Search

Three types of conditional lethal mutant were isolated from wild-type vesicular stomatitis virus, New Jersey serotype, after mutagenization by 5-fluorouracil: (i) conventional temperature-sensitive (ts) mutants, which form plaques at 31 C but not at 39 C; (ii) conventional host range mutants (hr CE), which grow in BHK but not in secondary chicken embryo cells; and (iii) temperature-dependent host range mutants (td CE), which form plaques both at 31 and 39 C on BHK cells but only at 31 C on chicken embryo cells. To determine whether the mutation in hr CE and td CE mutants affected the virion-associated RNA transcriptase, this enzyme was assayed in vitro at 31 and 39 C, and the results were compared with those obtained for the wild-type virus. The RNA trascriptase activity of hr CE mutants did not appear to be affected by the mutation. The td CE mutants fall into two classes: those that synthesized RNA at 39 C similar to the wild-type virus and those that did not. One mutant of the latter category, td CE 3, had heat-sensitive transcriptase regardless of whether it was grown in BHK or chicken embryo cells. A revertant to the wild-type phenotype isolated from this mutant had regained the ability to synthesize RNA at 39 C. These results strongly suggest that a polypeptide that is either the transcriptase itself or part of the transcriptase complex was made temperature sensitive by the mutation in the second class of td CE mutants. The inhibition of the transcriptase activity of the mutant td CE 3 was fully reversible by lowering the temperature of incubation from 39 to 31 C, and both inhibition and reactivation appeared to be instantaneous.

The sequences of the genes encoding the putative attachment (G) proteins of pathogenic (strain J3666) mouse lung-passaged and nonpathogenic (strain 15) tissue culture-passaged strains of pneumonia virus of mice (PVM) have been determined. In both cases the major polypeptide was synthesised from the second open reading frame (ORF), a feature also found in the G gene of respiratory syncytial (RS) virus, another pneumovirus. However, the ORFs of the G genes of the two PVM strains were initiated at different nucleotide positions in the mRNA and comparison of hydrophobicity profiles revealed the presence of the putative amino-terminal cytoplasmic domain in the strain J3666 G protein and its absence in the predicted G protein of PVM strain 15. In common with the G protein of RS virus, the gene product of both PVM strains contained a high serine, threonine, and proline content. Indirect immunofluorescence analysis of BSC-1 cells expressing the G gene products confirmed the surface location of the proteins. Thus, the absence of a cytoplasmic domain does not interfere with the translocation of the G protein of PVM strain 15. In vitro translation of mRNA from the two PVM genes directed the synthesis of a larger polypeptide with the G gene of PVM strain J3666 than was seen with the strain 15 G gene. In addition, a second protein was seen with strain J3666 mRNA which was the same size as the strain 15 G protein. Copyright 1995, 1999 Academic Press

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-tspseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing tsmutant of vesicular stomatitis virus New Jersey and tsmutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing tsmutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the Ngene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.

Seventy-seven temperature-sensitive (ts) mutants belonging to three antigenically distinct and geographically isolated members of the Bunyamwera complex--Batai virus, Bunyamwera virus, and Maguari virus--have been isolated after 5-fluorouracil treatment. High-frequency recombination was observed, and the mutants of each virus were classified into two groups, which were shown to be equivalent by heterologous recombination experiments. In most combinations heterologous recombination was less efficient than homologous recombination, but all crosses of group I and II mutants yielded viable recombinants. Recombination was an early event. Analysis by polyacrylamide gel electrophoresis of the proteins of the wild-type viruses and recombinant clones obtained from the six possible heterologous combinations of group I and II mutants indicated that recombination occurred by reassortment of genome subunits. Group I appeared to correspond to the genome subunit coding for the N protein, and group II corresponded to the G1/G2 determinant. The G1 (or G2 or both) protein was associated with neutralization specificity and plaque diameter, and the N protein was associated with plaque opacity. Complementation was observed between two nonrecombining mutants of Maguari virus belonging to group I, which may indicate that the N genome subunit codes for an additional protein. There appeared to be no genetic barrier to exchange of genetic material between Batai, Bunyamwera, and Maguari viruses in vitro, and it is concluded that the Bunyamwera complex is potentially a single gene pool if geographical and ecological constraints are discounted.

