The herpes virus family consists of more than hundred members that infect organisms, of which eight, differing markedly in the biology are known to infect humans. HSV- I is the most common one, causing oral lesions and sporadic encephalitis. These infections are highly prevalent affecting at least one in three individuals in the United States.The entry of the herpes virus into the cell is a two-step process. The initial step involves the cell surface heparan sulfate and glycoproteins in the viral envelope which enables the virus to penetrate into the cell. The second step is the fusion step. Depending on the nature of interaction and size of HS chain, a single chain may bind multiple viral ligands on a virion. There is substantial evidence showing that HS plays an important role in viral binding.HS is a heterogeneous, linear sulfated oligosaccharide composed of alternating glucosamine and uronic acid residues, which could specify distinct receptor for various viral ligands. HS, present on most exposed cell surfaces, make an ideal snare for the capture of most herpes viruses and may facilitate subsequent interactions with other co-receptors required for entry. Number of viruses, including HSV- I, HSV- II, HIV- I and dengue virus use sites of HS as receptors for binding to cells. Recently 2000 Liu et.al have characterized a HS based octasaccharide that binds to HSV-I gD. The distinguished feature in the composition of the octasaccharide is the presence of 3-O-sulfate glucosamine residue, which is an uncommon structural modification in HS. Its presence in the HSV-I gD binding sequence may confer specificity of interaction and assist HSV-I entry into the cell.Numerous sulfated molecules have been explored as mimics of HS in the inhibition of HSV-1 entry into cells. To date, most of the sulfated molecules screened for anti-viral activity have been carbohydrates. So, we reasoned that it should be possible to mimic critical interactions of HS with one or more viral glycoprotein using synthetic, non-polysaccharide, sulfated compounds. Further, it may be possible to mimic specific sequence(s) in HS, which play a role in HSV infection, with small synthetic, sulfated, non-carbohydrate molecules. In a search for synthetic mimics of HS as inhibitors of HSV-I infection, we screened a small, synthetic, sulfated flavonoids to discover a potent inhibitory activity arising from sulfation of a macromolecule present as an impurity in a crude natural product.The active principle was identified through an array of biophysical and chemical analyses as lignin sulfate, a heterogeneous; polydisperse network polymer composed of substituted phenylpropanoid monomers. Further, LC-MS with APCI in negative ionization mode, which have been reported in here for the first time for analysis of lignin, has been successfully used to deduce oligomeric structures present in the precursor of the active macromolecule based on the spectrum of the depolymerized lignin. This corroborates well with the structural information obtained using other analytical techniques. We hypothesize that the structural heterogeneity and polydispersity of lignin coupled with optimal combination of sulfate charge and hydrophobicity result in high potency. Given that the native lignin is inactive, lignin sulfate discovered here provides a variety of organic scaffolds that with the critical sulfate groups in space can mimic the HSV-I gD binding sequence.

Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

Yau, Kwok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 64-71). / Abstracts in English and Chinese. / Chapter Chapter 1: --- Introduction --- p.1 / Trichosanthin --- p.2 / Apoptosis --- p.10 / Herpes Simplex Virus --- p.16 / Conclusion --- p.28 / Chapter Chapter 2: --- Materials and Methods --- p.29 / Cell lines and virus --- p.30 / Infectivity assay --- p.30 / Treatment of cells and virus infection --- p.32 / MTT assay for cytotoxicity --- p.34 / Preparation of cell lysate --- p.35 / Bradford assay for protein concentration --- p.36 / Western blot analysis --- p.37 / ELISA for quantification of HSV-1 antigen --- p.38 / Statistical analyses --- p.39 / Chapter Chapter 3: --- Results --- p.40 / Cytotoxicity and anti-herpetic activity of TCS and CHX --- p.41 / Selective cytotoxicity of TCS toward HSV-1 infected cells --- p.44 / Selective cytotoxicity is implicated in the antiviral activity of TCS --- p.50 / The effect of selective cytotoxicity on TI value --- p.53 / Chapter Chapter 4: --- Discussion --- p.55 / References --- p.64

