Single Molecule analysis of KSHV/HHV8 DNA replication proteins - 7. Project Summary Effective antiviral therapeutics are needed for the treatment of KSHV infection. We propose to pinpoint novel KSHV antiviral targets by identifying virus specific protein functions and conformations. This project aims to study the molecular interactions (protein-DNA and protein-protein) that govern KSHV DNA synthesis and genome replication using a highly specialized electron microscopy (EM) approach. Previous studies used sequence homology between KSHV and related herpesviruses to determine conserved protein functions of the seven essential core DNA replication genes/proteins encoded by KSHV: ORF6(SSB), ORF9(POL), ORF40/41(PAF), ORF44(HEL), ORF56(PRI), ORF59(PF) and ORF50(RTA). However, protein sequence homologies only range from 20-50% and thus poorly predict protein function; therefore, an ultrastructural characterization of purified KSHV viral DNA replication proteins, individually and in concert are needed to identify their full range of functions (Aim 1). To evaluate the in vitro activities, electron microscopy (EM) will be used to directly visualize viral proteins and viral DNAs. Our EM approach produces qualitative data (heterogeneous protein complexes, oligomeric state, DNA architecture) and quantitative data (DNA mapping of protein binding locations, molecular size comparisons). The first aim of this proposal will characterize the molecular interactions and activities of a subset of already purified KSHV DNA replication proteins. 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 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 (PAF and PRI) 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. Finally, trainees (undergraduate and graduate students) will gain exposure and expertise in a wide range of biochemical and molecular biology approaches while also learning unique, highly-specialized EM techniques while completing Aims 1 and 2.
