Thesis (Ph. D.)--University of Chicago, Committee on Virology, June 2003. / Includes bibliographical references. Also available on the Internet.

Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 77-87).

Herpes Simplex Virus (HSV) requires the host cell secretory apparatus for the maturation and egress of newly synthesized viral particles. Not only do viral glycoproteins rely on the host ER and Golgi compartments for their proper processing, it is believed that enveloped particles are transported through these same organelles for their export out of the cells. Brefeldin A (BFA) is a compound that induces retrograde movement of material from the Golgi apparatus to the ER and causes the disassembly of the Golgi complex. In this study, the effects of BFA on the propagation of HSV-1 in infected cells were examined. Release of viral particles from infected cells was inhibited by as little as 1 µg/ml BFA. Further analysis revealed that BFA did not affect the normal assembly of viral nucleocapsids, but did block the movement of newly-enveloped particles from the nucleus into the cytoplasm. Naked nucleocapsids were found in the cytoplasm of infected cells treated with BFA, however, these particles were neither infectious, nor were they released from the cells. Although BFA altered the distribution of viral glycoproteins in infected cells, this alteration was reversed within 2 hours after the removal of BFA. In contrast, the BFA-induced blockage to viral release was not fully reversed after BFA was removed and cells were allowed to recover in fresh medium for 3 hours. These findings indicate that the BFA-induced retrograde movement of material from the Golgi complex to the ER early in infection arrests the ability of the host cell to support the maturation and egress of enveloped viral particles. Furthermore, exposure of infected cells to BFA during the exponential release phase of the viral life cycle can cause irreversible damage to the egressing particles. This suggests that productive growth of HSV-1 in infected cells relies on a series of events that, once disrupted by agents such as BFA, cannot be easily reconstituted. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Herpes simplex virus (HSV) is a clinically important virus, whose life cycle alternates between productive replication and latency. Infected cell protein 0 (ICP0) is generally believed to play a key role in determining the outcome of HSV infections. Synthesis of ICP0 promotes the productive replication of HSV, whereas absence of ICP0 renders HSV prone to establish latent infections. In the first part of the dissertation, I attempt to address the question how is ICP0 gene regulated. To tackle this question, we constructed recombinant HSV that encodes GFP-tagged ICP0 so that the regulation of ICP0 gene can be visualized in real time. Using this reagent, we found that ICP0 gene was subject to potent repression immediately following infection. Surprisingly, HSV's major transcriptional regulator, ICP4, was necessary and sufficient to repress ICP0 gene, and did so in an ICP4-binding-site dependent manner. Synthesis of ICP0 alleviated the ICP4-dependent repression of ICP0 gene. ICP4 co-immunoprecipitated with FLAG-tagged ICP0, thus, a physical interaction between ICP0 and ICP4 likely explains how ICP0 antagonizes ICP4's capacity to silence the ICP0 gene. Therefore, our findings suggest that ICP0 gene is differentially regulated by virus-encoded repressor ICP4 and virus-encoded antirepressor ICP0. In the second part of the dissertation, I attempt to address the question what function does ICP0 assume. Since the discovery of ICP0, the nuclear function of ICP0 has been the focal point of studies, whereas the cytoplasmic function of ICP0 is unknown. While pursuing our first study, we unexpectedly found that GFP-tagged ICP0 was predominantly localized to the cytoplasm during infections. Taking advantage of live-cell imaging, we found that ICP0 translocated from nucleus to cytoplasm during early phase of HSV infections, where it bundled and dispersed microtubules. Synthesis of ICP0 was proved to be necessary and sufficient to dismantle microtubules in HSV-infected and transfected cells. Therefore, our findings suggest ICP0 might play a previously unrecognized role in the cytoplasm through dismantling microtubule networks of the host cells. Furthermore, our study represents the first report showing a virus-encoded E3 ligase disrupts host cell microtubule networks, thus suggests a general function of many other viral E3 ligases.

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Eighty Four rabbits were studied over a period of 2 years for the effects of eye and head X-irradiation on their latent herres simplex ocular infection. Animals were infected intraocularly with herpes simplex virus and allowed to recover from the resultant keratitis. After the latency of the virus was established through daily culture of the eyes of the rabbits, the animals were irradiated. Irradiation was followed by periods of daily culture of both eyes. Eye irradiation of 200, 400, 1200, and 2890 roentgens produce no significant reactivation of the virus in the irradiated eye. On the other hand, head irradiation of 3000 roentgens produced recurrence of the virus in the eyes in a high percentage of the treated rabbits. This effect was reproduced repeatedly. Further experiments seemed to suggest that the virus was latent in the brain and could be reactivated in situ by X-irradiation of the brain. The infectious viral particles appeared to move preferentially to the site of initial infection, This effect may have importance in the fields of clinical medicine, radiation therapy and radiology, and in space travel safety parameters. Further research is planned. / 2031-01-01

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