Homotypic and heterotypic exclusion of vesicular stomatitis virus replication by high levels of recombinant polymerase protein L.
The recombinant polymerase protein L of vesicular stomatitis virus (VSV) expressed in COS cells is able to transcribe and replicate the viral genome, resulting in complementation of temperature-sensitive polymerase mutants of VSV at the restrictive temperature (M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985). Here we report that the efficiency of complementation is dependent on the level of L protein expression. Unexpectedly, only cells expressing low levels of recombinant L protein efficiently complemented tsL gene mutants, whereas cells with high levels of L protein did not. In fact, in all cells with high levels of L protein expression, which at 40 h posttransfection represented almost the total number of transfected cells, viral replication not only of the temperature-sensitive mutant but also of wild-type VSV was excluded. The inhibition of VSV appeared to occur at an early stage of the infectious cycle, and wild-type virus of the same serotype (Indiana) as the recombinant L protein as well as wild-type virus of a different serotype (New Jersey) was affected. Measles virus, on the other hand, was not arrested in cells with high levels of recombinant L protein, demonstrating that these cells were still capable of supporting a viral infection. The expression of high levels of only the amino-terminal half of the L protein from a recombinant mutant L gene that contains a small out-of-frame deletion in the middle of the L gene did not inhibit a VSV infection. Since the level of amplification for both L- and truncated L-encoding vectors is similar, we conclude that the arrest of VSV was caused by high levels of functional full-length L protein itself and not by high levels of vector-encoded L mRNA or other vector products or by side effects of vector amplification. These data strongly support the idea that the highly conserved gene order of nonsegmented negative-strand viruses and the sequential and attenuated mode of transcription are important regulatory elements which balance the intracellular concentration of viral proteins. They both assure that the L gene is the last and the least frequently transcribed gene, giving rise to low levels of L protein necessary for efficient replication.
D000818:Animals; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000032:analysis; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008459:Measles virus / Q000235:genetics / Q000502:physiology; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics* / Q000502:physiology; D018116:Vesiculovirus; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase