Herpesvirus viruses encode six proteins that come together in a highly regulated and orchestrated fashion to form a protective coat around the virus genome. Virus and phage protein coats have been studied as paradigms for how proteins interact and self-assemble into higher order structures. This proposal is based on our studies of the Kaposi's sarcoma-associated herpesvirus (KSHV) small capsid protein (SCP) which is encoded by open reading frame (ORF) 65. The KSHV SCP is essential for assembly unlike those of other herpesviruses. Thus, we propose to study and elucidate how this protein promotes self-assembly of icosahedral capsids. Our working hypothesis is that the gammaherpesvirus SCP is an important mediator of stable capsid shell assembly. We propose an unconventional mechanism but one that phage capsids also use. Thus, because of its location on the hexons which are made up of the major capsid protein (MCP) and because it must function at a stage prior to the formation of visible (by ultrastructural methods) assemblies we propose that it acts as an external scaffold or cross-link that strengthens and stabilizes hexon formation. In addition, genetic data from mutants of ORF65 that cannot assemble suggests possibly a novel mechanism whereby binding of SCP to the MCP creates an allosteric change that strengthens and reinforces the MCP interaction with the internal scaffold protein. We propose experiments that will test this hypothesis. If proven correct this would change the view that the SCP is simply a capsid decoration protein. It would reveal an important role for this small protein at the initial steps in the assembly pathway.
The Specific Aims proposed to achieve these goals are:
Specific Aim 1. Discover the interactions and properties of the KSHV SCP (ORF65). I. Nuclear assembly site localization. II. Interactions of ORF65. III. Dynamic functional state of ORF65.
Specific Aim 2. Use a cell-free system and in vitro methods to discover the novel functions of the SCP. I. Cell-free assembly. II. Sedimentation analysis of sub-assemblies. III. In vitro MCP-scaffold protein binding assay.
The major goals of this project are to elucidate the assembly pathway for Kaposi's sarcoma associated herpesvirus (KSHV). This will be investigated in an ex-vivo system namely an insect cell using recombinant baculoviruses for protein expression. The aims of this proposal address the unique role of the small capsid protein during the assembly of the KSHV capsid.
|Geoghegan, Eileen M; Zhang, Hong; Desai, Prashant J et al. (2015) Antiviral activity of a single-domain antibody immunotoxin binding to glycoprotein D of herpes simplex virus 2. Antimicrob Agents Chemother 59:527-35|
|Capuano, Christopher M; Grzesik, Peter; Kreitler, Dale et al. (2014) A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly. J Gen Virol 95:1755-69|
|Kalu, Nene N; Desai, Prashant J; Shirley, Courtney M et al. (2014) Nelfinavir inhibits maturation and export of herpes simplex virus 1. J Virol 88:5455-61|
|Bera, Alakesh; Perkins, Edward M; Zhu, Jian et al. (2014) DNA binding and condensation properties of the herpes simplex virus type 1 triplex protein VP19C. PLoS One 9:e104640|
|Hu, Shaohui; Feng, Yingzhu; Henson, Brandon et al. (2013) VirD: a virion display array for profiling functional membrane proteins. Anal Chem 85:8046-54|