Abstract
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a newly identified human pathogen with tumorigenic potential. The DNA polymerase (Pol-8) and processivity factor (PF-8) of KSHV were cloned recently. It was shown that PF-8 forms specifically a complex with Pol-8 in vitro and allows it to synthesize fully-extended DNA. Since both Pol-8 and PF-8 are apparently essential for viral DNA replication and since they cannot be substituted by any other cellular or viral proteins, they are potentially excellent antiviral targets. The development of a mechanistic plate assay is now described, which is suitable for rapid high-throughput screening of antiviral agents against Pol-8 and PF-8. The assay allows the measurement of not only total DNA synthesis activity (i.e. nucleotide incorporation) but also processivity (i.e. fully-extended DNA product). In this plate assay, any of the screen-compounds with an inhibitory effect against the total DNA synthesis activity and/or the processivity could be potential antiviral agents that target Pol-8 and/or PF-8. Particularly, since PF-8 is highly specific for Pol-8, the discovery of inhibitory agents against PF-8 may lead to specific antiviral therapies with minimal toxicity to host cells. This assay should be suitable for screening for inhibitory compounds against polymerases and processivity factors of other herpesviruses as well.
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