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.
The most recently discovered herpesvirus, Kaposi?s sarcoma herpesvirus (KSHV/HHV8) is known to cause three human cancers, Kaposi?s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman?s disease (MCD). No cure or vaccine exists for KSHV infection and its associated malignancies. To identify novel KSHV antiviral targets, we propose to identify virus specific protein functions and protein conformations by employing highly specialized electron microscopy (EM) methods to directly visualize purified KSHV DNA replication proteins and viral DNA sequences during the formation of the DNA replication fork at the viral origins of replication.
