RNA synthesis of vesicular stomatitis virus-infected cells: in vivo regulation of replication

Abstract

Pulse-labeling of vesicular stomatitis virus-infected HeLa and BHK cells with [3H]uridine throughout the infectious cycle demonstrated two peaks of uridine incorporation into virus-specific RNA molecules. By separating total RNA synthesis into replication and transcription products, we showed that replication occurs over a shorter period of time in one peak synthesis. The biphasic nature of uridine incorporation is in part due to a general membrane phenomenon of reduced metabolite transport during vesicular stomatis virus infection and in part due to the apparent uncoupling of replication and transcription. A change in the ratio of newly synthesized plus and minus strands of the genome length (42S) RNA was found as the infection proceeded. Early in the infection, plus-stranded 42S RNA comprised 40% of the total genome length RNA synthesis, whereas late in infection, only 15 to 20% of the 42S RNA synthesized was complementary to the virion minus strand. Our data suggest that the rate of synthesis of plus-stranded 42S RNA was constant throughout the infection. The rate of virus release was determined by monitoring the uptake of [3H]uridine into released virus particles. Virus maturation and release are closely associated with the assembly of 42S RNA-containing nucleocapsids.

In vitro reassembly of vesicular stomatitis virus skeletons

“…The abundant formation of VSV skeletons in vitro raises the possibility that similar structures may be formed in vivo. Calculations based on the data of Simonsen et al (16) show that the molar concentrations of nucleocapsids and M protein in VSV-infected HeLa cells are comparable to or greater than their concentrations in the reassembly experiments described here. Depending on the values assumed for the cell volume and the time after infection, the concentration of negative-strand nucleocapsids in infected cells falls in the range of 2 x 10-9 to 4 x 10-9 M and that of M protein is approximately 2…”

Transcription and replication of rhabdoviruses.

“…Moreover, the formation of these RNPs depends on continued protein synthesis, indicating that they are indeed replicating complexes. Simonsen et al (203) further demonstrated that, early in infection, positive-strand RNA composes 40% of the full-length RNA synthesized, whereas late in infection only 15 to 20% of the 42S RNA synthesized is positive strand. The rate of synthesis of the positive strand remains constant throughout infection.…”

Regulation of RNA Synthesis by the Genomic Termini of Vesicular Stomatitis Virus: Identification of Distinct Sequences Essential for Transcription but Not Replication

“…In turn, the 3Ј end of the positive strand (TrC) promotes synthesis of the genome-length negative strand. Replication is asymmetric, producing an approximately 5 to 10-fold excess of negative-sense genomes over positive-sense antigenomes in infected cells (24,38,40,42,48). This led to the suggestion that TrC is a more effective promoter of replication than Le.…”