Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation.
Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)
Ansardi
D C
DC
Department of Microbiology, University of Alabama at Birmingham 35294-0007.
Morrow
C D
CD
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005816:Genetic Complementation Test; D016679:Genome, Viral; D005973:Glutamine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009024:Morphogenesis; D009154:Mutation; D017955:Poliovirus / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014633:Valine / Q000378:metabolism; D014764:Viral Proteins
D011498:Protein Precursors; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D005973:Glutamine; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D014633:Valine