The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.

Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

The interferon (IFN) system is integral to antiviral innate immunity in vertebrate hosts. Inside a cell, viral pathogen associated molecular patterns (PAMPs) trigger the IFN response, comprised of IFN induction and an IFN-induced antiviral state. However, viruses have evolved strategies to counteract the IFN system. The E3 protein of vaccinia virus (VV), encoded by the E3L gene, impedes cytokine expression and suppresses the activation and function of antiviral proteins. Deletion of the E3L gene (VVΔE3L) produces an IFN sensitive mutant virus that is replication defective in most human cell lines. Due to the limited human cell lines available to support VVΔE3L replication, the capacity of E3 inhibition of human IFN-induced antiviral activities is not well defined. In this study, VVΔE3L was generated and characterized to facilitate the study of other viral IFN antagonists at modulating human IFN-induced antiviral responses. A human liver carcinoma cell line, Huh7, was found to support VVΔE3L replication. A comprehensive analysis of VVΔE3L IFN sensitivity revealed E3 inhibits all human type I and type II IFN-induced antiviral activities by modulation of the protein kinase R (PKR) pathway. Influenza non-structural protein 1 (NS1) is well-known to mediate the suppression of IFN induction and IFN action in influenza virus infections. However, the IFN antagonizing potential of influenza NS1 may be virus subtype and/or isolate specific. VVΔE3L was next applied as an expression vector to study influenza NS1 function in modulating IFN-induced antiviral activities and IFN induction in human cells. Recombinant viruses were generated to express influenza NS1 (from avian H5N1 and pandemic viruses 1918 pH1N1, 1968 pH3N2, and 2009 pH1N1) in replacement of E3. It was found that influenza NS1 inhibits human IFN-induced antiviral activity in a subtype and isolate specific manner. Moreover, influenza NS1 differentially regulates human IFN expression in a virus isolate-dependent manner. Altogether, this work highlights the potential of VVΔE3L as an excellent virus model system to study viral proteins modulating IFN expression and IFN-induced antiviral activities in human cells.

Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 85-92). Also available on the World Wide Web.

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Pathology. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Würzburg, Univ., Diss., 2008.

Master of Science / Biochemistry and Molecular Biophysics / Zhilong Yang / Poxvirus infections continue to threaten human health despite the eradication of smallpox, which was one of the most lethal infectious diseases in human history. Our objectives were to identify the host cell components/functions that are important for poxvirus infection and to gain insights into the molecular mechanism of poxvirus replication, ultimately guiding novel anti-viral development. Using vaccinia virus, the prototype poxvirus, we screened inhibitors of viral replication from over 3,000 chemical compounds, most of which have known cellular targets. This screening revealed numerous JAK/STAT3 inhibitors that could inhibit the replication of vaccinia virus. We further used multiple inhibitors of the JAK/STAT3 pathway and tested their effects on the replication of vaccinia virus in multiple primary and transformed cells through reporter assay and viral infectious particles measurement. The JAK/STAT3 inhibitors being tested were: SC144, an inhibitor of the interleukin 6(IL-6), a receptor of the JAK/STAT3 signaling pathway, AZ960 (a JAK2 inhibitor), Stattic and niclosamide (inhibitors of STAT3). Overall, our data indicate the JAK/STAT3 inhibitors could repressed vaccinia virus replication in multiple cell types, suggesting that the JAK/STAT3 signaling pathway is required for the efficient replication of vaccinia virus. Moreover, we observed that STAT3 was enriched in the cell nucleus, although the phosphorylation level of STAT3 was downregulated in vaccinia virus-infected cells during the early stages of infection. This study demonstrates an important role of the JAK/STAT3 signaling pathway in the replication of vaccinia virus, providing a possible novel direction by which to intervene in poxvirus infection and related diseases.

