M.P.H., Faculty of Health Sciences, University of the Witwatersrand, 2009 / Background: Herpes Simplex Virus type 2 (HSV-2) is a common cause of genital ulcers worldwide and has emerged as a co-factor in human immunodeficiency virus (HIV) acquisition and transmission. A study was conducted to determine the prevalence of HSV-2, its correlates, the accuracy of reported history of genital ulcer disease (GUD) to predict HSV-2 infection and the extent of symptom recognition in a clinic population in Johannesburg. Methods: 210 women aged 18 years or older were interviewed and socio-demographic, sexual behaviour and clinical information collected. Serological testing for HSV-2 and HIV infections was performed, but only where sera were available for the latter. Factors associations with HSV-2 infection were assessed using logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI). The sensitivity, specificity, predictive values and likelihood ratios of a history of GUD were calculated. Results: The estimated sero prevalence of HSV-2 was 73% (95% CI 67% - 79%). Few participants, 13/206 (6%) participants had knowledge of genital herpes. Only 9/203 (4%) participants recognised lesions of genital herpes following education and counselling about HSV-2 infection. HSV-2 infection was associated with older age(>25 years of age) OR 2.6 (95% CI 1.4-5.0), spending more than 2 nights away from home, OR 6.0 (95% CI 1.0-62.7), having more than 2 sexual lifetime partners, OR 2.2 (95% CI 1.1-3.9), a history of an STI in the past 3 months ,OR 3.6 (95% CI 1.2-9.5) and HIV infection, OR3.3( 95%CI 1.4-7.9). A history of genital ulceration performed poorly as a predictor of HSV-2 seropositivity; the sensitivity was 7% and specificity was 96%. Conclusion: HSV-2 prevalence was high and few participants were aware of their infection. HVS-2 infection was associated with risky sexual behaviour .A history of genital ulcer disease was not sufficient as a diagnostic tool for HSV-2 infection. Public health interventions should focus on behavioural modification and increasing awareness of genital herpes. HSV-2 management should be incorporated into HIV care and STI protocols.

Finally, NF-kappaB and another transcriptional regulator p53 are usually tightly related in their control of cell survival. Opposite to NF-kappaB, p53 mediates cell death signals, usually activated under DNA damage and subsequently involved in cell growth control, DNA damage repair or apoptosis. It was found in this study, DNA damage and cell cycle arrest responses tended to participate with the anti-HSV-1 activity. Although in HEp-2 cells, TCS induced more DNA damage ratio and S and G2/M phase arrest proportion than HSV-1 infected cells, but more p53 was expressed and activated by phosphorylating at Ser 15 by TCS in HSV-1 infected HEp-2 cells than uninfected ones. In the same time the activation of BAX, which promotes the apoptotic function of p53, increased during TCS treatment when infected with HSV-1, the p53 therefore regulates apoptosis in HSV-1 infected cell during TCS treatment. / Firstly, we demonstrated that TCS reduced HSV-1 antigen and DNA content, The IC50 (half maximal inhibitory concentration) of TCS on HSV-1 replication was 2.5 +/- 0.23 mug/ml. The anti-HSV-1 effect of TCS was related to interfering with viral replication during 3 to 15 hours after infection which coincide with early to late viral replication period. TCS had no effect on HSV-1 attachment, penetration or immediate early gene expression. However, the expression of early gene, late gene and virion release were diminished. / Fourthly, the role of the nuclear factor-kappaB (NF-kappaB) in the anti-HSV-1 effect of TCS was explored. NF-kappaB initiates cell survival pathways. It is widely involved in viral infection and replication to make sure virus overcomes the host cells immune response. We found HSV-1 enhanced the activity of NF-kappaB in HEp-2 cells by triggering its translocation from cytoplasm to nuclear. However, during the anti-HSV-1 effect of TCS, TCS suppressed HSV-1-aroused NF-kappaB translocation in HEp-2 cells, the inhibition of NF-kappaB activity in HSV-1-infected cells by TCS treatment tend to abolish the anti-apoptosis effect developed by HSV-1, so that the host cells suffered more extracellular stress and showed more apoptosis ratio than uninfected ones. / Taken together, this study demonstrated TCS interfered with HSV-1 early to late infection period. TCS selectively induced more HSV-1 