The kinetics of VP5 mRNA expression is not critical for viral replication in cultured cells.
We generated recombinant viruses in which the kinetics of expression of the leaky-late VP5 mRNA was altered. We then analyzed the effect of such alterations on viral replication in cultured cells. The VP5 promoter and leader sequences from positions -36 to +20, containing the TATA box and an initiator element, were deleted and replaced with a strong early (dUTPase), an equal-strength leaky-late (VP16), or a strict-late (U(L)38) promoter. We found that recombinant viruses containing the dUTPase promoter inserted in the VP5 locus expressed VP5-encoding mRNA with early kinetics, while virus with the U(L)38 promoter inserted expressed such mRNA with strict-late kinetics. Further, in spite of differences in its functional architecture, the VP16 promoter fully substituted for the VP5 promoter. Western blot analysis demonstrated that the amounts of VP5 capsid protein produced by the recombinant viruses differed somewhat; however, on complementing C32 and noncomplementing Vero cells, such viruses replicated to titers equivalent to those of the rescued wild-type virus controls. Multistep virus growth in mouse embryo fibroblasts, rabbit skin cells, and Vero cells also demonstrated equivalent replication efficiencies for both recombinant and wild-type viruses. Further, recombinant viruses did not show any impairment in their ability to replicate on serum-starved or quiescent human lung fibroblasts. We conclude that the kinetics of the essential VP5 mRNA expression is not critical for viral replication in cultured cells.
Lieu
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Program in Animal Virology, Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA.
Wagner
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D000818:Animals; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D036022:Capsid Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D016296:Mutagenesis; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010746:Phosphonoacetic Acid / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011755:Pyrophosphatases / Q000235:genetics; D012333:RNA, Messenger; D012367:RNA, Viral; D011817:Rabbits; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014779:Virus Replication
D036022:Capsid Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D012367:RNA, Viral; C082563:VP19 protein, Human herpesvirus 1; C442523:VP5 protein, Herpes simplex virus type 1; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D010746:Phosphonoacetic Acid