Herpes viruses are large DNA viruses whose life cycle can be characterized by transitions between active lytic infections and silent latent phases. The α herpes viruses are a subclass that includes herpes simplex virus types 1 and 2, as well as varicella zoster virus. These viruses typically conduct lytic infections in mucosal tissues of the skin, which causes localized tissue inflammation and cell death. Primary infection in mucosal tissues leads to infection of nearby sensory neurons, which transport virus to neuronal cell nuclei located within the sensory ganglia (trigeminal ganglia for HSV-1). The viral DNA, once inserted into the neuronal nuclei, is believed to be maintained in a transcriptionally repressed state during latency. Virus can reactivate from the latent state by initiating replication and transporting virus back to the site of primary infection causing recurrent disease. We have been studying the fate of the viral DNA within infected cells in both lytic and latent phases. The viral genome is 152,000 bp long and encodes at least 70 proteins. The genome exists in a linear form within the virion but circularizes once inserted into the nuclei of infected cells. The circular genome associates with cellular proteins including histones and other chromatin associated factors to become a unique type of mini-chromosome. The formation, maintenance and regulation of this mini-chromosome is believed to play crucial roles during latency and reactivation. Our current research is focused on the maintenance and repair of HSV genomic DNA during productive and latent infections. An overarching goal is to elucidate the complex relationship that exists between the virus and the many host cell proteins that function to coordinate the tasks required for lytic and latent phases of the virus infection.
Current Project:
The primary objective of this project is to determine how the HSV genome is managed and repaired during productive and latent infection. To study this, we use cell culture-based models of lytic and latent infection and an in vivo mouse model of latency. The primary techniques we employ to examine DNA integrity and damage are southern hybridizations and PCR based technologies. To mimic neuronal latency in cell culture, we are utilizing an NGF-differentiated PC12 cell system that supports HSV infection, genome circularization in cell nuclei, and maintenance of HSV DNA for at least 30 days in culture with minimal viral replication. Our studies in both quiescently-infected NGF-differentiated PC12 cells and latent murine trigeminal ganglia provided evidence that latent genomes acquire alkaline labile damage over the course of 30 days of quiescent infection. This suggests that viral DNA may be poorly repaired or attacked at a rate that otherwise exceeds repair. The alkaline lability suggests that the damage may include nicks in the DNA backbone as well as apurinic/apyrimidinic (AP site) damage, in which a DNA base is removed while keeping the backbone intact. It has been observed that primary neurons in general are somewhat impaired in their ability to repair damaged cellular DNA compared to proliferating cells, and this is believed to be related to neurons being more reliant on cellular transcription coupled DNA repair mechanisms as opposed to global repair systems. One of the hypotheses we are currently examining is that the accumulation of HSV DNA damage during quiescent infection may be related to the transcriptional status of HSV genes.
The work in quiescent and latent infections is being complemented by similar studies in cells that support lytic infection. It has been known for many years that ultraviolet (UV) irradiation damaged HSV can be induced to replicate by a host-derived mechanism. We have been examining the repair of UV damaged virion DNA in lytically infected Vero cells and preliminary data shows that UV damaged HSV DNA can be repaired during the first several hours of infection. The repair is partially blocked by the drug PAA, which is a viral DNA polymerase inhibitor. Currently, we are examining the specificity of the PAA repair inhibition for viral and cellular DNA polymerases. In the course of these studies, we are also interested in characterizing of the state of unirradiated virus DNA after infection in Vero cells. These preliminary results support a model in which the virion DNA is rapidly attacked (probably by a nuclease) within 30 – 60 min after inoculation of cultures, and that this period is followed by a phase of genome repair prior to DNA replication. This hypothesis is consistent with published data showing the recruitment of cellular DNA strand break repair proteins to viral pre-replication compartments. These studies should help to elucidate the interplay that exists between HSV and cellular DNA repair proteins.
As we move forward, we wish to look into genome maintenance in quiescent infections in human neuronal cells and primary neurons. The HSV natural host is the human, so studies in mice and other animals, though useful for research purposes, may not accurately depict the biology of the virus-host interaction in humans. Understanding the human biology of the virus-host interaction may be critical for developing new strategies for controlling HSV infection, as well as for providing additional insight into our general understanding of innate and acquired immunity
Selected Research Publications:
"Evidence that herpes simplex virus DNA derived from quiescently infected cells in vitro, and latently infected cells in vivo, is physically damaged"
Su Y.-H., Zhang X., Wang X., Song B.P., Zhu L., Oppenheim E., Fraser N.W., Millhouse S., and T.M. Block.
In revision, 2010.
"Recognition of Trimethylated Histone H3 Lysine 4 Facilitates the Recruitment of Transcription Postinitiation Factors and Pre-mRNA Splicing"
Sims III R.J., Millhouse S., Chen C.-F., Lewis B.A., Erdjument-Bromage H., Tempst P., Manley J.L., and D. Reinberg.
Mol. Cell, 28(4): 665-676, 2007.
"The C-terminal domain of RNA polymerase II functions as a phosphorylation-dependent splicing activator in a heterologous protein"
Millhouse S. and J.L. Manley.
Mol. Cell. Biol., 25(2): 533-544, 2005.
"Molecular circuitry regulating herpes simplex virus type 1 latency in neurons"
Millhouse S. and B. Wigdahl.
J. Neurovirology, 6: 6-24, 2000.
"HIV-1 LTR C/EBP binding site sequence configurations preferentially encountered in brain lead to enhanced C/EBP factor binding and increased LTR-specific activity"
Ross H.L., Gartner S., McArthur J.C., Corboy J.R., McAllister J.J., Millhouse S., and B. Wigdahl.
J. Neurovirology, 7: 235-249, 2001.
"Analysis of the HIV-1 LTR NF-kB-proximal Sp site III: Evidence for cell type-specific gene regulation and viral replication"
McAllister J.J., Phillips D., Millhouse S., Conner J., Hogan T., Ross H., and B. Wigdahl.
Virology, 274: 262-277, 2000.
"Sp1 and related factors fail to interact with the NF-kB-proximal G/C box in the LTR of a replication competent, brain-derived strain of HIV-1 (YU-2)"
Millhouse S., Krebs F.C., Yao J., McAllister J. J., Conner, J., Ross, H., and B. Wigdahl.
J. Neurovirology, 4: 312-323, 1998.
"ATF/CREB elements in the herpes simplex virus type 1 latency-associated transcript promoter interact with members of the ATF/CREB and AP-1 transcription factor families"
Millhouse S., Kenny J.J., Quinn P.G., Lee V., and B. Wigdahl.
J. Biomedical Sci., 5: 451-464, 1998.
"Upstream stimulatory factor (USF) family binds to the herpes simplex virus type 1 latency-associated transcript promoter"
Kenny J.J., Millhouse S., Wotring M., and B. Wigdahl.
Virology, 230: 381-391, 1997.
"Differential effects of IkB molecules on Tat-mediated transactivation of HIV-1 LTR"
Harhaj E., Blaney J., Millhouse S., and S. Sun.
Virology, 216: 284-287, 1996.
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