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V Stollar

Publications and source records attributed to V Stollar.

At least 37 records · Page 2Linked to original sources
PubMed1990

Both amino acid changes in nsP1 of Sindbis virusLM21 contribute to and are required for efficient expression of the mutant phenotype.

SVLM21 is a mutant of Sindbis virus which in contrast to the standard virus, SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. Previously, by making use of the infectious Toto plasmids, we had constructed recombinant viruses containing various SVLM21 sequences, and were thereby able to map the mutations associated with the SVLM21 phenotype to the gene for the nonstructural protein nsP1. Two mutations were found in the nsP1 gene of SVLM21. These led to predicted amino acid changes at residue 87 from Arg to Leu, and at residue 88 from Ser to Cys. In the work presented here, we assess the relative contributions of these two mutations to the SVLM21 phenotype using site-directed mutagenesis to obtain virus encoding only the change to Leu at residue 87 of nsP1 (SVMS319), or only the change to Cys at residue 88 (SVMS321). In addition we show that SVLM10, which was isolated during the selection procedure for SVLM21, encodes only the change at residue 88. In addition to its ability to grow in methionine-deprived mosquito cells, SVLM21 differs from SVSTD in two other respects: (1) it shows an increased sensitivity to neplanocin A (NPA) and (2) it generates increased levels of methyltransferase in infected cells. Whether we looked at resistance to low methionine, sensitivity to NPA, or levels of methyltransferase generated, SVMS319, SVMS321, and SVLM10 all expressed only a partial SVLM21 phenotype. Furthermore we were not able in these experiments to distinguish between these three viruses. We conclude therefore that both amino acid changes, i.e., at residues 87 and 88, are required to produce the full SVLM21 phenotype, and that both changes contribute equally.

Aedes↗
PubMed1990

Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures.

Cell-to-cell fusion of Aedes albopictus (mosquito) cells infected with dengue and St Louis encephalitis (SLE) flaviviruses was induced by exposure to low pH. The parameters of this low pH-induced fusion were examined. Syncytium formation was maximal in cultures 36 to 48 h post-infection and occurred when cultures were maintained at the acid pH for 15 min at 35 degrees C. The optimal pH range for fusion was 5.0 to 6.5 for dengue virus-infected cells and 5.0 to 5.5 for SLE virus-infected cells. Syncytia were not observed in vertebrate cells (Vero and BHK) under these conditions despite similar virus yields. Fusion was shown to be ATP-dependent and could be prevented by the addition of either polyclonal antiviral antibodies or monoclonal antibody to the envelope glycoprotein. The lysosomotropic amine ammonium chloride inhibited the replication of SLE virus in both mosquito and vertebrate cells, consistent with the idea that low pH-induced fusion is necessary for virus entry into both types.

Adenosine Triphosphate↗
PubMed1990

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner.

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.

Aedes↗
PubMed1989

Association of the Sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1.

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

Animals↗
PubMed1989

Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization.

In a previous paper (G. Winkler, V. B. Randolph, G. R. Cleaves, T. E. Ryan, and V. Stollar, 1988, Virology 162, 187-196) we showed that the newly synthesized dengue-2 virus nonstructural protein, NS1, exists briefly as a monomer and then undergoes dimerization. We demonstrate here that the dimerization of NS1 is associated with a change in hydrophobicity and sedimentability of this protein. Newly synthesized monomeric NS1 is a hydrophilic and water-soluble protein which cannot be pelleted at 75,000 g. After dimerization, however, NS1 showed increased hydrophobicity in a Triton X-114 phase partitioning system and was completely pelletable at 75,000 g; these findings are consistent with NS1 becoming membrane-associated. In experiments in which infected cells were treated with tunicamycin it was shown that the glycosylation of NS1 was not required for either the dimerization or the membrane association.

Animals↗
PubMed1989

SVLM21, a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase.

The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5' cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21 generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21 generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21 enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21 phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21 to methionine deprivation.

Animals↗
PubMed1989

Effect of Sindbis virus infection on induction of heat shock proteins in Aedes albopictus cells.

When Aedes albopictus cells (clone C7) were infected with Sindbis virus, the production of cytopathic effect CPE depended largely on the conditions under which the cells were cultured. We observed marked inhibition of cellular RNA and protein synthesis, as well as a loss of the ability to induce heat shock proteins, e.g., hsp70, under conditions which led to cytopathic effect. Infected cells in which heat shock proteins could no longer be induced contained much lower amounts of hsp70 mRNA after heat shock than did mock-infected cells which were similarly treated. It is suggested that this decreased level of hsp70 mRNA is due to a failure of these cells to synthesize hsp70 mRNA after heat shock.

