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2007 Scientific Report

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VARI | 2007 Research Interests The genetic information encoded in DNA must first be transcribed in the form of RNA before it can be translated into the proteins that do most of the work in a cell. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell. Others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event. Thus, regulation of gene expression is a key determinant of cell function. Our laboratory explores the mechanisms that regulate the first step in that flow, the process termed transcription. Over the past 20 years, my laboratory has used infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation. In the past 10 years, we have also asked similar questions in a very different biological context, the acclimation of plants to cold temperature. Transcriptional activation during herpes simplex virus infection Herpes simplex virus type 1 (HSV-1) causes the common cold sore or fever blister. The initial lytic or productive infection by HSV-1 results in obvious symptoms in the skin and mucosa, typically in or around the mouth. After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can “hide” in a latent mode for long times—essentially for the lifetime of the host organism. Occasionally, some trigger event (such as emotional stress, damage to the nerve from a sunburn, or a root canal operation) will cause the virus to reactivate, producing new viruses in the nerve cell and sending those viruses back to the skin to cause a recurrence of the cold sore. The DNA genome of HSV-1 encodes approximately 80 different proteins. However, the virus does not have its own machinery for expressing those genes; instead, it must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function it is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes). 63 VP16 recruitment of host cell transcription machinery The prevailing model for the mechanism of transcriptional activation is that a portion of an activating protein (such as VP16) called the activation domain (AD) can bind to the host cell RNA polymerase II or to its accessory proteins. In this manner, VP16 recruits or tethers accessory proteins to the genes that are to be activated. Over the years, several accessory proteins (also known as general transcription factors) have been implicated as potential targets for VP16. Of those, the evidence seems to point most directly at TFIID, a multi-protein complex that includes the TATA-binding protein (TBP). TBP itself can bind rather efficiently to the VP16 activation domain, and mutations in VP16 that disrupt transcriptional activation also disrupt the interaction with TBP. We have pursued the structure of the VP16-TBP interaction by methods including X-ray crystallography and nuclear magnetic resonance. We have also tested the hypothesis that VP16 can influence the orientation of TBP on the TATA-box DNA of a target gene promoter. This hypothesis, proposed by other laboratories, is based on the fact that both TBP and the TATA sequence to which it binds are quite symmetric, and yet TBP can effectively support transcription in only a single orientation. We developed a new quantitative method for assessing TBP orientation and using this method have now demonstrated that TBP binds in a well-oriented manner even in the absence of VP16. Moreover, on a TATA site engineered to be completely symmetric, to which TBP binds in both orientations, the VP16 activation domain has no significant influence. This work resolves a long-standing issue regarding TBP orientation and eliminates one hypothesis for the mechanism of transcriptional activation.

Van Andel Research Institute | Scientific Report Chromatin-modifying coactivators in herpes virus infections Eukaryotic DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins, and these spools are then further arranged into higher-order structures. This packaging creates an impediment to transcription, during which RNA polymerase must separate the two strands of DNA. The impediment can be overcome with the help of chromatin-modifying coactivator proteins, some of which alter the histone proteins by post-translational modifications (e.g., acetylation or methylation) and others of which can slide or remove the histone proteins to permit access by RNA polymerase to the DNA. Experiments using the VP16 activation domain in artificial contexts (for example, in yeast genetic assays) have indicated that VP16 can recruit various coactivator proteins to target genes. However, the HSV-1 viral DNA is not packaged with histones in the infectious virion, and prior evidence suggested the viral DNA remained largely chromatin-free during infection. Therefore, we wondered whether VP16 would recruit these coactivators to viral IE genes, and if so whether those coactivators would be acting on histone proteins (which didn’t seem to be present) or on some other target. Our results have clearly indicated that VP16 can recruit certain coactivators to IE genes during lytic infection. We have also shown that at least some histone proteins do associate with viral DNA, although perhaps not to the same extent as with cellular DNA. We are currently exploring further which histones associate with viral DNA, how quickly they are put in place, the mechanisms used to put them in place, and what VP16 and other regulatory proteins might do to counteract the repressive effects of chromatin, which could be considered a molecular defense mechanism. 64 Can a curry spice block herpes infections? Curcumin, the bright yellow component of the curry spice turmeric, affects eukaryotic cells in several ways. Another laboratory has reported that curcumin could block the histone acetyltransferase activity of two coactivator proteins, p300 and CBP. Because we had shown that VP16 can recruit p300 and CBP to viral IE gene promoters, we tested whether curcumin, as an inhibitor of p300 or CBP activity, would block viral IE gene expression and thus block HSV infection. Indeed, curcumin has dramatic effects on IE gene expression and substantial effects on virus infection (Fig. 1). We are now trying to determine whether that effect is indeed channeled through the p300 and CBP proteins or whether it arises from another of the biological activities of curcumin. Figure 1. - curcumin + curcumin Figure 1. HSV-1 infection of Vero cell monolayers. HSV-1 infection results in plaques or holes in a monolayer of cultured human cells (left). In the presence of curcumin (right), plaques are generally smaller and the cells within the plaques are not as completely obliterated. Photo by M. Roemer.

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