Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 98-104). Also available on the Internet.

The bovine papillomavirus type 1 E2 protein is a multifunctional early viral protein with roles in all phases of the cell cycle. E2 is required during G1 as a transcription factor, in S phase to initiate viral replication and during mitosis to tether the viral genome to dividing DNA. The viral genome contains 17 E2 binding sites, the majority of which are concentrated in the long control region (LCR), a regulatory region that is upstream of the viral coding sequence. The role of these binding sites has been explored in vitro using small plasmids and E1 and E2 proteins expressed in bacteria and insect cells. In this study we attempt to examine the placement of E2 on its binding sites during all phases of the cell cycle and in the context of a stably replicating viral system. As part of the examination of the role of E2 during mitosis, we have also examined the role of the cohesin protein Scc1 in viral tethering. Two groups have published disparate reports identifying the cellular protein that binds to the transactivation domain of E2 to stably maintain viral genomes during cell division. Our group has published that it is the DNA helicase ChlR1 that is required for viral tethering, while it has been reported that it is the bromodomain protein Brd4 that is responsible. In this study we contribute to a report that shows that the cellular protein Scc1 binds to the viral genome through a ChlR1 independent mechanism. The cohesin protein binds to BPV-1 E2 at intermittent stages of the cell cycle and may be a factor in viral genome tethering. This interaction may also be important for regulating viral transcription.

Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.

The replication cycle of polyomaviruses is, consistently with other viruses, fully dependent on host cells. Not only the cellular replicational and translational mechanisms are important for viruses, but also the virus infection is affected by other cellular proteins. This work is focused on the role of major cytoplasmic deacetylase, histone deacetylase 6 (HDAC6) in replication cycle of murine polyomavirus (MPyV). We showed that the presence of fully functional HDAC6 is essential for successful and productive infection. We found that HDAC6 affects not only early phase, but also late phase of infection. Cells with inhibited, or absent HDAC6 are infected with decreased effectivity and moreover lower amount of infectious viral particles is produced. On the other side, using cells with partially functional HDAC6, either in its deacetylase activity or in ubiquitin-binding activity, leads to increased ability of MPyV to infect those cells. Analysis of levels of early LT antigen and late structural protein VP1 in the infected cells showed, that viral proteins are affected by HDAC6. Our data suggest, that in the replication cycle of MPyV mainly the ubiquitin-binding domain of HDAC6 is required and the role of this domain in protein metabolism and degradation. In the second part of diploma project, we...

Le virus de la paralysie chronique de l'abeille (Chronic bee paralysis virus, CBPV) est l'agent étiologique d'une maladie infectieuse et contagieuse des abeilles adultes (Apis mellifera L.), appelée la paralysie chronique. Le CBPV est un virus à ARN simple brin positif qui contient 2 fragments d'ARN majoritaires. L'ARN 1 (3674 nt) et l'ARN 2 (2305 nt) codent respectivement 3 et 4 cadres ouverts de lecture (ORF). La séquence d'acides aminés de l'ORF 3 de l'ARN 1 partage des similitudes avec l'ARN polymérase ARN dépendante (RdRp) des virus des familles Nodaviridae et Tombusviridae. Par analogie avec ces familles virales, il a été suggéré que l'ARN 1 coderait les protéines non-structurales tandis que l'ARN 2 coderait les protéines structurales. Cependant, la réalité de ces protéines virales doit être démontrée expérimentalement afin d'étudier leurs fonctions, de mieux décrire ce virus et sa position taxonomique ainsi que d'améliorer les outils de diagnostic. Dans ce but, différentes approches expérimentales ont été utilisées. Une comparaison des protéomes d'hémolymphe d'abeilles non-infectées et infectées par le CBPV a été effectuée. Les protéines différentiellement exprimées ont été identifiées par empreinte peptidique massique (peptide mass fingerprint, PMF). Cette étude a permis d'identifier des protéines de l'abeille dont certaines contribueraient à une réponse immunitaire antivirale, mais aucune protéine virale n'a été identifiée par cette approche. Les ARN extraits du CBPV ont été utilisés dans des expériences de traduction in vitro. Malgré plusieurs essais réalisés en faisant varier les conditions expérimentales, cette approche s'est révélée infructueuse. / Chronic bee paralysis virus (CBPV) is the etiological agent that causes an infectious and contagious disease in adult bees (Apis mellifera L.), called chronic paralysis. CBPV is a positive single-stranded fragmented RNA virus which contains 2 major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode 3 and 4 putative open reading frames (ORFs), respectively. The amino acid sequence of ORF 3 on RNA 1 shares similarities with the RNA-dependent RNA polymerase (RdRp) of virus families Nodaviridae and Tombusviridae. By analogy with these viral families, it has been suggested that RNA 1 encodes non-structural proteins and RNA 2 encodes structural proteins. However, the reality of viral proteins needs to be experimentally demonstrated in order to study theirs functions, to describe CBPV biology and its taxonomic position and to improve diagnostic tools. With this aim, different experimental strategies have been used.A comparison of hemolymph proteomes between uninfected bees and bees infected with CBPV was performed. Differentially expressed proteins have been identified using peptide mass fingerprint method (PMF). This study allowed only identifying proteins of bees which could contribute to an antiviral immune response but viral proteins were not identified using this approach. Extracted CBPV RNAs were used for in vitro translation experiments. Despite several assays in varying experimental conditions, this approach has been unsuccessful. Another approach was to generate antibodies directed against different proteins or parts of viral proteins.

