Herpes Simplex Virus type-1 (HSV-1) is one of the eight human herpes viruses and it causes cold sores in infected individuals. HSV-1 infects epithelial cells, then travels to the trigeminal ganglion through retrograde transport in the peripheral neurons. During the initial lytic infection in the epithelial host cell, HSV-1 hijacks cellular RNA polymerase II (RNAP II) to sites of viral transcription for viral gene expression. Previous studies have found an intermediately phosphorylated form of RNAP II in HSV-1 infected cells starting approximately 5 hours post infection. This intermediately phosphorylated form of RNAP II appeared to be different from the usual hypo- and hyperphosphorylated forms of RNAP II normally found in uninfected cells. Subsequent studies had revealed that at late time in HSV-1 infection, elongating RNAP II, marked by phosphorylated serine 2 residues at the C-terminal domain (CTD), was degraded in infected cells, and this degradation could be prevented by inhibiting transcription. The loss of elongating RNAP II was in contrast to the high levels of viral transcription that occurs during late phase of HSV-1 replication cycle. We have previously proposed a collision model where the compact viral genome becomes overburdened by the high transcriptional activities at late time in HSV-1 infection, leading to arrested RNAP II complexes that then in turn, trigger a proteasome-mediated proteolysis of the polymerase to resolve stalled transcription. In this body of work, we first attempted to determine the if HSV-1 transcription is more similar to cellular transcription in that it requires phosphoserine-2 form of RNAP II and found that when we inhibited cdk9, the kinase responsible for phosphorylating serine 2 residues of the RNAP II CTD, viral transcription was also inhibited. Viral yield under cdk9 inhibition was also dramatically reduced, supporting the idea that like cellular transcription, HSV-1 transcription requires phosphoserine-2 form of RNAP II. We also attempted to substantiate our RNAP II collision model by assessing RNAP II occupancy on the HSV-1 genome using chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR) analysis. In our ChIP qPCR analysis of an Early HSV-1 gene cluster, we found that though ChIP signals from one of the antibodies were consistent with known expression patterns of the genes, we were unable to validate the results with phosphoform-specific ChIP results due to the high noise levels of these phosphoform-specific antibodies. When analyzing a Late HSV-1 gene cluster, we were unable to achieve satisfactory signal-to-noise ratios regardless of the antibody used. Therefore we were unsuccessful to support or refute our collision model at this time. Lastly, we investigated a possible link between HSV-1 induced RNAP II degradation and the cellular transcription-coupled nucleotide excision repair (TC-NER) pathway by examining the role of a general transcription factor TFIIS, a factor known to stimulate transcription after RNAP II becomes stalled, in HSV-1 transcription. Our initial immunofluorescence studies had shown that one of the isoforms of TFIIS (TCEA2) appeared to re-localize to HSV-1 transcription replicative compartments in infected cells. Subsequent confocal microscopy co-location studies however, showed a rather inconsistent localization patterns among the isoforms of TFIIS. When attempting to perform functional knock-down experiments of each of the TFIIS isoforms, we found that the antibodies we were using exhibited cross-reactivity to unknown antigens in Western blot analysis. More importantly, these antibodies failed to recognize FLAG-tagged TFIIS transiently expressed in HeLa cells, thereby overturning our initial re-localization results. At this time, we do not have evidence that support a functional involvement of TFIIS in HSV-1 transcription. We believe that further experimentation would be required to support our colliding RNAP II model during HSV-1 lytic infection.

Herpes simplex virus 1 ICP27 is an essential multifunctional regulatory protein that assumes different roles during infection. ICP27 disrupts multiple aspects of cellular gene expression, contributing to the shutoff of host protein synthesis. ICP27 also recruits RNA polymerase II to viral replication sites and mediates the export of viral mRNA. Furthermore, ICP27 has also been implicated in nuclear protein quality control, cell cycle control, activation of stress signaling pathways and apoptosis. ICP27 performs its activities by interacting with RNA and a myriad of proteins. However, how ICP27 is able to interact with so many different binding partners and how these interactions are regulated during the viral life cycle is not well understood. ICP27 has been reported to form homo-dimers and to undergo an intramolecular N- to C-terminal interaction. It’s hypothesized that these different configurations provide the flexibility it needs to interact with various binding partners and events or factors that promote one isomer over the other may serve to regulate different ICP27 activities. At present, how these configurations are related, and their specific functions, have not been well characterized. Moreover, the molecular details as to how ICP27 targets host RNA processing and the wider impacts its activities have on the host transcriptome have also not been detailed. In these studies, we (1) use a bimolecular fluorescence complementation and western blot-co-immunoprecipitation approach to perform a structural analysis in order to elucidate the functions of ICP27’s different conformations, (2) utilize a combination of biophysical and molecular techniques in order to detail the molecular mechanism of how ICP27 inactivates SR protein kinase 1, an important regulator of SR proteins, to inhibit cellular splicing, and (3) perform next-generation sequencing to globally Identify ICP27 modulations to host gene expression.

The results from our studies show: (1) ICP27’s conformational isomers utilize different protein domains for their assembly and these isomers can interact with different binding partners, (2) the ICP27 RGG box binds to the SRPK1 substrate docking groove in a similar manner and affinity as SRPK1’s cellular substrate SRSF1, and (3) ICP27 promotes accumulation of host antisense transcripts during infection. Together, the information gained in this dissertation expands our understanding of the molecular principles by which ICP27 functions.