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2015 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report 2015 Research Interests Our research explores the mechanisms that control how genes are expressed inside cells. Some genes must be expressed more or less constantly throughout the life of any eukaryotic cell; others must be turned on (or off) in particular cells at specific times or in response to specific signals or events. Regulation of gene expression helps determine how a given cell will function. Our laboratory explores the mechanisms that regulate the first step in that flow, the process of transcription. We use infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation in human cells. 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 the obvious symptoms in the skin and mucosa, typically in or around the mouth. Like all viruses, HSV-1 relies on the molecular machinery of the infected cell to express viral genes so that the infection can proceed and new copies of the virus can be made. This process is triggered by a viral protein known as VP16, which stimulates the initial expression of viral genes in the infected cell. Much of our work over the years has explored how VP16 activates these genes during lytic infection. After the initial infection resolves, HSV-1 finds its way into nerve cells, where the virus can remain in a latent mode for long periods of time—essentially for the entirety of the host organism’s life. Occasionally, some triggering event (such as emotional stress or damage to the nerve from a sunburn or a root canal operation) will cause the latent virus to reactivate, producing new viruses in the nerve cell and sending them back to the skin to cause a recurrence of the cold sore. We are investigating the role that VP16 might play during such reactivation. Chromatin-modifying coactivators in reactivating latent herpes simplex virus The strands of DNA in which the human genome is encoded are much longer than the diameter of a typical human cell. To help fit the DNA into the cell, cellular DNA is typically packaged as chromatin, in which the DNA is wrapped around “spools” of histone proteins and then further arranged into higher-order structures. When genes need to be expressed, they are partially unpackaged by the action of chromatin-modifying coactivator proteins, which either chemically change the histone proteins or physically slide the histones along the DNA. Transcriptional activator proteins such as VP16 can recruit these chromatin-modifying coactivator proteins to specific genes. We have shown that this process is not very important during lytic infection, because the viral DNA in either the viral particle or the infected cell is not effectively packaged into chromatin. However, in the latent state, few viral genes are expressed because the viral DNA is packaged much like the silent genes of the host cell. Our present hypothesis is that the coactivators recruited by VP16 are required to open up chromatin as an early step in reactivating the viral genes from latency. We are currently testing this hypothesis in quiescent infections of cultured human nerve cells. Regulating the regulatory proteins: posttranslational modifications of VP16 The activity of a given protein is not only dependent on being expressed at the right time, but also on chemical modifications of that protein. Proteins can be posttranslationally modified by adding chemical groups, including phosphates, sugars, methyl or acetyl groups, lipids, or small proteins such as ubiquitin. Each of these modifications can affect how the protein folds, how it interacts with other proteins, and how stable it remains in the cell. 56

Triezenberg We know that VP16 can be phosphorylated, and we have already defined several sites within the VP16 protein where this happens. We are now testing whether these or other modifications affect how VP16 functions, either as a transcriptional activator protein or as a structural protein of the HSV-1 virion. In some experiments, we make mutations that either prevent phosphorylation or that introduce an amino acid that mimics phosphorylation, and then we test the effects of these mutations on VP16 functions. In other experiments, we inhibit the enzymes, such as kinases, that apply the modifications. We expect that this work will lead to new ideas about ways to selectively inhibit the modification of VP16 using small-molecule drugs and thereby prevent or shorten infection. Other cellular regulators of HSV infections HSV, like all viruses, makes use of many cellular proteins to promote its infection. In response, the infected cells take defensive measures to inhibit the virus. We would like to find ways to block the cellular proteins that support the virus or boost the cellular proteins that inhibit it. We have found that some of the cellular proteins that normally repair damaged DNA in the host cell also contribute to the replication of viral DNA, but we are still working out just how that happens. We have also found that a number of protein kinases from the host cell help with early steps in the infection process. Some of those seem to be involved in the entry of the virus into the cell; others clearly affect viral infection, but we don’t yet know why. We are exploring each of these potential participants to find out what roles they play in virus infection and whether drugs that block these kinases might be useful in treating viral infection in humans. Recent Publication Danaher, Robert J., Ross K. Cook, Chunmei Wang, Steven J. Triezenberg, Robert J. Jacob, and Craig S. Miller. 2013. C- terminal trans-activation sub-region of VP-16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells. Journal of Neurovirology 19(1): 32–41. 57

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