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 gl ycine residue at the individual QG cleavage sites with valine (QG-->QV ). P1 cDNAs encoding the valine substitutions were created by site-dir ected mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 p recursors. The recombinant vaccinia virus-expressed mutant P1 polyprot eins were analyzed for proteolytic processing defects in cells coinfec ted with a recombinant vaccinia virus (WP3) that expresses the poliovi rus 3CD protease and for processing and assembly defects by using a tr ans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome tha t does not express functional capsid proteins (D. C. Ansardi, D. C. Po rter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substitu ted precursors were proteolytically processed at the altered sites bot h in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites we re slower than those of cleavage at the wild-type QG site in the precu rsor. Completely processed capsid proteins VP0, VP3, and VP1 derived f rom the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral s tructures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substituti on at the amino terminus of VP1 (VP1-G001V) assembled empty capsid par ticles but were deficient in assembling RNA-containing virions. The as sembly characteristics of the Vp1-G001V mutant were compared with thos e of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaar d and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletio n of the first four amino-terminal residues of VP1 (VP1-DELTA1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-DELTA1-4 prec ursor also occurred more slowly than complete cleavage of the wild-typ e precursor, and formation of virions was delayed; however, capsid pro teins derived from the VP1-G001V mutant assembled RNA-containing virio ns less efficiently than those derived from the VP1-DELTA1-4 precursor . These results demonstrate that maintenance of the glycine residues a t the QG cleavage sites of the poliovirus P1 precursor is required for assembly and RNA encapsidation events in addition to proteolytic proc essing and provide further evidence that the amino-terminal portion of VP1 plays a role in the RNA encapsidation process of poliovirus.