Aedes↗
PubMed1988

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer.

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NS1 (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NS1 is consistent with the idea that the high-molecular-weight form of NS1 is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NS1 is a general feature of flavivirus infection. It is formed between 20 and 40 min after NS1 is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NS1 are also found as the high-molecular-weight form.

Capsid↗
PubMed1988

SVLM21, a mutant of Sindbis virus able to grow in Aedes albopictus cells in the absence of methionine, shows increased sensitivity to S-adenosylhomocysteine hydrolase inhibitors such as neplanocin A.

Inhibition of S-adenosylhomocysteine (AdoHcy) hydrolase by compounds such as Neplanocin A (NPA) leads to the build-up of AdoHcy and the inhibition of methyltransferase enzymes. Whether assayed by efficiency of plaquing or virus yield, SVLM21, a mutant of Sindbis virus resistant to methionine deprivation, was more sensitive to NPA than was the standard virus (SVstd) from which it was derived. For example, whereas 10 micrograms NPA/ml depressed the yield of SVLM21 by more than 30-fold, the yield of SVstd was not significantly affected. Similar differences in sensitivities were shown to three other compounds which inhibit AdoHcy hydrolase. These results support the idea that SVLM21 codes for an altered RNA methyltransferase.

Adenine↗
PubMed1987

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells.

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.

Adenine↗
PubMed1987

Sindbis virus mutants resistant to mycophenolic acid and ribavirin.

Previous work from this laboratory has demonstrated a correlation between the inhibition by ribavirin (Rbv), mycophenolic acid (MPA), or 2-amino thiadiazole (TDA) of Sindbis virus replication in Aedes albopictus mosquito cells and a reduction in cellular GTP levels. This reduction in GTP results from the inhibition by these drugs of inosine monophosphate dehydrogenase (IMPDH), the first enzyme specific for the de novo synthesis of GMP. By serial passage of SV in A. albopictus cells in the presence of 25 microM MPA, we have now isolated viral mutants which are highly resistant not only to MPA but also to Rbv and TDA. For example, whereas 500 microM Rbv reduced the plaquing efficiency of SVSTD by at least 10(6)-fold, the same concentration of Rbv reduced the plaquing efficiency of the MPA-resistant mutants less than 5-fold. This is the first example of a viral mutant resistant to the antiviral compound Rbv.

Aedes↗
PubMed1986

Sequence analysis of the E2 gene of a hyperglycosylated, host restricted mutant of Sindbis virus and estimation of mutation rate from frequency of revertants.

SVap15/21, a strain of Sindbis virus (SV) derived from our standard laboratory strain of SV (SVstd) after repeated passage on Aedes albopictus cells, grows normally in mosquito cells but is host restricted (hr) in vertebrate cells. It is also temperature sensitive (ts) and produces pinpoint plaques on vertebrate cells (sp). E2 glycoprotein of SVstd differs from that of the more widely used SVHR (from which SVstd was derived) by an additional (i.e., third) N-linked glycan. The E2 of SVap15/21, in turn, differs from that of SVstd by the addition of a fourth glycan. We have determined the nucleotide sequence of the E2 genes of SVap15/21 and of SVstd, as well as that of our isolate of SVHR. The nucleotide sequence of the SVstd E2 gene predicted the occurrence of an additional N-linked glycan attachment site, not present in the SVHR E2, at Asn232 (Asp in SVHR). The sequence of the SVap15/21 E2 gene demonstrated three mutations relative to the SVstd gene, including one that predicted the occurrence of another potential N-linked glycan attachment site at Asn275. Sequence analysis of 15 revertants of SVap15/21 which are no longer host-restricted revealed that all had lost the glycosylation site at Asn275, confirming the connection between the hyperglycosylation and the host dependent block in assembly. Most of these revertants had also lost the temperature sensitivity and small plaque traits (i.e., were ts+ and lp). Each revertant of this class was characterized by one of three different mutations in two separate codons (Asn275 and Thr277), resulting in the loss of the glycosylation site at Asn275. A fourth mutation, resulting in an Asn275----Tyr substitution, was associated with a hr+ ts phenotype in three isolates. Finally an additional mutation in a different part of the E2 gene was found in two hr+ ts sp isolates that had also lost the glycosylation site at Asn275 through a mutation resulting in a Thr277----Ile substitution. Knowledge of the nucleotide sequences associated with the ts defect in SVap15/21 and with its reversion permit an estimation of the mutation rate of this virus. This calculation indicates a mutation rate of less than 10(-6) errors per base incorporation.

