Transcribing Complexes in Cells Infected by Vesicular Stomatitis Virus and Rabies Virus

Abstract

We analyzed cell extracts from BHK21 cells infected with vesicular stomatitis virus (VSV) and rabies virus for in vitro RNA polymerase activity. Cells infected with VSV B virions exhibited several complexes with in vitro RNA polymerase activity in sucrose gradients. These complexes synthesize VSV transcriptase product (4 to 18S) polyadenylated in RNA complementary to virion RNA. Cells infected with a high multiplicity of B virions and T particles show only one RNA polymerase complex active in vitro. This complex sediments at 110S aud makes only small (2S) RNA. Carrier BHK21 cells persistently noncytopathically infected with VSV contain several complexes active in RNA polymerase, but both exhibit very low activity. Cytoplasms of cells noncytopathically infected with rabies virus also show very low levels of a complex containing RNA polymerase activity. No transcriptase nor any other in vitro polymerase activity could be found associated with purified rabies virions, although they do carry out primary transcription in cells treated with actinomycin D and cycloheximide before infection.

Rabies virus protein synthesis in infected BHK-21 cells

“…A major difference between rabies virus and VSV has been the inability to detect RNA transcriptase activity in purified rabies virions (41,48). RNA transcriptase activity could not be detected in purified rabies virions by in vitro assays that were sensitive enough to detect RNA transcriptase with a specific activity 4,000 times less than that present in VSV virions (H. P. Madore and F. Sokol, unpublished observations).…”

“…The L protein, which is present in small amounts in intact VSV virions (16,50,51), has been shown to be necessary for RNA transcriptase activity (5-7, 10, 11, 31). In contrast, the presence of L protein in rabies virions has not been clearly established (40,42), and RNA transcriptase activity has not been detected in purified rabies virions (41,48), except possibly at extremely low levels (D. Bishop, personal communication). Although virus-specific RNA transcriptase activity has been detected in rabies-infected cells (48; H. P. Madore and F. Sokol, unpublished observations), the virus-specific polypeptide (L), which may be necessary for viral RNA transcriptase activity, had not been detected in our previous analysis of infected cells labeled under hypertonic conditions (26).…”

Transcriptase activity associated with rabies virion

“…Therefore, the requirement of a high concentration of a detergent is not fully understood. Concerning this point, an experiment concerning primary transcription when virus-infected cells have been pretreated with actinomycin D and cycloheximide (5,24) is of interest. These workers showed that purified rabies virion could carry out primary transcription in infected BHK cells, even though the activity was low.…”

“…Attempts to demonstrate this enzyme activity in the rabies virion in vitro have apparently been unsuccessful (5,22,24). Bishop reported that the highest activity observed incorporated only 5 to 10 pmol of UMP into the product per h per mg of protein (Abstr.…”

Serologic survey of selected rhabdoviruses

“…However, the rabies virus can carry out primary transcription in cells 6 which have been pretreated with actinomycin D and cycloheximide, suggest ing that the virion does possess some transcriptase activity (Villarreal and Holland, 1974).…”