AMINO-ACID SUBSTITUTIONS IN THE POLIOVIRUS MATURATION CLEAVAGE SITE AFFECT ASSEMBLY AND RESULT IN ACCUMULATION OF PROVIRIONS

The assembly of infectious poliovirus virions requires a proteolytic c leavage between an asparagine-serine amino acid pair (the maturation c leavage site) in VPO after encapsidation of the genomic RNA. In this s tudy, we have investigated the effects that mutations in the maturatio n cleavage site have on PI polyprotein processing, assembly of subvira l intermediates, and encapsidation of the viral genomic RNA. We have m ade mutations in the maturation cleavage site which change the asparag ine-serine amino acid pair to either glutamine-glycine or threonine-se rine. The mutations were created by site-directed mutagenesis of P1 cD NAs which were recombined into wild-type vaccinia virus to generate re combinant vaccinia viruses. The P1 polyproteins expressed from the rec ombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia viru s (VV-P3) that expresses the poliovirus 3CD protease. A trans compleme ntation system using a defective poliovirus genome was utilized to ass ess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Merrow, J. Virol. 67:3684-3690 , 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were proces sed by 3CD provided in trans from VV-P3. The processed capsid proteins VPO, VP3, and VP1 derived from the mutant precursor VP4-QG were unsta ble and failed to assemble into subviral structures in cells coinfecte d with VV-P3. However, the capsid proteins derived from VP4-QG did ass emble into empty-capsid-like structures in the presence of the defecti ve poliovirus genome. In contrast, the capsid proteins derived from pr ocessing of the VP4-TS mutant assembled into subviral intermediates bo th in the. presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins deri ved from the VP4-TS precursor encapsidated the defective genome RNA. H owever, the cleavage of VPO to VP4 and VP2 was delayed, resulting in t he accumulation of provirions. The maturation cleavage of the VPO prot ein containing the VP4-TS mutation was accelerated by incubation of th e provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and pr ovide evidence for the existence of the provirion as an assembly inter mediate.