Herpes simplex virus DNA replication and microRNA expression

Description

The interaction between the catalytic subunit, Pol, and the processivity subunit, U L 42, of herpes simplex virus 1 (HSV-1) DNA polymerase has been characterized structurally and mutationally, and is a potential target for novel antiviral drugs. Our laboratory developed and validated an assay for small molecules that could interrupt the interaction of U L 42 and a Pol-derived peptide, and identified one "hit", namely BP5, which inhibited U L 42-stimulated long-chain DNA synthesis by Pol in vitro , and exhibited little inhibition of polymerase activity by Pol alone. I showed that BP5 specifically inhibited the physical interaction of Pol and U L 42, and also inhibited viral replication at concentrations below those that caused cytotoxic effects. To investigate the antiviral mechanism of BP5, a resistant mutant was isolated after multiple-cycle selection. A candidate BP5-resistance (BP5 r ) mutation was identified by DNA sequencing. This point mutation in the TATA box of U L 42 was shown to confer BP5 r by genetic experiments using a bacterial artificial chromosome (BAC) of HSV-1 strain KOS that I generated and characterized. Structure-activity relationships of BP5 were studied and a less cytotoxic analog, which retains considerable antiviral activity via the same mechanism, was identified. This new compound may be useful for selection of other resistant mutants. MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. To identify mIRNAs encoded by HSV-1, in collaboration with others, I validated and applied a computational method to screen the complete genome of HSV-1, and identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate (HSV-1 miR-H1), which is encoded ~450 bp upstream of the transcription start site of the latency associated transcript, were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. I hypothesize that HSV-1 miRNAs regulate viral and host gene expression, have generated cell lines stably expressing HSV-1 miR-H1, and have constructed mutant HSV-1 not expressing miR-H1 using the KOS BAC.

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