Fifteen temperature-sensitive mutants of the RSN-2 strain of respiratory syncytial virus have been classified into six complementation groups, two of which appeared to be homologous with two of the three complementation groups of the A2 strain described by Wright et al. (P. F. Wright, M. A. Gharpure, D. S. Hodes, and R. M. Chanock, Arch. Gesamte Virusforsch, 41:238--247). Thus seven complementation groups of respiratory syncytial virus, designated A, B, C, D, E, F, and G, have been defined. The frequency and type of mutant isolated varied according to strain; group C was unique to the A2 strain, and groups D, E, F, and G were unique to the RSN-2 strain. The highest complementation indexes were obtained by preincubation for 7 h at permissive temperature, followed by incubation at restrictive temperature for 40 to 50 h in the case of A2 strain mutants or 80 to 90 h for RSN-2 strain mutants. Genetic recombination was not detected.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.

Only two recombination groups have been reported in genetic analyses of ts mutants of 10 different bunyaviruses from the Bunyamwera and California encephalitis serogroups, although three groups are expected from the tripartite structure of the genome of all members of the family Bunyaviridae. We describe now a ts mutant of Maguari virus, MAGts23(III), which recombined in both vertebrate (BHK-21) and invertebrate (Aedes albopictus) cells with mutants representing recombination groups I and II of this Bunyamwera serogroup virus. In addition, MAGts23(III) recombined with two mutants MAGts20 and MAGts21, provisionally identified as double mutants by their failure to recombine with group I or group II mutants, Mutant MAGts23(III) therefore represents a third bunyavirus recombination group. Mutant MAGts23(III) differed phenotypically from other bunyavirus mutants by growth restriction in BS-C-1 cells. Wild-type recombinants were obtained in the heterologous cross of MAGts23(III) and a group II mutant of Bunyamwera virus, but not in a cross with a group I mutant. The recombinants had the G protein of the Maguari virus parent and the N protein of the Bunyamwera virus parent. Analysis of the phenotypes of clones isolated at permissive temperature from the progeny of the other cross [MAGts23(III) and a group I mutant of Bunyamwera virus] indicated that recombination occurred in this cross, but that the possible recombinant phenotypes were not recovered with equal frequency. As a consequence, it has not been possible to obtain a gene assignment for group III from genetic data alone.

Nucleotide sequences of around 200 residues were determined adjacent to the 3' terminus of the genome RNA of vesicular stomatitis virus, New Jersey serotype, and adjacent to the 3'-terminal polyadenylic acid tract of the N protein mRNA of the same virus. These sequences were compared with the corresponding sequences previously determined for the Indiana serotype of vesicular stomatitis virus. The sequences obtained for the two strains were readily aligned, showing 70.8% homology overall. Examination of the sequences allowed identification of the translation initiation and termination codons for the N mRNA of each serotype. The deduced N-terminal and C-terminal amino acid sequences of the two N polypeptides were each similar, and most of the differences between them consisted of substitution by a clearly homologous amino acid. It was proposed that these nucleotide sequences, within limits imposed by their functions, comprise reasonably representative measures of the extent of sequence homology between the genomes of the two serotypes, and that this is higher than previously estimated, but with little exact homology over extended regions.

Fifty temperature-sensitive (ts) mutants of the rhabdovirus Chandipura virus have been classified into six complementation groups designated ChI to ChVI. Group ChI contains 44 mutants, group ChII contains 2 mutants, and the remaining groups have 1 mutant each. Mutants in groups ChI, ChIII, ChIV, and ChVI had RNA-negative phenotypes in experiments measuring amplification of RNA synthesis at restrictive temperature. The two mutants in group ChII had RNA-positive phenotypes, and the virions were thermolabile. Mutant ts Ch851 of group ChV was also RNA positive, and the M polypeptide of this mutant appeared to be unstable in cells incubated at restrictive temperature. It is likely, therefore, that complementation groups ChII and ChV represent the genes coding for the two viral proteins of the virion envelope. No precise assignment can be made in the case of the four RNA-negative groups, since all the mutants examined showed some polymerase activity in vitro at restrictive temperature. An attempt to obtain polymerase mutants by screening for sensitivity to rifampin was not successful. Six temperature-dependent host range mutants (the tdCE phenotype) of Chandipura virus failed to multiply in chicken embryo cells at restrictive temperature, but otherwise they differed in their host range properties from similar mutants of vesicular stomatitis virus.

