Cloning and functional characterization of DNA polymerases and processivity factors of human herpesvirus 6 and Kaposi\u27s sarcoma-associated herpesvirus

Herpesviruses encode all of their own proteins for the replication of their double-stranded DNA genome, including a viral DNA polymerase (Pol) and a processivity factor (PF). PFs associate with Pols and allow them to synthesize fully-extended DNA without dissociating from the template. Here, we describe the cloning and characterization of the Pols and PFs of two new human herpesviruses. Hunan herpesvirus 6 (HHV-6) is a T cell tropic herpesvirus and is an opportunistic pathogen in AIDS and transplant recipients. A 41 kDa early antigen of HHV-6, p41, was isolated previously and was shown to exhibit nuclear localization and DNA-binding activity. Here, p41 was observed to form a specific complex with the DNA polymerase of HHV-6 (Pol-6) both in vivo and in vitro. We then demonstrated that p41 is indeed the processivity factor of HHV-6 in that it allows Pol-6 to synthesize fully-extended DNA. Kaposi\u27s sarcoma associated herpesvirus (KSHV) is a newly identified herpesvirus with tumorigenic potential. Here, we cloned and expressed the putative DNA polymerase (Pol-8) and processivity factor (PF-8) of KSHV. Pol-8 and PF-8 also form a specific complex in vitro, and the DNA synthesis activity of Pol-8 greatly depends on the presence of PF-8. Interestingly, mutational analysis revealed that the C-terminal regions of both p41 and PF-8 are dispensable for their processivity function, suggesting that they may be involved in other functions. Moreover, in order to understand the mechanism by which herpesvirus PFs confer processivity, we demonstrated that PF-8, through its intrinsic DNA-binding ability, confers processivity by tethering Pol-8 to the DNA template. Two models were proposed for the mechanism of herpesvirus PFs and were investigated. In this study, we also demonstrated that herpesvirus PFs are highly specific in that they can only associate with their cognate Pols and confer processivity. Interestingly, a Pol8-PF8 fusion protein, but not a Pol8-p41 fusion, was able to achieve processive DNA synthesis. Finally, since both Pols and PFs are critical for herpesvirus DNA replication, we developed an assay for high throughput screening of antiviral agents against herpesvirus Pols and/or PFs, which may lead to the discovery of specific inhibitors of viral replication