The detection and characterization of multiple forms of SV40 nucleoprotein complexes

Abstract

A procedure has been devised that extracts multiple forms of SV40 nucleoprotein complexes from the nucleus of virus-infected cells. Four distinct complexes have been identified: (i) DNA-replicative intermediates, (ii) a 75S nucleoprotein complex which is the first product of replicative forms, (iii) a previrion structure, and (iv) mature virion. Employing continuous and pulse-chase labeling procedures that follow the incorporation of [ 3H]thymidine into viral nucleoprotein complexes, the kinetics of DNA synthesis and virion maturation are detailed. Replicating SV40 nucleoprotein sediments as a broad complex between 75 and 200 S. The newly synthesized viral DNA is assembled into a 75 S chromosome which appears to mature with time into a 200 S previrion particle. This previrion particle is probably the precursor of the mature virion (250 S). A temperature-sensitive SV40 mutant in the major viral structural protein, VP-1 (tsB-11), fails to produce the 200 S previrion and 250 S virion at the nonpermissive temperature and leads to the accumulation of the 75S nucleoprotein complex. In contrast to previous observations, labeled intracellular SV40 DNA can be efficiently (90%) chased into mature virions. Other published procedures for extracting nucleoprotein complexes ( Green et al., 1971 ; White and Eason, 1971; Varshavsky et al., 1976 ; Christiansen and Griffith, 1977) have not detected this efficient maturation of viral DNA into virions. The reason for this discrepancy is shown to be due to the disruption of mature virions by the buffers, chelating agents, and crude cell extracts employed by these procedures. Disrupted virions produce a heterogeneous set of nucleoprotein complexes that have previously been thought to be natural in vivo complexes.