Fifty temperature-sensitive (ts) mutants of Chandipura virus, a human rhabdovirus, have been classified into six complementation groups, designated ChI, ChII, ChII, ChIV, ChV, and ChVI and containing 44, 2, 1, 1, 1, and 1 mutants, respectively. Weak complementation was observed within group ChI, allowing the division of the group into subgroups ChIA and ChIB. Intragroup complementation was most extensive within subgroup ChIB, and one mutant in this subgroup complemented all but one (ts Ch598) of the mutants in group ChI. If ts Ch598 had been omitted from the analysis the number of complementation groups would have been increased to seven. Consequently, in circumstances where intragenic and intergenic complementation cannot be clearly distinguished, the number of complementation groups identified in rhabdoviruses could be overestimated. The identification of six complementation groups in three different rhabdoviruses need not imply the existence of an as yet unidentified sixth virus-specified polypeptide. The extensive intragroup complementation observed in Chandipura virus suggests that the functional form of one at least of the virion proteins of Chandipura virus is a multimer.

In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, tsE1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of tsE1, slightly less for tsE2, and only a little greater than that of wild-type virus NS polypeptide in the case of tsE3. Since the NS polypeptides of the revertant clones tsE1/R1 and tsE3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the tsmutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with 32Pito approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide.

The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (ts) mutants was assayed in vitro at the permissive (31°C) and restrictive (39°C) temperatures. RNA synthesis at 39°C by the RNA-negative tsA1 and the RNA-positive tsC1 and tsD1 mutants was similar to that of wild-type virus. The RNA-negative tsB1 synthesized only small amounts of RNA in vitro at 39°C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39°C: tsE1 synthesized very little RNA, tsE2 synthesized moderate amounts, and RNA synthesis by tsE3 was not inhibited. The two mutants of group F were also dissimilar, since tsF1 synthesized very little RNA at 39°C, whereas tsF2 synthesized as much RNA as wild-type virus. The revertant clones tsB1/R1, tsE1/R1, and tsF1/R1 synthesized RNA at 39°C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants tsB1, tsE1, and tsF1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant tsB1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31°C after 30 min of preincubation at 39°C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant tsE1 were heated for 5 h at 39°C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant tsF1 was also heat labile, although to a lesser extent than that of tsE1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.

Bunyamwera virus-induced polypeptide synthesis in BSC-1 cell has been studied using polyacrylamide gel electrophoresis and autoradiography. Four virus-induced polypeptides were identified. Their molecular weights were 200 X 10(6) (L), 128 X 10(6) (G1), 31 X 10(6) (G2), and 23 X 10(6) (N). Pulse-chase experiments, short labeling experiments, and experiments using amino acid analogs failed to show evidence of polypeptides processing by proteolytic cleavage. Analysis of the kinetics of synthesis of these polypeptides showed that a clear division into early and late categories could be made, the onset of synthesis of polypeptide N and L rapidly reached a peak and then declined. Polypeptides G1 and G2 were made for several hours; their rate of synthesis then declined. All four polypeptides then continued to be made in relatively small amounts for many hours.

