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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Thesis (M.A.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 107-115). / v, 115 leaves, bound 29 cm

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia / Made available in DSpace on 2013-06-25T18:52:35Z (GMT). No. of bitstreams: 1 311573.pdf: 7982077 bytes, checksum: 243e7ae4350af569490079c4e00e4810 (MD5) / Agaricus brasiliensis (syn A. subrufescens) é um fungo basidiomiceto nativo no Brasil, que apresenta paredes celulares ricas em polissacarídeos, tais como ?-(1?6)-(1?3)-glicanas nos corpos de frutificação e ?-(1?2)-glico-?-(1?3)-mananas no micélio. Neste trabalho, estes polissacarídeos foram isolados e modificados quimicamente gerando seus respectivos derivados sulfatados. Os polissacarídeos nativos (FR e MI) e sulfatados (FR-S e-MI-S) foram caracterizados quimicamente e avaliados quanto as suas atividades citotóxica e anti-herpética in vitro. Uma vez que FR-S e MI-S apresentaram uma promissora ação anti-HSV-1 (cepas KOS e 29-R) e anti-HSV-2 (cepa 333), seu mecanismo de ação foi determinado. A atividade anti-herpética in vivo do MI-S também foi testada utilizando-se modelos murinos de infecção ocular, cutânea e genital. FR-S e MI-S apresentaram duas novas bandas de absorção no espectro de infravermelho em 1253 e 810 cm-1, relacionados aos grupamentos C-S-O e S=O, respectivamente. A massa molecular do FR e MI foram estimados em 609 e 310 kDa, respectivamente. FR-S (127 kDa) e MI-S (86 kDa) apresentaram massas reduzidas provavelmente devido a hidrólise ocorrida durante a reação de sulfatação. FR-S e MI-S apresentaram ~14% de enxofre na análise elementar. Análise de RMN de 1H e 13C confirmaram as estruturas mencionadas anteriormente para FR e MI, sendo o FR-S totalmente sulfatado em C-4 e C-6, enquanto o MI-S parcialmente sulfatado em C-2, C-3, C-4 e C-6. Embora FR e MI não terem apresentado atividade, FR-S e MI-S exibiram promissora ação anti-HSV, com índices de seletividade (SI=CC50/EC50) superiores a 208. FR-S e MI-S não tiveram efeito virucida, mas significativamente inibiram a adsorção e penetração, provavelmente por interagir com glicoproteínas virais. Estudos de combinação revelaram que FR-S e MI-S atuam de forma sinérgica com o aciclovir. Além disso, MI-S inibiu a expressão das proteínas virais ICP27, UL42, gB e gD. Não foi observada toxicidade nos grupos de animais não infectados e tratados com MI-S. O tratamento tópico e oral com MI-S não foi efetivo na redução da doença ocular. A aplicação tópica de MI-S nas lesões cutâneas também não foi efetiva, mas os animais infectados cutaneamente e tratados oralmente com MI-S mostraram redução significativa dos escores da doença e cura acelerada das lesões pelo HSV-1 após o nono dia. A administração vaginal de MI-S (500 µg), 20 min antes do desafio viral, significantemente reduziu os escores da doença (dias 5 a 9), títulos virais (dias 1 e 3) e mortalidade em comparação aos grupos controles. A sulfatação aumentou em 5X o efeito citotóxico do FR contra células tumorais A549, enquanto foi essencial para a atividade do MI. A combinação de fatores como o alto grau de substituição e a massa molecular relativamente menor teve impacto positivo sobre a atividade citotóxica do FR-S e MI-S. Os resultados mostram que o MI-S tem potencial a ser utilizado na redução da severidade das lesões cutâneas pelo HSV-1 e principalmente como um microbicida evitando a transmissão sexual das infecções genitais pelo HSV-2. Ainda, os resultados promissores obtidos na triagem preliminar estimulam novos estudos para avaliação do mecanismo da atividade citotóxica do MI-S e FR-S.

