12,000 x g. The supernatant was back extracted with diethyl ether to a pH of 5, and the ribonucleotides were analyzed by high-performance liquid chromatography (9).

Determination of the K_ms of the viral replicase/transcriptase for UTP and CTP. Confluent mosquito cells were infected with either SV_STD or SV_CPC at an MOI of 1 PFU per cell. Twenty-four hours after infection, the p15 fraction was prepared as described by Lemm et al. (16). The p15 pellet isolated from one 100-mm-diameter dish was resuspended in 80 µl of storage buffer (10 mM Tris-Cl [pH 7.8], 10 mM NaCl, 15% glycerol). The polymerase assay was carried out as described by Lemm et al. (16). Briefly, 25 µl of the p15 fraction (equal to 9.4 x 10⁶ cells) was used in a 50-µl reaction mixture. The concentration of the nucleotide under study was varied from 0 to 500 µM. To determine the K_m for CTP, each reaction mixture contained 1 mM (each) ATP and GTP, 0.1 mM UTP, 5 µCi of [³H]UTP (36.2 Ci/mmol), and various concentrations of CTP. A similar reaction was set up to determine the K_m for UTP, except that in this case, the concentration of CTP was 0.1 mM, 5 µCi of [³H]CTP (20.5 Ci/mmol) was added, and the concentration of UTP was varied. The reaction mixtures were incubated at 30°C for 20 min. Incorporation of the labeled isotope was linear for up to 40 min. The labeled RNA products were captured on nitrocellulose membrane and quantified by liquid scintillation counting (33). Reactions were also carried out with p15 fractions from mock-infected cells. The data points in Fig. 4A and B represent values obtained after subtraction of the "incorporation" observed with p15 proteins from mock-infected cells. The K_m values were calculated with GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, Calif.).

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FIG. 4. Kinetic analyses of in vitro viral RNA synthesis by the SVCPC and SVSTD replicase/transcriptases. The assays were performed with p15 fractions from virus-infected mosquito cells as described in Materials and Methods. Nonlinear regression analysis with the GraphPad Prism version 3.0 software was used to calculate the Kms of the SV polymerase for CTP (A) and UTP (B). Each reaction was done in triplicate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of CPC on nucleotide pools. Figure 1 shows the effect of CPC on the pool size of the four rNTPs in A. albopictus cells. In both the zero time sample and the 8-h untreated sample, the ATP level was higher than that of any of the other rNTPs. After ATP, the pool sizes decreased in the order UTP > CTP > GTP. These results are similar to those we reported previously (29a), except that in the present studies the level of CTP is higher than the level of GTP. In extracts of cells treated with CPC for 8 h, the levels of ATP, UTP, and GTP all rose significantly whereas the level of CTP was drastically reduced. Thus, CPC selectively reduces the level of CTP.

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FIG. 1. Effect of CPC on the rNTP pools in C7-10 cells. rNTP concentrations are expressed as picomoles per 106 cells initially plated. Extraction and analysis of rNTPs were done as described in Materials and Methods.

Selection of an SV mutant resistant to CPC. As shown in Fig. 2, CPC markedly reduced the yield of SV_STD from mosquito cells. At a concentration of 3 µM CPC, the yield was reduced more than 100-fold, and at 10 µM, it was reduced more than 1,000-fold.

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FIG. 2. Effect of CPC on yield of SVSTD from mosquito cells. A. albopictus mosquito cells were infected with SVSTD at an MOI of 5 PFU per cell. After an adsorption period of 60 min, the virus was removed, the cultures were refed with medium, and CPC was added to the indicated concentrations. After incubation for 24 h at 34°C, samples of the medium were harvested and assayed for infectious virus by plaque formation on primary CEF.

