Mouse mammary tumor virus can mediate cell fusion at reduced pH

Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.     

Chemically-induced temperature sensitive mutants of dengue virus type 2

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.     

A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication

We have previously described the isolation of a RNA- temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The ts defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revertants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another ts mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39 degrees (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39 degrees. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39 degrees is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same ts phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revertants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the ts phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.     

St. Louis encephalitis virus temperature-sensitive mutants

Nine temperature-sensitive (ts) mutants of St. Louis encephalitis virus were isolated after "forced mutagenesis" with 5-fluorouracil or 5-azacytidine. The ts mutants could be grouped on the basis of RNA synthesis at 40 degrees C, the nonpermissive temperature and complementation analysis. Four complementation groups were identified. Members of two of the groups were negative for RNA synthesis at 40 degrees C while the remainder were positive.     

Isolation and basic properties of temperature--sensitive mutants of Venezuelan equine encephalomyelitis virus

Fifteen temperature-sensitive (ts) mutants were isolated from 4 Venezuelan equine encephalomyelitis virus clones by nitrous acid treatment or by growing the virus in the presence of 5-fluoruracil. Three of them were classified as RNA- mutants by their inability to synthesize RNA at nonpermissive temperature (1.5-3.3% in respect to the wild type). The remaining 11 mutants showed a slight decrease of RNA synthesis at the nonpermissive temperature (32-73+) and were referred to the RNA+ phenotype. One mutant possessed RNA +/- phenotype (18%). Five complementation groups were determined by complementation analysis of the mutants.     

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.     

Requirement for ICR-like sequences in the replication of brome mosaic virus genomic RNA.

Previous studies using a brome mosaic virus (BMV) RNA-2 deletion mutant (pRNA-2 M/S) and additional derivatives as reporters established that viral sequences resembling internal control regions (ICRs) 1 and 2 of tRNA gene promoters are vital to (+)-strand replication in protoplasts. Transfer of these mutations to genomic RNA-2 and functional analysis in protoplast, local lesion, and systemic infections revealed a sequence-specific requirement for bases within the ICR2-like motif. Despite the low (generally less than 20% of wild-type) and sometimes undetectable levels of replication of these RNA-2 mutants, sufficient p2a protein was produced to support at least modest levels of RNA-1, -3 and -4 replication in protoplasts. However, only those RNA-2 ICR2 mutants supporting substantial replication of the viral genome in protoplasts were capable of establishing local lesions in Chenopodium hybridum and systemic infections in barley, further establishing the essential role of the ICR-like sequences in viral infectivity. Upon passage through a second set of barley plants, accumulation patterns for progeny from inocula containing certain RNA-2 mutants paralleled those from wild-type inocula, indicating repair of the introduced mutations. RNA stability and translatability were shown to be unaffected by the introduced mutations. BMV RNA-3 contains several ICR-like sequences, each of which was individually deleted. Whereas deletion of the 5'-terminal ICR2-like motif had little effect on RNA-3 accumulation in protoplasts or local lesion formation, it debilitated systemic spread in barley. Deletion of an internal ICR2-like motif at position 1100 decreased (+):(-) strand asymmetry from greater than 100:1 to 14:1, reduced RNA-3 replication in protoplasts to less than 15% of wild-type, and abolished local lesion and systemic infectivity.     

Macromolecular synthesis in cells infected with frog virus 3. III. Virus-specific protein synthesis by temperature-sensitive mutants.

Six temperature-sensitive (ts) mutants of frog virus 3, (five DNA⁺ and one DNA^?) representing six separate complementation groups, were examined for their intracellular patterns of virus-specific protein synthesis both at permissive (23°) and nonpermissive (30°) temperature. At permissive temperature protein synthesis and its regulation by each mutant was similar to wild type. At nonpermissive temperature all proteins were detected but some had altered rates of synthesis, indicating defective regulation by three of the six ts mutants.The six mutants can be divided into three categories based upon the time and nature of the ts defect during the replication cycle. The first category includes three mutants, each representing a separate complementation group in which virus-specific protein synthesis and its regulation is apparently normal at nonpermissive temperature. These mutants have defects in the virus maturation and assembly process suggesting the participation of several frog virus 3 genes in the assembly process. The second category consists of a single mutant that has an early defect in the regulation of viral protein synthesis; consequently a late pattern of virus-specific protein (VSP) synthesis is absent in cells infected with this mutant at nonpermissive temperature. The third category includes two mutants; these mutants are unable to regulate the rate of synthesis of certain VSP but have some features of the late pattern of virus-specific protein synthesis.The data presented in this report confirm and extend the evidence for temporal control of the rate of viral protein synthesis during frog virus 3 infection. This control appears to be mediated through several viral regulatory proteins.     

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.