infected HEp-2 cells to apoptosis than uninfected ones, the selectivity of TCS was due to apoptotic signaling pathway switching from CD95 (Fas/Apo-1)-mediated type I to type II apoptotic pathway. Furthermore, during TCS induced-apoptosis in HSV-1 infected cells, TCS suppressed NF-kappaB activation that triggered by HSV-1 infection. At the meanwhile, p53 participated in the TCS-induced apoptosis regulation in infected cell. / TCS is toxic to cell because its RIP activity, killing of the viral host cells certainly inhibits virus expansion, only when it kills more infected cells, the material could be considered as an anti-viral agent. It was found TCS induced losing of cell viability and enhancing in apoptosis in HEp-2 cells and HSV-1 infected HEp-2 cells. The decrease of cell viability and increase of apoptosis ratio were enhanced when HEp-2 cells were infected with HSV-1 compared with uninfected ones. The 50% of effect concentration (EC50 ) in cytotoxicity and apoptosis were decreased from 24.64 +/- 1.17 mug/ml and 37.57 +/- 1.47 mug/ml in uninfected HEp-2 cells to 3.01 +/- 1.30 mug/ml and 3.89 +/- 1.31 mug/ml in HSV-1 infected HEp-2 cells respectively. / The reason of type I to type II apoptosis pathway transition might due to the activity change of death receptor on HEp-2 cells. The type I apoptotic pathway induced by TCS was related to CD95 (Fas/Apo-1) system activation and signaling pathway. When HEp-2 cells were infected with HSV-1, the CD95 (Fas/Apo-1) was suppressed by HSV-1 infection. As a result, TCS triggered a less CD95 (Fas/Apo-1) dependent type II apoptotic pathway in the infected cells. / Thirdly, TCS activated different apoptotic pathways, namely type I and type II apoptotic pathways, between uninfected and infected cells. The type I apoptotic pathway bypasses the dependence on the mitochondrial but quickly activates a large amount of caspase-8 at the CD95 (Fas/Apo-1) formed death inducing signaling complex (DISC), which amplifies the signal. By contrast, the formation of the DISC in the type II apoptotic pathway is strongly reduced. It depends on loss of the mitochondrial transmembrane potential (DeltaPsi m) and release of cytochrome c and capase-9 activation to mediate apoptosis signal transduction. We found in HSV-1 infected an uninfected HEp-2 cells, TCS induced the loss of DeltaPsim, this DeltaPsim losing was increased when HEp-2 cells were infected with HSV-1. Furthermore, when there were no HSV-1 infection, TCS induced caspase-dependent type I apoptosis pathway that quickly activated large amount of caspase-8 after TCS treatment. However, when infected with HSV-1, this pathway turned into mitochondrial dependent type II pathway involving caspase-9 response, whose apoptosis ratio was diminished by over expressed Bcl-2, which is a hallmark defining type I or type II apoptosis. / Trichosanthin (TCS) is a type I ribosome inactivating protein (RIP), it was found to inhibit human simplex virus type 1 (HSV-I) but the anti-HSV-I mechanism is unclear. HSV-1 is a widely distributed DNA virus, it causes large range of human diseases. During the lytic life cycle of HSV-1, highly regulated cascade of genes are expressed to interfere with host cell metabolism and immune response. In this context the anti-HSV-1 mechanism of TCS in human epithelial carcinoma HEp-2 cells was studied. / He, Dongxu. / Advisers: Wing Ho Yung; Siu Cheung Tam. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-05T17:52:22Z No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-06-05T17:54:37Z (GMT) No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) / Made available in DSpace on 2017-06-05T17:54:37Z (GMT). No. of bitstreams: 1 LianaCostaPereiraDissertacao2014.pdf: 1788731 bytes, checksum: aaf1eef40e98ed7a21c08304f7dce33b (MD5) Previous issue date: 2014-05-29 / Viral infections affect every living organism. Health care programs and vaccines have been developed to control and prevent those infections, but there is still the occurrence of severe diseases and with great social-economic importance, whose only alternative is the treatment with antivirals drugs. Those compounds possess different mechanisms of action and are specific for each virus. Nevertheless, with many viruses resistant strains rise, a growing interest in novel antivirals development or in the improvement of existing drugs have been observed. In the last few years, antimicrobial peptides have shown antiviral activities making them candidates for antiviral drugs. The