Biomedical subjects

V Stollar

Publications and source records attributed to V Stollar.

At least 19 recordsLinked to original sources

PubMed2003

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus.

We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5' and 3' non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Aedes↗

PubMed2000

A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase.

Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of virus was reduced 100- to 1000-fold. By serial passage of our standard SV(STD) in Ae. albopictus cells in the presence of increasing concentrations of PZF, a mutant, SV(PZF), was derived, which was not inhibited by PZF. SV(PZF) is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease levels of UTP and CTP. Analysis of chimeric viruses containing sequences from the SV(PZF) and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region identified four mutations (nt 5750, 6627, 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T in nsP4. Characterization of viruses containing one or more of these mutations demonstrated that all three mutations in the nsP4 coding region are required to produce full resistance to PZF. Using a molecular model of nsP4 based on the structure of HIV reverse transcriptase, we located amino acid change M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these three amino acid changes in nsP4 alter the geometry of the NTP binding pocket so as to increase the affinity of the enzyme for CTP and UTP.

Adenosine↗

PubMed1999

An amino acid change in the exodomain of the E2 protein of Sindbis virus, which impairs the release of virus from chicken cells but not from mosquito cells.

In order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SV(STD)) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SV(LM17), did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SV(LM17) in greater detail. In yield assays, the titer of SV(LM17) produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SV(STD), in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SV(LM17) was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SV(LM17), viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SV(LM17)-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SV(LM17) was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype.

Aedes↗

PubMed1999

SVMPA, a mutant of sindbis virus resistant to mycophenolic acid and ribavirin, shows an increased sensitivity to chick interferon.

SVMPA is a mutant of Sindbis virus, selected for its ability to replicate in mycophenolic acid (MPA)-treated mosquito cells. SVMPA has another phenotype: although able to replicate normally in primary cultures of chick embryo fibroblasts (CEF), its replication is restricted in secondary cultures prepared from aged primary CEF cultures. The mutations responsible for these phenotypes mapped to the region of the viral genome that codes for nsP1. We report here that SVMPA has yet another phenotype. Relative to our standard Sindbis virus (SVSTD) from which it was derived, SVMPA shows an increased sensitivity to chick interferon, both crude interferon prepared from virus-infected cells and recombinant interferon. Characterization of viral mutants obtained after site-directed mutagenesis indicated that the same mutations responsible for the host restriction of SVMPA in secondary cultures of CEF were also responsible for its increased sensitivity to chick interferon.

Animals↗

PubMed1999

Synthesis and methyltransferase activity of nonstructural protein nsP1 in Sindbis virus-infected Aedes albopictus cells.

To investigate the synthesis and turnover of the nonstructure protein, nsP1, in Aedes albopictus cells, we labeled infected cells with [35S]-methionine and immunoprecipitated nsP1 with a polyclonal monospecific rabbit antibody. Synthesis of nsP1 in mosquito cells could be detected 2 hr after infection and continued as long as 24 hr post-infection, regardless whether the infected cells were maintained at 28 degrees C , 34.5 degrees C , or 37 degrees C. Whereas the time pattern of nsP1 synthesis varied with temperature. Nonstructural protein 1 synthesis at 28 degrees C was maximal at 4 hr, then decreased. At 37 degrees C synthesis reached a high level at 2 hr and remained constant for 24 hr. Pulse-chase experiments showed that nsP1 in mosquito cells, whether made as early as 5 hr post-infection or as late as 24 hr, were stable during that time, the longest period tested. Enzyme activity reached a maximum at 10 hr and remained almost the same level until 24 hr. The yield of Sindbis virus from mosquito cells was higher at 34.5 degrees C and 37 degrees C than at 28 degrees C. Methyltransferase activity was needed for modification of positive-strand genomic and subgenomic RNAs. The activity of methyltransferase observed until late in the replication cycle probably accounted for both the continued synthesis and minimal turnover of nsP1.

Aedes↗

PubMed1997

Methyltransferase activity of the insect orbivirus JKT-7400: photoaffinity labeling of a minor virion protein, VP4, with S-adenosylmethionine.

JKT-7400 virus is an orbivirus originally isolated from Culex mosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor. The apparent Km of the MTase for S-adenosylmethionine (AdoMet) was 25 microM. Photoaffinity labeling experiments in which 3H-[methyl]-AdoMet was incubated with virions or viral cores demonstrated labeling of VP4, a minor protein present in the viral core, suggesting that this protein is the viral MTase. Labeling of VP4 was inhibited by addition of unlabeled AdoMet or S-adenosylhomocysteine (AdoHcy).

