Study of the Coordinated Assembly of Kshv Lytic Dna Replication Proteins

Eukaryotic DNA replication is a highly regulatory process that requires multiple replication enzymes assembled on the DNA. Due to the lack of an easy assay system for cellular DNA replication, most of what we know about cellular DNA replication has come from the study of viral systems. Here, we focus our study on the lytic DNA replication of Kaposi’s sarcoma-associated virus (KSHV). Although the lytic DNA replication of herpesviruses has been uncovered over several decades, the control of assembly of the viral DNA replication machinery is mostly unknown. Like other herpesviruses, KSHV encodes six conserved core replication proteins essential for viral lytic DNA synthesis. These six replication proteins include ORF6 (single-stranded DNA-binding protein, SSB), ORF9 (DNA polymerase, POL), ORF40/41 (primase-associated factor, PAF), ORF44 (helicase, HEL), ORF56 (primase, PRI), and ORF59 (polymerase processivity factor, PPF). Based on the current views of KSHV lytic DNA replication, the ORF44, ORF56 and ORF40/41 proteins can form a heterotrimeric helicase-primase subcomplex, whereas the ORF9 and ORF59 proteins form a heterodimeric replison subcomplex. In our preliminary results, we unexpectedly found that KSHV has several distinct surveillance mechanisms operating to control the assembly of the multi-subunit core replication complex. These control mechanisms include alteration of the subcellular localization, protein stability, or different actions of a given replication component or subcomplex when the other replication components (or subcomplexes) are not yet available in cells. Understanding the coordinated assembly of all six core replication components in KSHV may be important not only for gaining insights into viral DNA replication but also for providing useful information for antiviral drug development. Herein, we propose six specific aims to study the viral core replication complex assembly. These specific aims include: 1) determining the protein-protein interactions between KSHV core replication proteins; 2) elucidating the multi-subunit assembly of the viral core replication complex; 3) exploring the mechanism and significance for the cytoplasmic retention of specific core replication components; 4) studying how ORF59 increases ORF9 protein abundance; 5) investigating the nuclear import and export mechanisms of the ORF44-ORF56-ORF40/41 subcomplex; 6) searching for specific drugs against the assembly of the viral core replication complex.

Project ID:PC10907-0958
External Project ID:MOST109-2320-B182-027-MY3