SV40 MORPHOGENESIS: PATHWAY AND EFFECT ON THE STRUCTURE OF SV40 CHROMATIN (VIRUS ASSEMBLY, PROTEIN-DNA INTERACTIONS, NUCLEOPROTEIN COMPLEX, TEMPERATURE-SENSITIVE MUTANT)

The Simian Virus 40 (SV40) morphogenetic pathway was studied by analyzing the nucleoprotein complexes assembled in cells infected with a wild-type SV40 virus (wt776) and mutants temperature-sensitive in virus assembly: tsB and tsC. The tsB and tsC mutations map to the gene coding for the major capsid protein VP1. At the nonpermissive temperature (40(DEGREES)C), the 75 S viral chromatin accumulates in tsC-infected cells whereas, in tsB-infected cells, the viral chromatin associates with the capsid proteins VP1, VP2, and VP3 to form semiassembled virus particles (SAP) which sediment heterogeneously from 100 to 160 S. The structural and biochemical properties of the tsB mutants support the hypothesis that SV40 assembly proceeds by the gradual addition of the capsid proteins onto chromatin. Further characterization of the tsB mutants by pulse-chase kinetic studies and temperature shift experiments revealed a critical relationship between VP1 structure and its function in the capsid shell polymerization process. On the other hand, the tsC mutants seem to be defective in the initiation of shell assembly on viral chromatin. Micrococcal and DNAase I digestion analyses were performed on tsC chromatin, wt776 chromatin and tsB SAP to study the organization of histones on SV40 DNA and the possible effect of virus assembly on this organization. Micrococcal nuclease digestion analysis revealed that tsC chromatin assembled at 40(DEGREES)C possesses a shorter average nucleosomal repeat length (179 base pairs) compared to that observed for wt776 chromatin (195 base pairs) or tsB SAP (198 base pairs). Further studies demonstrated that the shorter tsC chromatin repeat length observed results from an alteration in a VP1 function specific to tsC mutants. These findings provide the first genetic evidence involving a nonhistone protein in the determination of the nucleosomal repeat length in nucleohistones, and lead to a model in which the capsid proteins play a role in a nucleosomal rearrangement process that functions not only in virus assembly but also in controlling viral gene expression. Further characterization of the SV40 nucleoprotein complexes by DNAase I digestion analysis indicated preferred nucleosome positioning sites on SV40 DNA. Some of these sites were shared by tsC, wt and tsB nucleoprotein complexes