We established previously that the temperature-dependent host range mutant, td CE 3, of vesicular stomatitis virus (VSV) New Jersey possesses temperature-sensitive RNA transcriptase activity. In this paper, we describe dissociation and reconstitution experiments designed to determine which VSV polypeptide is affected by the td CE 3 mutation. Wild-type VSV New Jersey (ts+), the temperature-dependent host range mutant (td CE 3), and the revertant of this mutant (td CE/R1) were used. Transcribing nucleoprotein preparations, isolated from purified virus particles, were treated in the presence of digitonin with either 0.9 M LiCl to produce supernatants containing virtually only the L polypeptide or 2.0 M LiCl to produce ribonucleoprotein pellets containing only the polypeptides N and NS. Supernatant and pellet fractions synthesized either no or only trace amounts of RNA in vitro. Reconstitution of the supernatants with the pellets in all combinations at 31 degrees C restored much of the transcriptase activity of the transcribing nucleoprotein preparations. RNA synthesis occurred at 39 degrees C when the three pellets were reconstituted with wild-type and revertant supernatants. However, supernatant of the mutant td CE 3 reconstituted with any of the three pellets resulted in little or no detectable transcriptase activity at 39 degrees C. This implies that the polypeptide affected by the td CE 3 mutation is the L polypeptide.

The requirement of the presence of a nucleus for the replication of vesicular stomatitis virus and influenza virus has been examined by following the growth and development of these viruses in enucleate BS-C-1 cells. Vesicular stomatitis virus replicates normally in enucleate cells with the rate of production of infectious virus, the amount of virus-specific protein synthesis, and the type of proteins produced being essentially the same in nucleate and enucleate cells. Influenza virus does not replicate in enucleate cells, no virus gene products can be detected, and there is no inhibition of cellular protein synthesis.

Forty-eight temperature-sensitive (ts) mutants have been isolated from a wild-type strain of the New Jersey serotype of vesicular stomatitis virus (VSV) after exposure to the base analogue mutagen 5-fluorouracil. Of these mutants, 47 have been classified into 6 nonoverlapping complementation groups containing 21, 17, 4, 3, 2, and 1 mutant, respectively (1 mutant remaining unallocated). The ribonucleic acid (RNA) phenotype of 23 of these mutants has been established. Four of the six groups contain one or more mutants unable to synthesize detectable amounts of viral RNA under restrictive conditions (39 C). No complementation was observed in mixed infection with tsmutants from the five established complementation groups of the Indiana serotype of VSV.

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual tsmutants. The defective particles associated with some of the tsmutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.

Temperature-sensitive (ts) mutants of Cocal virus (VSV Cocal) were isolated after treatment with the base analogue mutagen, 5-fluorouracil. These mutants could be classified into four mutually complementing groups. Weak complementation was detected between certain pairs of VSV Cocal tsmutants and tsmutants of vesicular stomatitis virus (VSV) Indiana, but no complementation was observed with tsmutants of VSV New Jersey. Two complementing tsmutants of Chandipura virus, an unrelated rhabdovirus, did not complement any VSV mutant, Thus, ability to complement in the VSV group appears to be correlated with serological relationships.The RNA and protein-synthesizing capacities of these tsmutants have been determined, and it is possible to establish a correspondence between the VSV Cocal and the VSV Indiana complementation groups.

One hundred and seventy-five temperature-sensitive (ts) mutants of vesicular stomatitis virus (type Indiana-C) induced by 5-fluorouracil (FU), 5-azacytidine (ACR), and ethyl methane sulfonate (EMS) have been assigned to four complementation groups by a qualitative test. Group I contains 151 mutants; group II, 2 mutants; group III, 1 mutant; and group IV, 15 mutants; 6 are unclassified. FU was much more effective as a mutagen than either ACR or EMS. The proportion of the mutants belonging to groups I and IV, however, was similar in the case of all three mutagens. Fifteen mutants from groups I and IV have been used to obtain quantitative complementation data. Both groups appear to be homogeneous. Complementation yields increase with increasing multiplicity, but the number of particles per cell required to elicit maximal complementation is small. The pattern of genetic recombination parallels that of complementation. No recombination could be detected in crosses within group I (<0.001%) or group IV (<0.07%), whereas recombination (0.31 to 3.4%) was observed in crosses between groups I and IV. Recombination frequency did not increase with multiplicity above an input of 0.6 plaque-forming units per cell. Many group I mutants have very low reversion rates, and BHK 21 clone 13 cells infected with one of these mutants have been “cured” of infection by prolonged exposure at the restrictive temperature.