present study aimed to evaluate the possible antiviral activities of synthetic peptides clavanin MO, LL-37, Cn-AMP1 and variants of the Pa-MAP1 against HSV-1 and the Aichivirus replication. The peptides cytotoxic effects against Vero cells were evaluated by MTT method, whereas the clavanin MO, the Pa-MAP 1.8 and Pa-MAP 18br showed more cytotoxicity, with concentrations higher than 15,4 ??M and 12 ??M, respectively. Considering the antivirals assays, it was observed that the peptide Pa-MAP 1 causes 90 % of HSV-1 replication, with a SI higher than 4.8 and a virucidal mechanism of action, but null against the Aichivirus. Futhermore LL- 37 presented 90 % of Aichivirus inhibition, with a SI of 3.4. Others peptides tested showed percentages below 41 %. Future studies are need in order to elucidate which HSV-1 structure does Pa-MAP1 interacts and futher extend to in vivo tests. In summary, this study it is the first one to describe antiviral tests for the treatment of the Aichivirus infection. / As infec????es virais acometem todos os organismos vivos. Para o controle e preven????o dessas infec????es, programas de sa??de p??blica e vacinas t??m sido desenvolvidos, mas ainda h?? a ocorr??ncia de doen??as graves e de grande impacto socioecon??mico cuja ??nica alternativa ?? o tratamento com antivirais. Estes possuem diferentes mecanismos de a????o e s??o espec??ficos para cada tipo viral. Entretanto, o surgimento de variantes resistentes de muitos tipos virais tem levado h?? um crescimento no interesse de desenvolver novos antivirais ou at?? mesmo melhorar os existentes. Nos ??ltimos anos, estudos sobre pept??deos antimicrobianos relatam a atividade antiviral desses compostos, tornando-os candidatos a medicamentos antivirais. O presente estudo teve como objetivo avaliar a poss??vel atividade antiviral dos pept??deos sint??ticos clavanina MO, LL-37, Cn-AMP1 e variantes da Pa-MAP contra o Herpes v??rus humano 1 e Aichiv??rus. O efeito citot??xico dos pept??deos em c??lulas Vero foi avaliado pelo m??todo do MTT, sendo que a clavanina MO, a Pa- MAP 1.8 e Pa-MAP 18br demonstraram maior citotoxicidade em concentra????es maiores que 15,4 ??M, e 12 ??M, respectivamente. Considerando os ensaios antivirais, observou-se que o pept??deo Pa-MAP1 apresentou 90 % de inibi????o da replica????o do HHV-1, com um IS maior que 4,8, sendo que seu mecanismo de a????o foi definido como virucida, por??m contra o Aichiv??rus o PI foi nulo. Al??m disso, LL-37 apresentou 90 % de inibi????o do Aichiv??rus com um IS de 3,4. Os outros pept??deos testados apresentaram percentuais abaixo de 41 % contra ambos os v??rus. Futuros estudos moleculares s??o necess??rios para se determinar com qual estrutura do HHV-1 a Pa-MAP1 interage e para estender os testes para modelos in vivo. Este estudo ?? o primeiro a relatar testes antivirais para o tratamento do Aichiv??rus.

A atividade antiviral de compostos flavonoídicos tem sido amplamente investigada nos últimos anos. Nesse contexto, a primeira etapa da presente tese teve por objetivo avaliar a atividade anti-herpética in vitro dos principais isoflavonóides de Glycine max (soja): genisteína, daidzeína, gliciteína e cumestrol. Dentre os isoflavonóides investigados, a genisteína e o cumestrol mostraram uma interessante atividade frente aos vírus HSV-1 (cepa KOS e 29R, sensível e resistente ao aciclovir, respectivamente) e HSV-2 (cepa 333), interferindo em diferentes etapas do ciclo de replicação dos vírus. Devido à reduzida hidrossolubilidade desses isoflavonóides, foi proposta a sua incorporação em nanoemulsões de uso tópico. As formulações foram otimizadas através de um experimento fatorial completo do tipo 23 . O efeito dos fatores tipo de óleo (óleo de rícino ou miristato de isopropila), co-tensoativo iônico (oleilamina ou ácido olêico) e fosfolipídeo (DSPC ou DOPC) sobre as propriedades das nanoemulsões e a retenção da genisteína na pele de orelha suína foi investigado. Nanoemulsões compostas de miristato de isopropila/ oleilamina/DOPC apresentaram um menor diâmetro de gotícula e uma maior retenção de genisteína na pele de orelha suína enquanto que a combinação miristato de isopropila/oleilamina/DSPC mostrou o menor índice de polidispersão. A viscosidade das formulações selecionadas foi ajustada através do uso de hidroxietilcelulose visando à obtenção de produtos de uso tópico, obtendo-se hidrogéis de comportamento pseudoplástico. Na sequência, um conjunto de resultados obtidos demonstrou que a composição das