Functional characterization of UL15, an essential gene involved in DNA cleavage/packaging of herpes simplex virus type-1

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Microbiology

Degree

Ph.D.

Abstract

At least seven viral genes, UL6, UL15, UL17, UL25, UL28, UL32 and UL33 have been implicated in the DNA cleavage and packaging process of herpes simplex viruses type 1 (HSV-1). The goal of this dissertation is to define the role of UL15 in this process. We constructed two HSV-1 UL15 insertion mutants and demonstrated that they are defective in DNA cleavage/packaging. We detected two forms of UL15 with an $\alpha$-UL15 polyclonal antibody: a full length 81-kDa form and a 30-kDa truncated form (UL15.5) in HSV-1-infected cells. These two proteins are the products of separate in-frame translation events from the UL15 gene. The failure of the UL15 insertion mutants to synthesize 81-kDa UL15 indicates that it is required for DNA cleavage and packaging. UL15.5, which is translated from Met$\sp{443}$ within the UL15 ORF, however, is not required for viral growth in cell culture. On the other hand, the conserved putative ATP-binding motif is indispensable for function of UL15.^ We demonstrated that UL15 localizes to the nucleus in the absence of any other viral proteins, indicating that UL15 contains its own nuclear localization signal. UL15 colocalizes with replication compartments at early times post-infection, supporting the hypothesis that DNA replication, cleavage and packaging occurs in the same intranuclear compartments. We also investigated the ability of various cleavage/packaging proteins to associate with viral capsids. Consistent with previous studies, we detect UL6 and UL25 in both B and C capsids. In contrast, both UL28 and 81-kDa full length UL15 were found predominantly in B capsids (intermediate) but not in C capsids (mature) during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in capsids and in virions. While UL6, UL25 and UL28 are able to associate with B capsids in the absence of other cleavage/packaging proteins, the 81- and 87-kDa UL 15-related proteins do not associate efficiently with B capsids lacking UL6 or UL28, suggesting that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28. ^

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