Our research is focused on the mechanisms that control whether genes are turned on or turned off inside cells. The genetic information encoded in DNA must first be copied, 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, while others must be turned on (or turned off) in particular cells either at specific times or in response to a specific signal or event. 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 known as transcription. We use infection by herpes simplex virus as an experimental context for exploring the mechanisms of transcriptional activation in human cells.
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. 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 latent 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, the virus must divert the gene expression machinery of the host cell. That process is triggered by a viral regulatory protein designated VP16, whose function is to stimulate transcription of the first viral genes to be expressed in the infected cell (the immediate-early, or IE, genes).
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 space of a cell, 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 elaborate packaging creates a problem when access is needed to the information carried in the DNA, such as when particular genes need to be expressed. This problem is solved in part by chromatin-modifying coactivator proteins, which either chemically change the histone proteins or else slide or remove them.
Transcriptional activator proteins such as VP16 can recruit these chromatin-modifying coactivator proteins to target genes. We have shown this to be true for the viral genes that VP16 activates during an active infection. Curiously, however, the DNA of herpes simplex virus is not wrapped in histones inside the viral particle, and it also seems to stay relatively free of histones inside the infected cell. That observation leads to a paradox: why would VP16 recruit chromatin-modifying coactivators to the viral DNA, if the viral DNA doesn’t have much chromatin to modify?
We took several approaches to test whether the coactivators recruited to viral DNA by the VP16 activation domain really play a significant role in transcriptional activation. In some experiments, we knocked down expression of given coactivators using short interfering RNAs (siRNAs). In other experiments, we used cell lines that have mutations disrupting the expression or activity of a given coactivator. We expected to find that viral gene expression was inhibited, but the experiments yielded unexpected results: in each case, expression of the viral genes was essentially unaffected. We were forced to conclude that our initial hypothesis was wrong; the coactivators, although present, are not required for viral gene expression during lytic infection.
The death of one hypothesis, however, gives life to new ideas. After the initial infection of a cold sore subsides, herpes simplex virus establishes a life-long latent infection in sensory neurons. In the latent state, the viral genome is essentially quiet; very few viral genes are expressed. Moreover, the viral genome becomes packaged in chromatin much like the silent genes of the host cell. So our new hypothesis is that the coactivators recruited by VP16 are required to reactivate the viral genes from the latent or quiescent state. We now have evidence that VP16 is likely the very first viral gene to be expressed during the reactivation process. We want to test whether the ability of VP16 to recruit coactivators is essential for subsequent events of reactivation. We will test this hypothesis both in quiescent infections in cultured cells and in animal models with genuinely latent herpesvirus infections.
The activity of a given protein is not only dependent on being expressed at the right time, but also on chemical modifications of its amino acids and on its interactions with other proteins. Proteins can be post-translationally modified by adding chemical groups including phosphates, sugars, methyl or acetyl groups, lipids, or small proteins such as ubiquitin. Each of these modifications might affect the protein in different ways, including how the protein folds, how it interacts with other proteins, and how stable it remains in the cell.
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 modifications matter for how VP16 functions, either as a transcriptional activator protein or as a structural protein of the HSV-1 virion. In some experiments, we create 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 that apply the modifications (for phosphorylation, these enzymes are known as protein kinases). We expect that this work will lead to new ideas about ways that we can selectively inhibit modification of VP16 using small-molecule drugs, and thereby prevent or shorten the infection process by HSV.