Replication of vesicular stomatitis virus.

The replication of the RNA genome of the rhabdovirus vesicular stomatitis virus (VSV) was studied in vivo and in vitro. The products of replication, (+) and (-) strand 42S RNA, were separated from the mRNA species in CsCI gradients, allowing the determination of the rates of 42S RNA synthesis throughout the infectious cycle. It was found that 42S RNA synthesis and virus release occurred over a narrow time span and were maximal at t hours (h) post-infection (PI). The ratio of (+): (-) strand 42S RNA shifted from 40:60 at 2 h PI to 20:80 at 4 h PI. This shift was shown to be due to an increase in the rate of (-) strand 42S RNA synthesis while the rate of (+) strand 42S RNA synthesis remain constant. The apparent constance of the rate of (+) strand synthesis suggests that there are separate regulators of (+) and (-) strand synthesis. The replicate complex from VSV-infected HeLa cells was isolated in Renografin gradients. By pulse-labeling cells for 5 min at 5 h PI, we were able to isolate a complex while contained radiolabeled RNA molecules which ranged in size from 18S to 42S. Longer times of pulse-labeling demonstrated that the radiolabeled RNA species accumulated as 42S RNA. The characteristics of this complex were examined in several ways. We demonstrated that the nascent RNA species contained in the RNP complexes were ribonuclease-resistant and single-stranded, which indicated that the RNA species were associated with protein. The radiolabeled RNP complexes had an equilibrium density in CsCI identical in the viron nucleocaposid. Hybridization analyses of the nascent RNA species demonstrated that the ratui if (+): (-) RNA contained in the replicative complex was higher at 2 h PI than at 6 h PI, just as we observed with the rates of synthesis of 42S RNA in vivo. The actual ratios of the RNA contained in the replicative complex and the 42S isolated in CsCI gradients were similar as well. The nascent (-) strand RNA which was approximately 18S in size was shown to be enriched for sequences identical to the 5' end of the genome RNA. These results strongly suggest that the RNP complexes we have isolated are in fact replicative complexes. The in vitro synthesis of genome-size 42S RNA was observed using lysates prepared from infected cells, but not when virion RNPs were used in the reaction. The synthesis of several smaller unique RNA species was also observed in the crude cell extract system. The 42S RNA which was synthesized in vitro was bound by oligo(dT) column which suggests that the 42S RNA contained poly (A). The synthesis of the 42S RNA was prevented when the cells were treated with cycloheximide prior to preparation of the cell extract. The synthesis of full-length RNA was maximal at 5 h PI, the same time at which replication in vivo was shown to be maximal. Preliminary hybridization analyses of the 42S, 2S and 20S RNA species which were synthesized in vitro by the cell extracts indicated that these RNA species were (+) strand in sence.