Orientador: Tereza Cristina Cardoso / Coorientador: Alício Martins Júnior / Banca: Fernanda da Cruz Landim e Alvarenga / Banca: Eunice Oba / Resumo: Este estudo foi delineado com objetivo de verificar os efeitos da exposição experimental do sêmen fresco bovino ao BoHV-5, previamente à congelação, através de análises in vitro e de produção de embriões. Os seguintes testes foram avaliados no Grupo I (sêmen não exposto ao vírus) e II (sêmen exposto ao vírus): motilidade e movimento espermático, através de análise computadorizada, integridade da membrana plasmática/acrossomal (IMPA) e função mitocondrial (FM), utilizando sondas fluorescentes, translocação da fosfatidilserina (PS) na membrana do espermatozóide, com Anexina-V, desenvolvimento embrionário in vitro, evidenciado pela proporção de embriões gerados, detecção viral em espermatozóides e embriões, através do emprego da técnica de hibridização in situ (ISH) e PCR. Nenhuma diferença significativa foi observada para IMPA e FM entre os grupos. Entretanto, resultados da translocação da PS no Grupo II foram significativamente diferentes (P=0,00005) dos encontrados no Grupo I; valores maiores (P<0,05) para velocidade média (VAP), velocidade curvilínea (VCL) e amplitude de deslocamento lateral da cabeça (ALH) e menores para a retilinearidade (STR) e linearidade (LIN) foram obtidos no Grupo II quando comparados com o Grupo I. Concluindo, a ISH revelou a presença de DNA viral nos espermatozóides e embriões infectados; além disso, o BoHV-5 levou à alterações funcional e morfológica nos espermatozóides, sem comprometer o desenvolvimento embrionário, mesmo após ser carreado para os embriões, através da infecção dos oócitos na fertilização in vitro / Abstract: This study was designed to verify the effects of experimental exposure of fresh bull semen to BoHV-5, previously to the freezing process, on in vitro sperm evaluations and embryo production. The following tests were performed in groups I (semen not exposed to virus) and II (semen exposed to virus): sperm motility and movement by computerized analysis (CASA), plasma/acrosomal membrane integrity (PAMI) and mitochondrial function (MF) by fluorocromes dyes, sperm membrane phosphatidylserine (PS) transposition using Annexin-V, in vitro embryonic development evidenced by the proportion of yielded embryos, and viral detection in sperm and embryos performed by in situ hybridization (ISH) assay and PCR. No significant difference was observed for PAMI and MF between groups. However, results of PS transposition in group II were significantly different (P=0.00005) from those found in group I; higher values (P<0.05) for mean velocity (VAP), curvilinear velocity (VCL) and lateral head displacement (ALH) and lower values for straightness (STR) and linearity (LIN) were obtained in group II when compared to those observed in group I. In conclusion, the ISH assay showed the presence of viral DNA on spermatozoa and embryos; besides, BoHV-5 induced functional and morphological sperm alterations, but did not compromise the embryonic development even after being carried to the embryos through the IVF infected oocytes / Mestre

Made available in DSpace on 2014-06-11T19:27:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-29Bitstream added on 2014-06-13T18:31:15Z : No. of bitstreams: 1 souza_dg_me_araca.pdf: 1858727 bytes, checksum: 571362381118ee9810c1695198ec90f5 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este estudo foi delineado com objetivo de verificar os efeitos da exposição experimental do sêmen fresco bovino ao BoHV-5, previamente à congelação, através de análises in vitro e de produção de embriões. Os seguintes testes foram avaliados no Grupo I (sêmen não exposto ao vírus) e II (sêmen exposto ao vírus): motilidade e movimento espermático, através de análise computadorizada, integridade da membrana plasmática/acrossomal (IMPA) e função mitocondrial (FM), utilizando sondas fluorescentes, translocação da fosfatidilserina (PS) na membrana do espermatozóide, com Anexina-V, desenvolvimento embrionário in vitro, evidenciado pela proporção de embriões gerados, detecção viral em espermatozóides e embriões, através do emprego da técnica de hibridização in situ (ISH) e PCR. Nenhuma diferença significativa foi observada para IMPA e FM entre os grupos. Entretanto, resultados da translocação da PS no Grupo II foram significativamente diferentes (P=0,00005) dos encontrados no Grupo I; valores maiores (P<0,05) para velocidade média (VAP), velocidade curvilínea (VCL) e amplitude de deslocamento lateral da cabeça (ALH) e menores para a retilinearidade (STR) e linearidade (LIN) foram obtidos no Grupo II quando comparados com o Grupo I. Concluindo, a ISH revelou a presença de DNA viral nos espermatozóides e embriões infectados; além disso, o BoHV-5 levou à alterações funcional e morfológica nos espermatozóides, sem comprometer o desenvolvimento embrionário, mesmo após ser carreado para os embriões, através da infecção dos oócitos na fertilização in vitro / This study was designed to verify the effects of experimental exposure of fresh bull semen to BoHV-5, previously to the freezing process, on in vitro sperm evaluations and embryo production. The following tests were performed in groups I (semen not exposed to virus) and II (semen exposed to virus): sperm motility and movement by computerized analysis (CASA), plasma/acrosomal membrane integrity (PAMI) and mitochondrial function (MF) by fluorocromes dyes, sperm membrane phosphatidylserine (PS) transposition using Annexin-V, in vitro embryonic development evidenced by the proportion of yielded embryos, and viral detection in sperm and embryos performed by in situ hybridization (ISH) assay and PCR. No significant difference was observed for PAMI and MF between groups. However, results of PS transposition in group II were significantly different (P=0.00005) from those found in group I; higher values (P<0.05) for mean velocity (VAP), curvilinear velocity (VCL) and lateral head displacement (ALH) and lower values for straightness (STR) and linearity (LIN) were obtained in group II when compared to those observed in group I. In conclusion, the ISH assay showed the presence of viral DNA on spermatozoa and embryos; besides, BoHV-5 induced functional and morphological sperm alterations, but did not compromise the embryonic development even after being carried to the embryos through the IVF infected oocytes

In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)