Aedes↗

PubMed1997

Characterization of JKT-7400, an orbivirus which grows in Aedes albopictus mosquito cells: evidence pointing to a minor virion protein, VP6, as the RNA guanylyltransferase.

JKT-7400 virus, an orbivirus originally isolated from Culex mosquitos, was plaque purified and adapted to Aedes albopictus mosquito cells. Conditions which enhance viral cytopathic effect and optimize plaque formation are described. In contrast to bluetongue virus, the prototype orbivirus, no replication of JKT-7400 virus in vertebrate cells was observed. The core particle of JKT-7400 virus contains 10 segments of dsRNA and three minor proteins, VP1, VP4, and VP6. The inner shell contains two major proteins, VP2 and VP7, and the outer shell consists of the other two major proteins, VP3 and VP5. Evidence is presented suggesting that the viral protein associated with the capping of virus mRNA, i.e., the guanylyltransferase, is VP6, one of the core proteins.

Aedes↗

PubMed1997

Complementation of and interference with Sindbis virus replication by full-length and deleted forms of the nonstructural protein, nsP1, expressed in stable transfectants of Hela cells.

Stable transfectants of Hela cells were isolated which expressed either the full-length 540-amino-acid Sindbis virus (SV) nonstructural protein, nsP1 (the form encoded by the mutant, SV(LM21)), or one of four forms with a carboxyl-terminal deletion. SV(LM21), in contrast to standard SV (SV(STD)), can replicate in Hela cells maintained in low-methionine (LM) medium. Expression of full-length nsP1(1-540), nsP1(1-492), or nsP1(1-448) resulted in complementation of SV(STD) when infected Hela cells were kept in LM medium after infection. In contrast, when cells were infected with SV(LM21) and maintained in LM medium, stable expression of any of the deleted forms of nsP1 interfered with the replication of virus. The ability of "cellular" nsP1 to complement SV(STD) in LM medium correlated with its methyltransferase activity.

Animals↗

PubMed1996

Mutagenesis of the Sindbis virus nsP1 protein: effects on methyltransferase activity and viral infectivity.

It has been suggested that four amino acids which are absolutely conserved in th nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a "methyltransferase motif." In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249. Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lower Km for S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 in Escherichia coli, and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249 were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, in E. coli. Changing His39, Arg91, Asp94, or Tyr249 to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369 to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activity in vitro. However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity.

Base Sequence↗

PubMed1995

A mutant of Sindbis virus which is released efficiently from cells maintained in low ionic strength medium.

Experiments many years ago showed that reducing the ionic strength of the medium Sindbis virus-infected chick embryo fibroblasts were maintained prevented the release of progeny virus into the medium. It was proposed at that time that the susceptibility of virus release to inhibition by low ionic strength (LIS) medium might be determined by the properties of the viral envelope proteins. We thought that if such were the case, it might be possible to obtain a viral mutant which is resistant to the inhibitory effect of LIS medium. We report here that we were able to isolate such a mutant of Sindbis virus, and that we identified two amino acids (Glu23 and Thr76) which, when present in the E2 protein, made possible the release of infectious progeny virus from cells incubated in LIS medium.

Animals↗

PubMed1994

Mutations in the nsP1 coding sequence of Sindbis virus which restrict viral replication in secondary cultures of chick embryo fibroblasts prepared from aged primary cultures.

SVMPA, a mutant of Sindbis virus (SV), which is able to replicate in Aedes albopictus cells treated with mycophenolic acid (MPA) or ribavirin, also has a host range phenotype. This phenotype is most clearly demonstrated by means of efficiency of plaquing (EOP) assays on secondary chick embryo fibroblasts (CEF) prepared from aged primary CEF. For example, in one experiment in which standard SV (SVSTD) had a relative EOP (EOP on primary CEF divided by EOP on secondary CEF) of 1.6 the corresponding value for SVMPA was 2340. The host restriction of SVMPA was also seen with similarly prepared secondary cultures of duck embryo fibroblasts, but not with established lines of quail cells. The finding that the accumulation of viral RNA was much lower in SVMPA-infected secondary CEF than in SVSTD-infected CEF indicated that the replication of SVMPA in these cultures was blocked at an early step. Revertants of SVMPA were isolated which were no longer host-restricted but had retained their resistance to MPA. Of the three mutations [nucleotide (nt) 120, 127, and 963] in the nsP1 coding sequence of SVMPA, which lead to amino acid changes, these revertants had retained the nt 127 and nt 963 mutations but had lost the nt 120 mutation. This, along with earlier findings, indicated that only the nt 127 and nt 963 mutations are required for resistance to MPA. This result also associated the nt 120 mutation with the host restriction phenotype. In other experiments derivatives of pToto1101 (a plasmid from which infectious Sindbis virus RNA can be transcribed) were constructed by site-directed mutagenesis and used to test the effect of specific mutations on the viral phenotype. Although we were unable to obtain viable virus with the nt 120 mutation alone, virus with the nt 120 and nt 127 mutations was viable and host-restricted. We suggest that the nt 120 mutation by itself is lethal and that the nt 127 mutation suppresses the lethal effect of the nt 120 mutation. The SVMPA mutation at nt 120, which is associated with the host range phenotype, changes Gln21 of nsP1 to Lys. When a more conservative change was engineered, i.e., to Asn, the virus was not host-restricted. Although the reason for the restriction of SVMPA replication in secondary CEF is not known, some possible explanations are discussed.