Amino Acid Sequence↗
PubMed1986

Dominance of the CPE(+) phenotype in hybrid Aedes albopictus cells infected with Sindbis virus.

The effect of Sindbis virus (SV) infection was analyzed in hybrid Aedes albopictus cells formed by fusing ouabain-resistant CPE(+) cells to CPE(-) alpha-amanitin resistant cells. Although the 24-h yields of virus from the parental CPE(+) and CPE(-) clones were similar, the rates of viral RNA synthesis and virus release at early times post-infection were higher in the CPE(+) cells. In all eight hybrid clones studied, the CPE(+) phenotype was dominant. In addition, the kinetics of viral RNA synthesis and virus release in the hybrids closely resembled what was observed in the CPE(+) parent clone. These data indicate that both in the parental and in the hybrid cells, expression of SV-induced CPE is associated with a high level of viral RNA synthesis and a rapid production of virus during the early period after infection. It is suggested that CPE(+) cells contain some factor or activity which is lacking or less abundant in the CPE(-) cells and that this activity is important in the regulation of viral RNA synthesis.

Aedes↗
PubMed1985

Sindbis virus mutants able to replicate in methionine-deprived Aedes albopictus cells.

Previous work from this laboratory has shown that the replication of Sindbis virus (SV) in Aedes albopictus cells is sensitive to methionine withdrawal. This sensitivity is thought to reflect a diminished concentration of S-adenosylmethionine (Ado Met) resulting from methionine starvation. Serial passage of SV on Ae. albopictus cells maintained in low concentrations of methionine gave rise to a population of mutants whose replication in mosquito cells was resistant to methionine starvation. In vertebrate cells, these mutants were also resistant to inhibition by cycloleucine. We favor the hypothesis that the adaptation to low methionine reflects the accumulation of mutations resulting in a viral RNA "cap" methyltransferase with an increased affinity for Ado Met.

Aedes↗
PubMed1984

Host-dependent mutants of Sindbis virus whose growth is restricted in cultured Aedes albopictus cells produce normal yields of virus in intact mosquitoes.

Two host-dependent (hd), temperature-sensitive (ts) mutants of Sindbis virus (SV), clones 35 and 58, which are restricted in their ability to grow at 34 degrees in cultured Aedes albopictus cells [K.J. Kowal and V. Stollar (1981). Virology 114, 140-148] were not restricted in their ability to grow at 28-34 degrees after intrathoracic inoculation of Ae. albopictus, Ae. dorsalis, Ae. epactius, Anopheles freeborni, and Culex tarsalis mosquitoes. Compared to standard SV (SVSTD), the growth of SV clone 35 was somewhat restricted in parenterally infected C. pipiens females. Titers of SVSTD and the two mutants were modulated to relative low levels by some Ae. albopictus and Ae. dorsalis females after 192 hr incubation at 34 degrees but not at 28 degrees, suggesting that under some conditions mosquitoes have the ability to control the replication of SV. The hd and ts phenotypes of SV mutants were retained during growth at 31 degrees for 36 hr and 72 hr in Ae. epactius and Ae. dorsalis females, respectively, and at 28 degrees or 34 degrees for 192 hr in Ae. albopictus females. The demonstration that mutant viral clones, which are restricted in cultured mosquito cells, can produce normal yields of virus in intact mosquitoes indicates that considerable caution must be exercised in extrapolating results obtained with cultured cells to the level of the intact organism.

Aedes↗
PubMed1984

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2.

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Aedes↗
PubMed1984

Evidence that ribavirin is transported in Aedes albopictus cells by the nucleoside transport system.

Aedes albopictus cells (CAE-3-6) deficient in the transport of nucleosides showed a markedly impaired ability to transport the antiviral compound, ribavirin. Furthermore, in order to inhibit either cell growth or viral replication, the transport negative CAE-3-6 cells required significantly higher concentrations of ribavirin than were needed for a similar degree of inhibition in the parental C7 cells. Although ribavirin did lower the level of GTP in CAE-3-6 cells the effect was slower and less striking than in the parental cells. These results indicate that ribavirin is transported across the plasma membrane of Aedes albopictus cells by the same transport system which functions to transport naturally occurring nucleosides.

Aedes↗