formulações (especialmente o fosfolipídeo DOPC e o agente espessante hidroxietil celulose) pode influenciar a liberação e a retenção dos isoflavonóides em mucosa esofágica suína. A integridade da mucosa também desempenha um papel no aumento da permeação/retenção do cumestrol, conforme ilustrado nas imagens de microscopia confocal, utilizando vermelho do Nilo como marcador fluorescente. De maneira geral, a incorporação dos isoflavonóides nas nanoemulsões aumenta a atividade anti-herpética desses compostos in vitro. O conjunto dos resultados demonstra que as formulações desenvolvidas são potenciais carreadores de uso tópico para genisteína e cumestrol no tratamento das infecções herpéticas. / The antiviral activity of flavonoid compounds has been extensively investigated in recent years. In this context, the first step of this study was to evaluate the antiherpes activity in vitro of the main isoflavonoids of Glycine max (soybean): genistein, daidzein, glycitein and coumestrol. Among the investigated isoflavonoids, genistein and coumestrol showed interesting activity against HSV-1 (KOS and 29R strains, which are acyclovir-sensitive and acyclovir–resistant strains, respectively) and HSV-2 (333 strain), interfering at different stages of the virus replication cycle. Due to the low hydrosolubility of these isoflavonoids, their incorporation into topical nanoemulsions was proposed in this study. The formulations were optimized through a 23 full factorial design. The factors effect of oil type (castor oil or isopropyl myristate), ionic co-surfactant (oleylamine or oleic acid) and phospholipid type (DSPC or DOPC) on physicochemical properties and genistein retention into porcine ear skin was investigated. Nanoemulsions composed of isopropyl myristate/ DOPC/oleylamine showed the smaller particle size and higher genistein retention into skin, whereas the formulation composed of isopropyl myristate/DSPC/oleylamine exhibited the lower polydispersity index. The viscosity of selected formulations was adjusted with hydroxyethyl cellulose to obtain topical products, which showed nonNewtonian behavior. In sequence, a set of results showed that formulations composition (especially the phospholipid DOPC and the thickening agent hydroxyethyl cellulose) could influence the release and retention of isoflavonoids in porcine esophagus mucosa. The integrity of mucosa plays a role on the increase of coumestrol permeation/retention, according to confocal microscopy images, using Red Nile as fluorescent label. In general terms, the incorporation of isoflavonoids into nanoemulsions increases the antiherpes activity of these compounds in vitro. The overall results show that the formulations developed in this study are potential topical carriers for genistein and coumestrol in the treatment of herpes infections.

Publicación a texto completo no autorizada por el autor / Desarrolla y caracteriza films poliméricos cargados de aciclovir (ACI) para el tratamiento tópico del herpes simple. Complejos interpolielectrolito - fármaco (CIPEF) basados en chitosan (Ch), ácido hialurónico (AH) y aciclovir (ACI) fueron desarrollados mediante el método de coacervación en medio acuoso empleando una cantidad de ACI y AH suficientes para neutralizar el 50 y 10% de los equivalentes de grupos amino ionizables del chitosan, respectivamente; obteniéndose un complejo de Ch - ACI50 - AH10. Adicionalmente, los films fueron obtenidos utilizando el método de casting, mediante la adición de glicerina al 2% p/v y caracterizados posteriormente. El diámetro hidrodinámico (Dh) y el potencial zeta (PZ) de las dispersiones fueron medidos mediante espectroscopía de correlación de fotones (DLS). Complejos con y sin fármaco exhibieron tamaños de 834,3 ± 59 nm y 662,3 ± 29 nm respectivamente. El potencial zeta de los CIPEF presentaron valores de 59,6 ± 0,79 mV. El desplazamiento iónico, mediante la adición de NaCl 0,9%, mostró un ligero cambio en el pH debido a la liberación del grupo amino por sustitución con sodio indicando interacción iónica. Por el contrario, el agregado de glucosa al 5 % no produjo cambios. La calorimetría exploratoria diferencial (DSC) reveló ausencia de pico de fusión a 250 °C del ACI evidenciando ausencia de fármaco libre. El análisis termogravimétrico (TGA) mostró que los polielectrolitos (PE), ACI y los complejos son estables a altas temperaturas (~200°C) indicando estabilidad térmica en comparación con la mezcla física. La difracción de rayos X (Dx) y microscopía óptica de los CIPEF revelaron que los complejos no presentan picos en el difractograma, evidenciándose ausencia de fármaco (F) libre. Los films mostraron coeficiente de variación (Cv)<5%, evidenciando un proceso de secado homogéneo y una distribución uniforme del fármaco en el film, respectivamente. In vitro, films presentaron una liberación controlada de ACI. En conclusión, los resultados mostraron que los films obtenidos presentan buenas propiedades y podrían constituirse en alternativas prometedoras para el tratamiento tópico del herpes. / Tesis

