STRUCTURE AND ASSEMBLY OF SV40 TSBC MUTANTS AND MUTANTS DELETED IN THE AGNOGENE (MORPHOGENESIS, MATURATION)

SHI CHUNG NG, Purdue University

Abstract

Morphogenesis of SV40 is thought to proceed through the rapid deposition of viral capsid proteins VP1, VP2, and VP3 onto the 75 S chromatin. Little is known about the initial stages of assembly possibly because of the cooperativity is assembly and hence the rapidity of shell polymerization. This question was approached by investigating the various forms of nucleoproteins that accumulate in cells when the assembly pathway is blocked due to temperature sensitivity of the major capsid protein VP1. In cells infected with tsBC mutants (with the exception of tsBC11), two classes of SV40 DNA-containing particles accumulate under restrictive conditions. The first class sediments at 75 S and corresponds to SV40 chromatin. The second class appears to be heterogeneous and sediments primarily from 90 to 160 S (this is referred to as heterogeneously sedimenting nucleoproteins, HSN). Biochemical analysis shows that HSN are enriched in viral capsid proteins as well as core histones, and they contain monomer-supercoiled SV40 DNA. When analyzed by electron microscopy, HSN appear as viral chromatin to which a protein cluster 200-250 (ANGSTROM) in diameter is attached. Nuclease endogenous to BSC cells digests the tsBC nucleoproteins at restrictive temperatures. Mapping results indicate that the cleavage sites are located around the open region. Therefore, tsBC nucleoproteins extracted under restrictive conditions have structural features in common with wild-type SV40 chromatin. Studies on the stability of various forms of nucleoproteins accumulated from wild-type- or mutant-infected cells indicate that both disulfide bonds and electrostatic interactions are important in stabilizing capsid-capsid and capsid-chromatin interactions. DNA sequence analysis indicate that all of the mapped tsBC mutants are point mutations, and six of them are identical. Data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced mature virions more slowly then cells infected with wild-type virus. These findings, taken together with the data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, strongly suggest that agnoprotein plays a role in expediting virion assembly.

Degree

Ph.D.

Subject Area

Genetics

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