To obtain a mutant resistant to CPC, a serial undiluted passage of SV in mosquito cells was carried out in the presence of a gradually increasing concentration of CPC. The passage was begun with SV_STD and continued for 34 passages; the first four passages were carried out in the presence of 1 µM CPC, and the last seven passages were done with 10 µM CPC. Intermediate concentrations of CPC were used for the intervening passages. For each passage, virus was harvested 20 to 24 h after infection and infected cells were maintained at 34°C. At various passage levels, the stocks were assayed for resistance to CPC. The desired level of resistance in the passaged virus populations was not obtained until more than 30 passages had been carried out. After 34 passages the virus was plaque purified and a working stock was prepared on primary CEF. This stock was designated SV_CPC and used in all of the experiments described in this report. In contrast to SV_STD, which produced large plaques (3 mm in diameter), the plaques produced by SV_CPC were only 1 mm in diameter.

Figure 3 shows that whereas 5 µM CPC reduced the yield of SV_STD by almost 300-fold, the same concentration of CPC reduced the yield of SV_CPC by only 4- to 5-fold. Thus, SV_CPC shows good resistance to CPC, implying that it can replicate well in cells with a markedly reduced level of CTP.

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FIG. 3. Effect of CPC on yields of SVSTD and SVCPC from mosquito cells. The procedure was the same as that described for Fig. 2, except that the MOI was 2 PFU per cell.

Do SV_PZF and SV_CPC show cross-resistance? Mosquito cells infected with SV_STD, SV_CPC, or SV_PZF were treated with 5 µM CPC or 3 µM PZF or left untreated. Medium was harvested after 24 h and assayed for infectious virus by plaque assay on chick cells. As shown in Table 1, the yields of SV_STD from CPC- and PZF-treated cells were reduced approximately 350- and 600-fold, respectively. In contrast, neither PZF nor CPC reduced the yield of SV_PZF. Consistent with the results shown in Fig. 2, SV_CPC showed the expected resistance to CPC; however, it was as sensitive as SV_STD to PZF. Thus, SV_PZF is cross-resistant to CPC but SV_CPC is not cross-resistant to PZF.

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TABLE 1. Cross-resistance of SVPZF to CPCa

Identification of the mutation(s) responsible for resistance to CPC. Since we had shown earlier that the mutations responsible for the resistance of SV_PZF to PZF, i.e., to decreased levels of UTP and CTP, were in the nsP4 coding sequence, it was likely that the mutations responsible for the resistance of SV_CPC to CPC, i.e., to decreased CTP, would also lie in this region. Accordingly, we prepared a chimeric plasmid of pToto1101 that contained the full-length wild-type SV genome sequence, but with the cDNA sequence from nt 5262 to nt 7999 replaced with the SV_CPC sequence (20), and used it to generate infectious RNA. The sequence from nt 5262 to nt 7999 contains all of the coding information for nsP4 and approximately half of nsP3. The virus (SV_{Toto:CPC:5262-7999}) obtained from this infectious RNA, like SV_CPC, was resistant to CPC (data not shown).

Sequencing of the region from nt 5262 to nt 7999 revealed two mutations. The first, A5750C, changed the opal codon near the end of the nsP3 coding sequence to Cys, and the second, A7523C, changed Leu 585 of nsP4 to Phe. Since earlier work had shown that a change of the opal codon to an amino acid codon did not lead to any obvious phenotype or to an increase in the level of nsP4 (18), and since the mutation found in the opal codon of SV_PZF was not required for the resistance of that mutant to PZF, it was likely that the drug resistance phenotype of SV_CPC was due to the A7523C mutation. When this single mutation was introduced into SV_Toto, the resulting virus showed a resistance to CPC similar to that of SV_CPC (Table 2). Thus, in contrast to SV_PZF, which required three amino acid changes to produce resistance to PZF, SV_CPC required only a single amino acid change to produce resistance to CPC. It is notable that whereas SV_CPC produced small plaques, SV_{Toto:nsP4:L585F} gave rise to large plaques.

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TABLE 2. Phenotypes of recombinant virus and virus with the SVCPC mutationa

When the plaque purification was done that gave rise to SV_CPC (after the 34 serial undiluted passages described above), other plaques were also picked. Three of these were characterized, and like SV_CPC, they were resistant to CPC; furthermore, all three had the same A7523C mutation as SV_CPC. According to the molecular model of nsP4 that we proposed earlier (20), residue 585 of nsP4 should be located in the thumb domain of nsP4.