The nuclear lamina consists of a mesh-like network of lamin proteins anchored to the inner nuclear membrane by interactions with integral membrane proteins such as emerin. Emerin binding to lamin A/C is one of the interactions that connect the inner nuclear membrane to the lamina. Infection by herpesviruses results in changes in the organization of the nuclear lamina, perhaps in order to facilitate envelopment of capsids at the inner nuclear membrane. In HSV-1 infected cells, alterations to the lamin proteins have been shown to involve pUL34, pUL31, and pUS3 proteins, which are also required for normal nuclear envelopment. We tested hypotheses about the mechanism and significance of lamina disruption. This thesis presents the following data. Infection of multiple cell types induced emerin hyperphosphorylation that was dependent on the presence of pUL34 and kinase active pUS3 proteins. The pUL34-dependent component was also sensitive to Rottlerin treatment suggesting that cellular kinases sensitive to Rottlerin were involved in emerin modification. LAP2 (another lamin associated protein) was de-modified (perhaps de-phosphorylated) in a pUS3 and pUL34 independent manner. Emerin was not required for growth of HSV-1. Hyperphosphorylation of emerin was required for its disassociation from the lamina. PKC family members have been implicated in the disruption of the nuclear lamina during herpesvirus nuclear egress. To test the hypothesis that PKC activity was required for viral replication, PKC activity was blocked with PKC inhibitors and dominate negative PKC constructs. Chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta did not inhibit viral growth. In addition to lamin associated proteins, lamin localization is also disrupted during herpesvirus infections. Emerin and lamin A/C are binding partners and the localization of both proteins is disrupted by pUS3 and cellular kinase mediated phosphorylation. To test the hypothesis that HSV-1-induced lamin A/C disruption is mediated via a mechanism similar to emerin's, pUS3 and Rottlerin Sensitive Kinases were inhibited and lamin A/C localization was observed. Unlike emerin, HSV-1-induced disruption of lamin A/C was not altered by Rottlerin Sensitive Kinase inhibition suggesting that HSV has multiple mechanisms for disruption of the lamina. Phosphorylation of lamina components, by Rottlerin Sensitive Kinases, may be a required event prior to primary envelopment. To test this hypothesis, growth of HSV-1 was tested in Rottlerin treated infected cells. Although the inhibitor Rottlerin, did reduce viral growth, it was also was also associated with severe depression of viral late-gene expression. TEM analysis suggested that Rottlerin Sensitive Kinases(s) were required for: (i) nuclear egress and (ii) capsid accumulation or formation supporting the hypothesis that the capsids were made in the presence of Rottlerin were unable to leave the nucleus. pUS3 is a multi-functional protein in alpha-herpesviruses. It has been implicated in lamina disruption, protecting the infected cell from apoptosis, and de-envelopment at the outer nuclear membrane. In BT-549 cells, a breast cancer cell line with low PKC delta expression, the hypothesis was tested that in the absence of cellular lamina disrupting kinases, an US3-null virus would be blocked at the lamina disruption step. In BT-549 cells, the US3-null (vRR1202) virus was 10-fold decreased above the typical 10-fold decrease, compared to WT virus, to produce a 100-fold decrease in infectious PFU yet apoptosis was not increased. Lamin A/C disruption occurred via similar mechanism in both breast cancer cell lines: BT-549 and MCF-7. Interestingly, in the BT-549 cells, emerin was extensively hyperphosphorylated in an US3-null infection, yet was not redistributed along the NE. These data support a model that one or more specific residue(s) must be phosphorylated for emerin disconnection from lamina.

