Viruses are obligate parasites that use the host cellular machinery to produce progeny virions. The host responds to this invading pathogen by induction of the immune system; however, the virus employs a variety of strategies to overcome these attacks. The complexity of the virus-host interaction is of great interest to researchers with aims to both characterize the relationship and target steps of the viral life cycle to hinder infection. Many targeted tactics employ single protein analysis; however, approaches that examine the whole set of virus/host interactions are available. Transcriptional alterations within host cells have been determined for many virus- host interactions by micro-array techniques; however little is known about the effects on cellular proteins. This study uses a quantitative mass spectrometric-based method, SILAC, to study differences in a host cell's proteome with infection by a virus. Mammalian reoviruses and herpes simplex viruses are prototypical viruses commonly studied to determine virus life cycle and interactions with hosts. Using three strains of reoviruses and one HSV1 strain, cells were infected to identify differentially regulated proteins at different times. Thousands of proteins were identified for each virus type, some up or down regulated after infection. Biological functions and network analyses were performed using online networking tools. These pathway analyses indicated numerous processes including cell death and inflammatory response are affected by T1L reovirus infection. Comparing reovirus strains revealed a greater overall proteomic change in host function when infected with the more pathogenic T3DC strain. For the HSV infection, host proteins altered during the different immediate early, earlyand late phases of infection helped characterize the host-virus interaction parallel to the virus life cycle. Overall, my study has characterized proteomic changes in different virus infection systems, identifying numerous novel cellular functional pathways and specific proteins altered during virus infections, specifically the secretogranin II protein that had opposite types of regulation in reoviruses and HSV and was examined for its effects on virus replication. Further studies on the novel proteomic characteristics may provide greater understanding to the complex virus-host interactome, leading to possible antiviral targets. / October 2015

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There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.

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Herpes Simplex Virus Type 1 (HSV-1) establishes latent infections in neurons in vivo and lytic infections in epithelial cells and fibroblasts. During latent infections, HSV-1 transcription is restricted and the genomes are not replicated. Latent HSV-1 genomes are chromatinized, such that digestion with micrococcal nuclease (MCN) releases DNA fragments with sizes characteristic of nucleosomal DNA. During lytic infections, in contrast, all HSV-1 genes are expressed, the genomes are replicated, and their digestion produces primarily heterogeneously sized fragments. However, as evaluated by ChIP assays, HSV-1 DNA interacts with histones during lytic infections, although in most cases only a small percentage of HSV-1 DNA co-immunoprecipitates with histones (or is cleaved to nucleosome sizes following MCN digestion). Therefore, although current models propose that chromatin regulates HSV-1 transcription, it remains unclear how the association of histones with only a small percentage of HSV-1 DNA can globally regulate viral transcription. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. My objective was therefore to evaluate the biophysical properties of the HSV-1 DNA-containing complexes during lytic infection. Differing from pervious studies, however, I used classical chromatin purification techniques. I show that most HSV-1 DNA is in unstable nucleoprotein complexes and, consequently, more accessible to MCN than DNA in cellular chromatin. This HSV-1 DNA is protected from MCN redigestion only after crosslinking, similar to unstable cellular nucleosomes. HSV-1 DNA is in such complexes throughout lytic infection. Using unrelated small-molecule inhibitors, I further show that inhibition of HSV-1 transcription is associated with a decrease in MCN accessibility of HSV-1 DNA. Roscovitine, a cyclin-dependent kinase inhibitor, prevents activation but not elongation of IE, E, and L HSV-1 transcription. Consistent with a functional association between accessibility and transcription, roscovitine only decreases the accessibility of DNA templates of which it also inhibits transcription, independent of specific promoter sequences. In summary, I show that most HSV-1 DNA is in unstable nucleosome-like complexes during lytic infection and that accessibility to HSV-1 DNA likely plays a key role in regulating HSV-1 transcription.

The viral family Herpesviridae consists of large double stranded DNA viruses including eight species that infect humans with varying pathology from benign rashes to cancerous cell transformation. From three subfamilies, alpha-, beta- and gammaherpes, the alphaherpes contains the genera iltovirus, mardivirus, varicellovirus and simplex, two of which, the human simplex viruses I and 2 (HSV) induce life-long infections that have appeared to have coevolved with their hosts from the origins of our species. Unique features of the simplex genus are latency, tropism in dorsal root ganglia neurons, extraordinary high GC content ranging from 65 to 77%, and nucleosome formation of their genomes within the host's nucleus without integration. Reviewing the basic molecular and genetic characteristics of herpes simplex will introduced in Chapter 1, followed by the introduction of a newly sequenced, de novo assembled and predicatively annotated herpes simplex virus, Leporid Herpes Virus-4 (LHV4). Isolated from a virulent outbreak in domesticated rabbits, LHV4 has the smallest reported simplex virus to date at roughly 125,600 base pairs and presents similar pathology seen in rabbit models infected with HSV. Comparative genomics revealed a high degree of sequence similarity and genome synteny between LHV4 and other simplex viruses. Four genes were not computationally predicted in our annotation and may be absent in the LHV4 genome. The absent proteins correspond to: UL56, ICP34.5, US5 and US12 and have postulated roles in membrane trafficking, neurovirulence, apoptotic control and MHC I presentation respectively. The solved genome structure leads to how this compacted genome functions with the noted absences to produce a similar pathology in rabbits to that of HSV and whether other biological correlates will continue to be found in in vitro and in vivo infection. The inverted repeat regions (IR), duplicated and inverted to simplex virus' two larger blocks of protein-coding regions are described in Chapter 3. The similarities and differences in critical genes from the IR that balance latency and replicative viral cycles are compared. A two-fold reduction in IR content indicates the ability for a simplex virus to maintain infectivity despite this large truncation. The appendix describes the eukaryotic phylogeny of two initiating proteins of the mismatch repair (MMR) pathway. MMR proteins are present in the replicative foci of productive herpes virus infection and this analysis may indicate adaptive pressures involved in both genomic fidelity and host tropism. The emerging era of state-of-the-art genome sequencing and computational power advances this newly characterized herpes virus, along with its model host organism, as excellent candidates for systems interaction, and experimental biology. / Graduation date: 2012

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Bibliography: leaves 141-182. / xi, 182, [36] leaves, [12] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to analyse the molecular events associated with CD8+ T lymphocyte activity in HSV infected sensory ganglia. The role of CD8+ T cells in cytokine responses to ganglionic HSV infection is investigated, with particular reference to the Th1/Th2 paradigm and a known anti-viral mediator, IFN-[gamma]. A non-directed method of mRNA analysis is applied to HSV infected ganglia with the specific aim of identifying transcripts that may be associated with CD8+ T cell activity in the nervous system. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997

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?