Wang Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 124-135). / Abstracts in English and Chinese. / Acknowledgements / Abstract / 摘要 / Table of contents / List of figures / List of tables / List of abbreviations / CHAPTER / Chapter 1. --- Introduction / Chapter 1.1 --- Background of SARS and epidemiology / Chapter 1.2 --- SARS symptoms and infected regions / Chapter 1.3 --- SARS virus / Chapter 1.4 --- Treatment for SARS at present / Chapter 1.5 --- Vaccine development is a more effective way to fight against SARS / Chapter 1.6 --- Vaccine candidates / Chapter 1.6.1 --- Truncated S protein as a vaccine candidate / Chapter 1.6.2 --- Full-length N protein as a vaccine candidate / Chapter 1.7 --- E.coli expression system / Chapter 1.8 --- Baculovirus expression system / Chapter 1.8.1 --- Characteristics of baculovirus / Chapter 1.8.2 --- Infection cycle of baculovirus / Chapter 1.8.3 --- Control of viral gene expression in virus-infected cells / Chapter 1.8.4 --- Merits of baculovirus expression system / Chapter 1.9 --- Aim of study / Chapter 2. --- "Bacterial expression and purification of rS1-1000(E), rS401-1000(E) and rN(E)" / Chapter 2.1 --- Introduction / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Reagents for bacterial culture / Chapter 2.2.2 --- Reagents for agarose gel electrophoresis / Chapter 2.2.3 --- 2'-deoxyribonucleoside 5'-triphosphate (dNTP) mix for polymerase chain reaction (PCR) / Chapter 2.2.4 --- Sonication buffer / Chapter 2.2.5 --- Reagents for immobilized metal affinity chromatography (IMAC) purification / Chapter 2.2.6 --- Reagents for gel filtration chromatography / Chapter 2.2.7 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) / Chapter 2.2.8 --- Reagents for Western blotting / Chapter 2.3 --- Methods / Chapter 2.3.1 --- General techniques in molecular cloning / Chapter 2.3.2 --- "PCR amplification of the S1-400,S401-1000" / Chapter 2.3.3 --- Construction of clone pET-S 1-400 and PET-s401-1000 / Chapter 2.3.4 --- Construction of clone pAC-N / Chapter 2.3.5 --- Expression / Chapter 2.3.6 --- Inclusion bodies preparation / Chapter 2.3.7 --- Inclusion bodies solubilization using urea / Chapter 2.3.8 --- Protein refolding by rapid dilution and dialysis / Chapter 2.3.9 --- Purification of recombinant protein by nickel ion chelating Sepharose fast flow column (IMAC) / Chapter 2.3.10 --- Gel filtration chromatography for further purification / Chapter 2.3.11 --- Bradford assay for the protein concentration analysis / Chapter 2.3.12 --- Protein analysis / Chapter 2.4 --- Results / Chapter 2.4.1 --- SDS-PAGE analysis of the expressed proteins / Chapter 2.4.2 --- Western blot analysis of the bacterial cell lysate / Chapter 2.4.3 --- Protein purification by IMAC / Chapter 2.4.4 --- Purification of rS401-1000(E) by gel filtration / Chapter 2.4.5 --- Determination of production yield of recombinant fusion proteins / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Expression vector selected for rS1-400(E) and rS401-1000(E) expression / Chapter 2.5.2 --- Protein expression in E.coli / Chapter 2.5.3 --- Purification process / Chapter 3. --- Baculovirus expression and purification of rS401-1000(ACN) and rN(BMN) protein / Chapter 3.1 --- Introduction / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Reagents for insect cell culture and virus work / Chapter 3.3 --- Methods / Chapter 3.3.1 --- "PCR amplification of N and cloning of S401-1000, N genes into the transfer vector pVL1393" / Chapter 3.3.2 --- Cloning of S401-1000 into transfer vector pFastBac HT B / Chapter 3.3.3 --- Virus works / Chapter 3.3.4 --- Identification of recombinant BmNPV or AcMNPV / Chapter 3.3.5 --- Manipulation of silkworm / Chapter 3.3.6 --- Mouse immunization for polyclonal antibody against rN(E) protein / Chapter 3.4 --- Results / Chapter 3.4.1 --- Expression of rN(BMN) in baculovirus / Chapter 3.4.2 --- Expression of rS401-1000(BMN) and rS401-1000(ACN) in baculovirus / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- The expression level of rN(BMN) in both in vitro and invivo / Chapter 3.5.2 --- The rS401-1000(ACN) protein expression level in vitro / Chapter 3.5.3 --- Failure in generating rS401-1000(BMN) / Chapter 3.5.4 --- Purification process of rN(BMN) by IMAC / Chapter 4. --- "Characterization of recombinant rS1-400(E), rN(E), rN(BMN), rS401_1000(E) and rS401-1000(ACN)" / Chapter 4.1 --- Introduction / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Reagents for enzyme-linked immunosorbent assay (ELISA) / Chapter 4.2.2 --- Reagents for purification of human IgG / Chapter 4.2.3 --- Source and identity of Immune sera / Chapter 4.3 --- Methods / Chapter 4.3.1 --- ELISA / Chapter 4.3.2 --- Purification process of human IgG / Chapter 4.4 --- Results / Chapter 4.4.1 --- Validation of Immune sera using SARS viral lysate / Chapter 4.4.2 --- Immunoreactivities of rS1-400(E) and rN(E) against pooled patients sera and normal human serum / Chapter 4.4.3 --- Immunoreactivity comparison of rN(E) and rN(BMN) / Chapter 4.4.4 --- Comparison of the immunoreactivities of rS401-1000(E) and rS401-1000(ACN) / Chapter 4.4.5 --- Immunoreactivity of SARS related proteins against Anti-SARS Antibody (Equine) / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Comparison of the immunoreactivities of SARS related proteins expressed in the present study / References

