Title

Analysis of the intranuclear localization of essential herpes simplex virus type 1 replication proteins

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Cell|Biology, Microbiology

Degree

Ph.D.

Abstract

Herpes simplex virus type 1 (HSV-1) is a large, double-stranded DNA virus which replicates in the nuclei of infected cells. Seven virus-encoded proteins are essential for replication; a heterotrimeric helicase-primase complex (UL5, UL8, UL52), a heterodimeric polymerase complex (UL30, UL42), an origin-binding protein (UL9) and a single-stranded DNA binding protein (UL29). During infection, viral replication occurs in nuclear domains called replication compartments (RCs). RCs had previously been shown to contain UL29, UL30, UL42 and UL9. I have shown that they also contain UL5, UL8, UL52. Early during infection and in cells treated with polymerase inhibitors, UL29 localizes to numerous punctate sites, prereplicative sites (PRs), which have been proposed to be RC precursors. My thesis deals with determining which factors are necessary for PR and RC formation. To determine which viral factors are necessary for PR formation, I examined the localization of UL29 in cells infected with viral mutants which failed to express UL5, UL8, UL52 or UL9. Cells infected with these mutants contained UL29 in a diffuse staining pattern that was converted to a PR pattern by adding polymerase inhibitors. To determine if there was redundancy amongst the proteins for PR formation, viruses lacking both UL5 and UL9 or both UL5 and UL30 were generated. Analysis of cells infected with these double mutants suggested that neither UL5 nor UL9 are necessary for PR formation and that certain polymerase inhibitors stimulate the localization of UL29 to PRs by affecting cellular polymerases. I determined the minimal requirements for RC formation by transfecting cells with constructs expressing the essential viral replication proteins and a plasmid containing a viral origin of replication. RCs formed which were morphologically identical to those found in infected cells and their formation did not require UL9 or an origin of replication. These structures localized adjacent to preexisting nuclear matrix sites known as ND10s, as is the case during viral infection. I also found that UL29 localizes independently to the vicinity of ND10s and so may play a role in organizing the viral replication apparatus at the nuclear matrix. ^