Identifying novel activities of KSHV/HHV8 DNA replication proteins

Project Summary
Viruses are the causative agents of approximately 12% of human cancers. The most recently discovered
herpesvirus, Kaposi’s sarcoma herpesvirus (KSHV/HHV8) is known to cause three human cancers. Effective
antiviral therapeutics are needed for the treatment of KSHV. Viral DNA replication and the KSHV DNA replication
proteins are essential to successful viral replication and, thus, appealing therapeutic targets. The focus of this
proposal is characterizing the functions and molecular interactions of the core KSHV DNA replication proteins in
order to develop potential therapeutic strategies against KSHV infection. Previous studies used sequence
homology between KSHV and related herpesviruses to determine conserved protein functions of the six essential
core DNA replication proteins encoded by KSHV, ORF6(SSB), ORF9(POL), ORF40/41(PAF), ORF44(HEL),
ORF56(PRI), ORF59(PF) and the origin binding protein, ORF50(RTA). However, protein sequence homologies
only range from 20-50% and thus poorly predict protein function; therefore, an ultrastructural characterization
and in-depth biochemical analysis of individual KSHV viral DNA replication proteins, or in combinations, is
needed to identify their full range of functions (Aim 1). To evaluate the in vitro protein activities, high resolution
electron microscopy (EM) will be complemented by biochemical analysis. Direct visualization via EM produces
qualitative data (heterogeneous protein complexes, oligomeric state, DNA architecture) and quantitative data
(DNA mapping of protein binding locations, molecular size comparisons). Our preliminary finding provide novel
insights into the activities of KSHV proteins. The first aim of this proposal will characterize the molecular
interactions and activities of a subset of already purified KSHV DNA replication proteins (ORF6, ORF9, ORF44,
ORF59 and ORF50). The findings will provide valuable insights into KSHV replication and inform future studies
of proteins purified from a human cell culture system to directly compare the impact of viral protein post-
translational modifications of proteins produced from insect cells with human cell native modifications. The
second aim of this proposal is focused on developing a system for generating viral proteins in physiologically
relevant human cell lines. We have previously produced five of the seven KSHV replication proteins using an
insect Sf9 cell system, but commercial and lab attempts to express and purify the remaining two proteins
(ORF40/41 and ORF54) from non-mammalian cells have been unsuccessful. We hypothesize that by utilizing
relevant human cell types, we will overcome the challenges of producing viral proteins in non-human cell lines
and enhance the functionality of the purified proteins (Aim 2). This in-depth molecular study of the core DNA
replication proteins will advance the general understanding of KSHV biology and gamma-herpesvirus replication
and the data generated from this proposal will provide the foundation for future proposals aimed at identifying
virus specific inhibitors to prevent KSHV infection.