We next determined the effects of other amino acid changes at position 585 of nsP4. Accordingly, mutations were introduced into pToto that were predicted to change Leu 585 of nsP4 to Tyr, Arg, His, Trp, Val, or Ala. As shown in (Table 3), only SV_{Toto:nsP4:L585F} and SV_{Toto:nsp4:L585Y}, viruses with the Phe and Tyr substitutions, respectively, were resistant to CPC. Like SV_CPC, SV_{Toto:nsP4:L585Y} produced small plaques, but as noted above, SV_{Toto:nsP4:L585F} produced large plaques. Virus with His or Val in place of Leu 585 of nsP4 was sensitive to CPC. However, whereas the former produced large plaques, the latter produced a mixture of large and small plaques. None of the mutant viruses were resistant to PZF. We were not successful in obtaining viable virus when Trp, Arg, or Ala was substituted for Leu 585 of nsP4 and assume that these substitutions were lethal.

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TABLE 3. Phenotype of SV with different amino acids at position 585 of nsP4a

Kinetic constants of the replicase/transcriptases encoded by SV_CPC and SV_STD. We next determined the kinetic parameters for the viral replicase/transcriptases encoded by SV_CPC and SV_STD. Since the p15 fractions from SV-infected cells make both genomic and SG viral RNAs (2, 3), the RNA-synthesizing activity we measured is referred to as a replicase/transcriptase. The K_ms for CTP and UTP were measured as described in Materials and Methods.

Figure 4A shows the replicase/transcriptase activities (as measured by incorporation of [³H]UTP) of the SV_CPC and SV_STD enzymes made in infected mosquito cells as a function of the concentration of CTP. The K_ms for CTP as determined from the plot were 3.0 and 16.3 µM, respectively, for the SV_CPC and SV_STD replicase/transcriptases. Thus, the K_m for CTP of SV_CPC polymerase is approximately 5.4-fold lower than that of SV_STD, consistent with the idea that the SV_CPC replicase/transcriptase has a higher affinity for CTP than does the SV_STD replicase/transcriptase. Figure 4B shows a plot of the replicase/transcriptase activities made in SV_CPC- and SV_STD-infected cells as a function of the concentration of UTP. The K_ms for UTP of the two enzyme activities were quite similar, 10.1 and 12.5 µM, respectively. Similar K_m values were obtained when the data were analyzed by Lineweaver-Burk plots.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although rNTPs are the immediate precursors for the synthesis of viral RNA, there have been few studies of how manipulation of the rNTP pools affects the replication of RNA viruses. Critical questions include the following: how much can the rNTP pools be reduced before the synthesis of viral RNA is affected, what are the relative sensitivities of viral and host RNA syntheses to depletion of the rNTP pools, does the size of the rNTP pools vary from cell type to cell type, and could such variation influence the efficiency of viral replication and perhaps even the permissiveness of a cell for viral replication?

In this report we have shown that CPC, a compound that interferes with the synthesis of CTP, markedly reduced the size of the CTP pool in A. albopictus mosquito cells; this reduction in the size of the CTP pool was associated with a significant lowering of the yield of virus from treated cells. It is reasonable to assume that the level of CTP in the CPC-treated cells was too low to support efficient synthesis of viral RNA by the viral replicase/transcriptase.

Earlier work in our laboratory over a period of years has shown that by depleting mosquito cells of a metabolite that is the substrate of a virus-coded enzyme, it is possible to isolate viral mutants able to replicate in cells with low levels of that substrate. To the best of our knowledge, there are only five low-molecular-weight metabolites that are substrates for enzymes encoded by SV. These are ATP, GTP, UTP, and CTP, which are substrates for the viral RDRP (nsP4), and S-adenosylmethionine, which is a substrate for the viral RNA methyltransferase. GTP is a substrate not only for the RDRP but also for the viral RNA guanylyltransferase or capping enzyme. Thus, we isolated SV_LM21, which is able to grow in cells presumably depleted of S-adenosylmethionine (8), and SV_MPA, which is able to grow in cells depleted of GTP (25). The findings that the mutations responsible for both the SV_LM21 and SV_MPA phenotypes mapped to the nsP1 coding region provided the first evidence associating nsP1 with the capping and methylation of the viral mRNAs (21, 26). Similarly, SV_PZF was isolated on the basis of its ability to grow in cells depleted of UTP and CTP. As expected, the mutations responsible for the ability of SV_PZF to grow under these conditions were in the nsP4 coding sequence (20).

