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Searching for RNAP II on the Compact HSV-1 Genome


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.

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