Background: Genital herpes is one of the most common sexually transmitted infections in the United States. As genital herpes is incurable and contagious, individuals with genital herpes face the decision to disclose their status to potential sexual partners with each new relationship formed. Such disclosure places individuals with genital herpes in a position to face rejection, which is commonly reported as one of the most concerning aspects of having genital herpes. The present study seeks to further understand the nature of genital herpes disclosure by addressing two core aims: 1) to understand determinants of and reasons for disclosure and non-disclosure and 2) to explore the relationship between past partner reactions to a disclosure and future intentions to disclose. Methods: Data on genital herpes disclosure experiences were collected via an online questionnaire, which was distributed through a variety of online channels including social media websites and email lists. Individuals who self-identified as having genital herpes and were 18 years and older were eligible for participation. Results: In examining Aim 1, the majority of participants (80.4%) disclosed to their last sexual partner. Age, relationship length, type of relationship, and expectations of a partner's response were significantly associated with the decision to disclose at the bivariate level. Expectations of a partner's reaction (AOR = .20, 95% CI .074-.539) and relationship type (AOR = 8.31, 95% CI 1.96-35.32) remained significant in multivariable modeling, explaining 45.2% of the variance in disclosure. Respondents who reported being in socially committed relationships and those who expected more positive partner reactions to a disclosure were more likely to disclose. Disclosure was also significantly associated with many romantic relationship building activities (e.g., establishing an exclusive relationship) but largely not associated with the sexual progression of a relationship. The decision to disclose was commonly multi-faceted, with the majority of participants reporting more than one reason that they did or did not disclose. Primary reasons for disclosure included "I wanted to be honest", "To protect my partner from getting herpes", and "It's my partner's right to know", while the most common reasons for non-disclosure were "I was concerned my partner would react badly", "I was ashamed", and "I was concerned that my partner would have rejected me". Regarding Aim 2, participants reported low levels of negative reactions and perceived rejection in response to their last disclosure experience. Intentions to disclose in the future were high among those who anticipated future sex partners. Discussion: The decision to disclose is often multi-faceted, and relationship characteristics play a key role in the decision to disclose. Among those who did disclose in this study, the majority did not report negative repercussions, including bad partner reactions and rejection. Future studies should examine if individuals are able to accurately assess potential partner reactions in order to better understand the differences between those who choose not to disclose and those who choose to disclose but experience a negative partner reaction or rejection.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Bibliography: leaves 92-118. / xii, 118, [20] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports a method for the study of HSV-1 transcripts during latency. High resolution non-isotopic in situ hybridization (ISH) is used to study the intracellular location of HSV-1 latency associated transcripts (LATs) in primary sensory neurons of latently infected mice and humans. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>

Transcription and DNA replication of herpes simplex virus type 1 (HSV-1) occur in nuclear domains adjacent to structures named ND10. The HSV-1 single-stranded DNA binding protein ICP8 localizes to these nuclear domains to direct the assembly of the pre- and replication compartments. Inhibition of cyclin dependent kinases with roscovitine inhibits HSV-1 DNA replication, even in the presence of all required HSV-1 proteins, at an unidentified step. Here I show that roscovitine inhibits the localization of pre-expressed ICP8 to new replication sites. Therefore, the inhibition of HSV-1 DNA replication occurs at a step prior to initiation. I next evaluated the mechanisms of inhibition of proper ICP8 localization. ICP8 was extracted at lower salt concentrations from roscovitine-treated than untreated cells, but the affinity of ICP8 for ssDNA in vitro was not affected. I propose that roscovitine inhibits HSV-1 DNA replication by inhibiting DNA accessibility. I also discuss alternative mechanisms.

Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing. We show that ICP27 inhibits pre-mRNA splicing in vitro in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. In order to learn more about how splicing is organized in the cell nucleus in vivo, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.

L'encéphalite herpétique (EH), causée par l'herpès simplex virus-1 (HSV-1), peut résulter de défauts monogéniques de l'immunité médiée par TLR3. L'induction d'interférons (IFNs)-α/β ou -λ via TLR3 est cruciale à la protection après infection primaire avec HSV-1 dans le système nerveux central (SNC). Nous décrivons deux enfants avec l'EH portant différentes mutations hétérozygotes (D50A et G159A) dans TBK1, encodant TANK-Binding Kinase 1, une kinase aux carrefours de multiples voies de signalisation induisant des IFNs. Les deux allèles mutants de TBK1 sont perte-de-fonction par des mécanismes différents: instabilité de la protéine (D50A) ou perte d'activité kinase (G159A). Ces allèles sont associés à un trait autosomal dominant (AD) par des mécanismes différents: haplotype-insuffisance (D50A) ou dominance négative (G159A). Un défaut de réponses à poly(I:C) par TLR3 est observable dans les fibroblastes hétérozygotes pour G159A, et non pour D50A TBK1. Néanmoins, la réplication virale et la mortalité cellulaire après infection par deux virus dépendants de TLR3 (HSV-1 et VSV) étaient élevées dans les fibroblastes des deux patients. Ces phénotypes peuvent être sauvés par IFN-α2b. De plus, la production d'IFNs en réponse à des agonistes et virus indépendants de TLR3 est maintenue dans les PBMCs et fibroblastes des patients. Le phénotype cellulaire restreint, partiel représente ainsi le phénotype clinique de ces patients, limité à l'EH. Ces données identifient la déficience partielle AD de TBK1 comme une nouvelle étiologie génétique de l'EH de l'enfance, et indiquent que TBK1 est essentiel pour le contrôle de HSV-1 dans le SNC, médié par TLR3 et dépendant des IFNs