In this report we describe the isolation and properties of SV_CPC, a mutant selected for its ability to grow in cells depleted only of CTP. In contrast to the situation with SV_PZF, only one amino acid change was needed to enable the virus to grow in CTP-depleted mosquito cells, but as was the case with SV_PZF, the amino acid change was in nsP4. However, the amino acid residue that was changed in SV_CPC was different from those changed in the case of SV_PZF.

It is notable that both SV_PZF and SV_CPC were found to have a mutation near the end of the nsP3 coding sequence eliminating the opal termination codon. However, abolishing this termination codon does not appear to increase the amount of nsP4 in infected cells (18) but would give rise to a slightly longer form of nsP3. Furthermore, site-directed mutagenesis clearly indicated that resistance to PZF or CPC did not depend on elimination of the opal codon.

Since no structure has been described for the alphavirus nsP4 protein, when we isolated SV_PZF, we generated a molecular model of this protein. Although molecular models have obvious limitations, especially when the test protein and the template protein are not closely related, they may be useful in localizing amino acid residues to specific domains of a protein. In the case of SV_PZF, we localized one changed residue to the finger domain and the other two to the thumb domain. In the case of SV_CPC, the single changed amino acid was localized to the thumb domain. If our molecular model is valid, it is noteworthy that in neither case were the mutations in the catalytic site, i.e., in the palm region of nsP4.

We undertook the isolation of SV_PZF and SV_CPC with the expectation that the replicase/transcriptase enzymes encoded by these viruses would have an increased affinity for UTP and CTP in the first case, and perhaps only for CTP in the second case. This idea is supported by our finding that the SV_CPC replicase/transcriptase has a K_m for CTP approximately fivefold lower than that of the SV_STD replicase/transcriptase, whereas the K_ms for UTP are very similar. This is the first report of an SV mutant (or possibly of any positive-strand RNA virus mutant) that encodes a replicase or transcriptase with a decreased K_m for one of its rNTP substrates.

The question arises of whether these are the only mutations that can give rise to the phenotypes we were seeking. We have shown that in addition to a Phe substitution for Leu, a Tyr substitution can also result in resistance to CPC. Of particular interest is our finding that SV_PZF is cross-resistant to CPC but SV_CPC is not cross-resistant to PZF. This suggests that specific amino acid changes may selectively alter the affinity of the viral RDRP for individual rNTP substrates and that each of these rNTPs makes contact with different amino acid residues of the RDRP. Although it might be expected that changes in the RDRP that alter its affinity for the rNTP substrates would occur mainly in amino acid residues that make direct contact with these substrates or lie in proximity to the residues that do, there is evidence that amino acid changes in the finger and thumb domains can alter the conformation of the catalytic site or the specificity of a polymerase for nucleoside analogs of the rNTPs (5, 15).

We have identified specific amino acid residues that are altered in the nsP4 proteins encoded by SV_PZF and SV_CPC. Future work will be directed at identifying other amino acid changes in nsP4 that permit the virus to replicate in cells with decreased levels of one or more of the rNTP substrates. SV_PZF and SV_CPC are novel viral mutants that were selected for their ability to replicate in cells with reduced levels of rNTPs. Further study of these mutants should lead to important information bearing on viral replication and should provide insights useful for the development of viral vectors (6) and novel inhibitors of viral replication.

 
We thank Michael Newlon for help with the figures and Yan Hu for technical support.

This work was funded by U.S. Public Health Service grant AI-49273.

 
* Corresponding author. Mailing address: Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854. Phone: (732) 235-4596. Fax: (732) 235-5223. E-mail: stollar{at}umdnj.edu.

Present address: Wyeth-Lederle Vaccines, Pearl River, NY 10965.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, September 2004, p. 9645-9651, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9645-9651.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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