The human c-myc gene is a cellular proto-oncogene composed of three exons and two introns. Transcription of c-myc is controlled by two promoters, Pl and P2. The activity of these promoters is regulated by many factors, such as cellular transcription factors E2F, YYl, and HSV-1 immediate-early proteins, ICPO, ICP4. Many regulatory elements located both upstream of and between P 1 and P2 have been identified, and some of these are required for optimum expression of c-myc. In this thesis research, a region downstream from P2 in the c-myc exon 1 was identified by its response to transactivation by HSV-1 immediate-early proteins, ICPO and ICP4. The purpose of this research was to examine this region for regulatory sites that respond to HSV-1 infection. I hypothesized that after HSV-1 infection, ICPO and ICP4 activate c-myc expression, in part, through regulatory sequences present in exon 1. To test for this hypothesis, reporter plasmids containing (I) the c-myc promoter (from - 101 bp relative to Pl) and exon 1 coupled to the bacterial CAT gene were constructed. (ii) The c-myc exon sequences used were either intact (wild-type) or they were constructed with various deletions. The activities of these plasmids were examined in transient expression assays. To analyze protein binding, electrophoretic mobility shift assay (EMSA) and completion EMSAs were carried out. The results from these experiments lead to the following conclusions: (i) ICP4 and ICPO serve as activators, whereas ICP27 inhibits c-myc gene expression. (ii) The region from +332 to +513 within the c-myc exon 1 contains an important element required for transactivation of the c-myc gene by HSV-1 proteins. (iii) Cellular proteins, including factor YYl, bind to the region from +332 to +513 in the c-myc exon 1. Although the exact mechanism by which HSV-1 immediate-early proteins regulate cmyc gene expression is still not clear, it gives rise to a possibility that this regulation is caused by turning on or activation of the cellular regulatory proteins by ICP4 and ICPO. The cellular proteins in turn activate the c-myc gene expression by interacting with the ciselement downstream from P2.

A regulatory element involved in the transcriptional activation of the major capsid protein (VP5) of herpes simplex virus type 1 was identified and characterized in this research project. Gel mobility shift assay with nuclear extracts from both uninfected and HSV-1 infected HeLa cells identified two major protein-DNA complexes involving the VP5 promoter. No viral specific complex found. DNase I and orthophenanthroline-cu+ footprint analyses in the same laboratory revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, mutated VP5 promoters with deletion and insertion around LBS were constructed and linked to a reporter gene, bacterial chloramphenicol acetyltransferase gene. The effect of mutations were tested in transient expression assay. Deletion of LBS resulted in seven to eight-fold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV immediate-early genes. These results indicated LBS is involved in the maximal transactivation of the VPS gene. A search of published gene sequences found the homologs of LBS exist in a number of HSV-1 By promoters, and other viral promoters, as well as cellar promoters. Some of these homologs have found involved in the transcription regulation.

The herpes simplex virus 1 (HSVl) VP5 gene codes for the major viral capsid protein. Understanding of the mechanism of how the VP5 gene is regulated in host cells will help us to understand the molecular action of the HSV 1 life cycle and its interplay with the host cell gene expression machinery (transcription and translation). This may ultimately provide scientific bases for both better prevention and cure of HSV 1 caused diseases. Previous work from Dr. Robert L. Millette' s laboratory has indicated that a 164 base pair region of the VP5 promoter gene could activate the transcription of an attached reporter gene (bacteria CAT gene). Furthermore, a 12 bp (GGCCATCTTGAA) cis-acting element situated within the 164 bp promoter region was required for the promoter activity. To understand the function of this cis-element in the regulation of VP5 transcription and to identify the trans-acting factors interacting with this element, gel mobility shift assays were first carried out using the fragment containing the 12 bp site as the probe. A cellular factor, YY 1, was found to bind to this site in a sequence specific manner. Based on the oligonucleotide competition assays, partial protease digestions, and antibody supershift assays, it became clear that two cellular factors bound to the VP5 promoter. These were related, if not identical, to the previously identified Yin-Yang- 1 factor (YY 1), and transcription factor the SPl. Site-directed mutagenesis studies indicated that these two factors bind to distinct sites on the 164 bp fragment. Point mutations studies on the 12 bp YYl binding site demonstrated that seven of the 12 bp were required for YY 1-DNA complex formation and the first four bp in the 12 bp were very important for VP5 gene regulation. Also, it was found that YY 1 performs both positive and negative regulator function in VP5 gene regulation. In conclusion, two cellular transcription factors, YY 1 and SPl, play a major role in VP5 gene expression.