Plant viruses have been instrumental in our understanding of the biophysical properties pertaining to non-enveloped icosahedral virus particles. A substantial amount of research has been performed over five decades on Turnip yellow mosaic virus (TYMV), arguably one of the most extensively studied icosahedral plant viruses and the type-member of the Tymovirus plant virus genus. Even with a substantial body of published scientific literature, little is known about the role of specific coat protein (CP) residues in TYMV assembly, disassembly and disencapsidation. We have shown through our mutagenesis studies that the N-terminal region of the CP that is involved in the formation of an annulus structure and is disordered in A-subunit pentamers is not essential in vivo, but annulus-forming residues are critical in ensuring virion stability and low accessibility after virus is purified (Chapter 2). We have shown that a range of amino acid residue types is tolerated within the CP N-terminus in vivo, although they can greatly affect the stability of virions and empty particles, most notably at low pH (Chapter 3). Unlike full-length CP, N-terminal deletion and substitution mutants fail to reassemble into particles in vitro (Chapter 2, 3) suggesting a critical determinant for the N-terminus in reassembly (discussed Chapter 7). This is the first documented in vitro reassembly reported for a member of the Tymoviridae family and should provide a framework for further studies. We have identified a new way to create empty artificial top component (ATC)-particles through treatment with EDTA (Chapter 6) and we also show that tymoviruses can be engineered with altered pH-dependent enhanced stability (Chapter 4). In collaboration with the Qian Wang laboratory from the University of South Carolina we have shown that an RGD (Arg-Gly-Asp) motif can be genetically engineered within the CP of TYMV, resulting in infectious particles with attractive stem-cell adhesion properties (Chapter 5). With focus on basic viral mechanisms, we have crystallized the TYMV virion and ATC particle at pH 7.7 and collected data to less than 5 Å resolution (Chapter 4, supplementary). These structures represent the first tymovirus-based structures solved above pH 5.5 and will provide insight into the N-terminal conformations within the TYMV particle. Finally, we have characterized an N-terminal CP cleavage seen after ATC formation (Chapter 4) suggesting an additional and yet uncharacterized feature associated with decapsidation. / Graduation date: 2012

Ph.D. / Molecular Microbiology and Immunology / Both poxviruses and γ2-herpesviruses share the K3-family of viral immune evasion proteins. These proteins are characterized by an amino-terminal RING-CH domain followed by two transmembrane domains. We analyzed several human homologues of the K3-family termed membrane-associated RING-CH (MARCH) proteins. All MARCH proteins localized to subcellular membranes while several reduced surface levels of known K3-family substrates. Thus, MARCH proteins appear to be structurally and functionally homologous to viral K3 proteins. One of the major challenges in determining the function of this family is the identification of their physiological substrates. To overcome this we created a quantitative proteomics approach which can be used to identify novel substrates for both the K3- and MARCH-families. Using stable isotope labeling by amino acids in cell culture, we compared the proteome of plasma membrane, golgi, and endoplasmic reticulum membranes in the presence and absence of K5 and MARCH-VIII. Quantitative mass spectrometric protein identification from these fractions revealed that CD316 (bone marrow stromal antigen 2), CD166 (activated leukocyte cell adhesion molecule) and syntaxin-4 were consistently underrepresented in the plasma membrane of K5 expressing cells, while CD44, CD81 (TAPA-1) and B-cell receptor-associated protein 31kDa (Bap31) were consistently underrepresented in the plasma membrane of MARCH-VIII expressing cells. Furthermore, downregulation of each of these proteins was independently confirmed. Our results both identify and characterize a novel family of human ubiquitin ligase enzymes and elucidate a novel technique which can analyze this family and be easily adapted to the analysis of other cellular enzymes viral immune modulators.