Submitted by MARINA GEA PERES null (marinageavet@yahoo.com.br) on 2016-08-27T20:12:24Z No. of bitstreams: 1 TESE MARINA 2016 COMPLETA.pdf: 2431076 bytes, checksum: b3001086cd643632e3ea43f4cd826018 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-30T16:35:33Z (GMT) No. of bitstreams: 1 peres_mg_dr_bot.pdf: 2431076 bytes, checksum: b3001086cd643632e3ea43f4cd826018 (MD5) / Made available in DSpace on 2016-08-30T16:35:33Z (GMT). No. of bitstreams: 1 peres_mg_dr_bot.pdf: 2431076 bytes, checksum: b3001086cd643632e3ea43f4cd826018 (MD5) Previous issue date: 2016-08-01 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo teve por objetivo verificar positividade ao Vaccinia virus em amostras de animais domésticos, selvagens e humanos provenientes de áreas com e sem histórico de surto, na região centro oeste do estado de São Paulo. Foram amostradas aleatoriamente 47 propriedades produtoras de leite, sendo 10 em Torre de Pedra (que já registrou dois surtos prévios ao presente estudo), 15 em Bofete e 22 em Anhembi (com histórico de surtos desconhecido). Avaliou-se a presença de DNA viral em amostras de sangue coletadas de bovinos, outros mamíferos domésticos e selvagens, bem como de humanos e de DNA viral em amostras de fezes e urina de pequenos roedores silvestres capturados nas áreas de mata das 47 propriedades analisadas. O DNA viral foi detectado em amostras de sangue de quatro vacas, um cavalo, três gambas (um Didelphis albiventris e dois Didelphis aurita), bem como em amostras de fezes e urina de pequenos roedores silvestres, sendo seis amostras positivas para DNA viral nas fezes (dois Oligoryzomys flavescens, dois Oligoryzomys nigripes e dois Sooretamys angouya), e uma para DNA viral na urina (Oligoryzomys flavescens). A análise das sequencias do gene vgf mostras nossas amostras agrupadas com amostras de Vaccinia vírus Brasileiras e vacinais, nos permitindo classificar o vírus detectado nas amostras de sangue, fezes e urina, como Vaccinia vírus. Relata-se pela primeira vez a detecção de DNA viral em amostras de sangue, fezes e urina de animais sadios, naturalmente infectados, e sugere-se que o Vaccinia vírus circula de forma subclínica na região amostrada. Adicionalmente sugere-se que roedores silvestres naturalmente infectados e aparentemente saudáveis são potenciais fontes de infecção evidenciada por eliminação de Vaccinia virus em amostras de fezes e urina. / The present study aimed to verify positivity for Vaccinia virus in samples from domestic and wild animals, and humans from areas with and without outbreaks history, in the central west region of São Paulo State. Was randomly sampled 47 milking farms: 10 in Torre de Pedra (which registered two previous outbreak), 15 in Bofete and 22 in Anhembi (both without histories of outbreak). We assessed the presence of viral DNA in blood samples collected from cows, other domestic and wild mammals, as well as from humans and in feces and urine sample from wild rodents captured in forest areas in the 47 sampled farms. The viral DNA was detected in blood samples of four cows, one horse, three opossums (one Didelphis albiventris and two Didelphis aurita), as well as in feces and urine samples of wild rodents: six samples PCR-positive for viral DNA in feces (two Oligoryzomys flavescens, two Oligoryzomys nigripes and two Sooretamys angouya), and one for viral DNA in urine (Oligoryzomys flavescens). The sequences analysis of vgf gene show our samples clustered with Brazilian and vaccine Vaccinia virus, which allowed us to classify our samples as Vaccinia virus. Reports for the first time the detection of viral DNA in blood, feces and urine samples of healthy animals, naturally infected, and suggests that Vaccinia virus is subclinical circulating in the region sampled. Additionally suggests that wild rodents naturally infected and apparently healthy are potential infection source evidenced by elimination of Vaccinia virus in feces and urine samples. / FAPESP: 2013/07693-1