In Vivo Transcription and Protein Synthesis Capabilities of Bunyaviruses: Wild-Type Snowshoe Hare Virus and Its Temperature-Sensitive Group I, Group II, and Group I/II Mutants

The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive ( ts ) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280 :213-215, 1975; M. Bouloy and C. Hannoun, Virology 69 :258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16 :1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 μg/ml), α-amanitin (25 μg/ml), or rifampin (100 μg/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40°C) for virus replication gave values comparable to those obtained at permissive temperatures (33°C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33°C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40°C than those of the SSH group I ts mutants or wild-type SSH virus at 40°C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40°C were significantly higher than their primary transcription capabilities (as measured at 33°C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using 32 P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33°C (primary and secondary transcription) or 40°C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28 :417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30 :767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33°C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40°C, low levels of N and G1 polypeptides were evident. Images