Aedes↗

PubMed1993

Insect-transmitted vertebrate viruses: alphatogaviruses.

Alphatogaviruses, of which Sindbis virus (SV) is the prototype, replicate to high titer in the laboratory both in mosquito cells and in vertebrate cells. By studying the replication of SV in mosquito cells as well as in vertebrate cells, we were able to obtain several viral mutants which have novel phenotypes and have contributed to our basic knowledge of this virus family. These include three host range mutants: SVAP15/21 which replicates normally in mosquito cells but is restricted in vertebrate cells and SVCL35 and SVCL58, which are restricted in mosquito cells but replicate normally in vertebrate cells. As well, two other mutants are described here: SVLM21, which can replicate in methionine-starved mosquito cells and SVMPA, which can replicate in mosquito cells treated with mycophenolic acid or ribavirin. The causal mutations of both SVLM21 and SVMPA are within the sequence encoding the nonstructural protein nsPl; these and other findings have enabled us to associate the capping and methylation of the viral mRNAs with the nsPl protein. Our work serves to emphasize that it is both worthwhile and important to study the replication of arthropod-borne viruses in cells derived from the arthropod host as well as in cells derived from the vertebrate host.

Aedes↗

PubMed1993

Reversal of the antiviral activity of ribavirin against Sindbis virus in Ae. albopictus mosquito cells.

Earlier work in our laboratory has shown that the replication of Sindbis virus in Aedes albopictus mosquito cells is inhibited by ribavirin (Rbv) and mycophenolic acid (MPA) (Sarver and Stollar (1978) Virology 91, 267-282; Malinoski and Stollar (1980) Virology 102, 473-476). We report here that the antiviral effect of Rbv and MPA can be reversed by depriving infected cells of methionine or isoleucine, or by treating them with fluorodeoxyuridine (FUdR) or cycloleucine. We suggest that, as was the case when the antiviral activity of Rbv was reversed by actinomycin D (Malinoski and Stollar (1981a) Virology 110, 281-291), these effects may be mediated by changes in the GTP pools of treated cells.

Aedes↗

PubMed1992

The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses.

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.

Amino Acid Sequence↗

PubMed1991

A mutant of Sindbis virus with altered plaque morphology and a decreased ratio of 26 S:49 S RNA synthesis in mosquito cells.

When our stock of standard Sindbis virus (SVSTD) is assayed by plaque formation on Aedes albopictus mosquito cells, about 1-2% of the plaques appear much clearer and sharper than the majority of the plaques. One of these clear plaques was picked, grown into a viral stock (SVCP), and used to prepare viral cDNA. Making use of the infectious Sindbis virus plasmid, Toto 1101 (Rice et al., 1987), we mapped the causal mutation for the clear plaque phenotype to a region between nt 7334 and 7716, and by sequencing of the viral RNA identified a mutation at nucleotide 7592. This mutation lies in the junction region of the viral genome, specifically at nucleotide -6, with reference to the initiation site for 26 S RNA synthesis. In SVCP-infected mosquito cells, but not in SVCP-infected chick cells, the ratio of subgenomic 26 S to 49 S (genomic) RNA synthesis was decreased relative to that observed in SVSTD infected cells. In terms of amino acid coding, the SVCP mutation is silent.

Aedes↗

PubMed1991

Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.

We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.

Capsid↗

PubMed1991

Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.

Aedes↗

PubMed1990

Acidotropic amines inhibit proteolytic processing of flavivirus prM protein.

Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weight, glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM occurs in the acidic post-Golgi vesicles.

Ammonium Chloride↗