Mammals are constantly challenged by numerous pathogens that pose a threat to their health. Upon infection, retroviruses quickly integrate their genome into that of their host thereby permanently modifying it. Protein members of the APOBEC3 (A3) family exhibit cytidine deaminase activity that specifically acts on single-stranded DNA to deaminate deoxycytidine bases into deoxyuridines. This process is potentially mutagenic because uracil directs the insertion of adenine on the opposite DNA strand. High levels of mutations induced by A3 proteins in the retroviral genome ultimately inactivate progeny viruses. However, under conditions where low levels of A3 proteins are present, sub-lethal mutagenesis can occur and is generally believed to be beneficial for the virus. Powerful and affordable techniques designed to detect rare deamination events generated by these deaminases along the full length of retroviral genomes are therefore essential. Through the course of my studies, I developed such a new tool that I called HyperHRM which was instrumental to my project’s success. In addition to the antiretroviral affects of their catalytic activity, some members of the A3 family have the ability to hinder reverse transcription independently of their enzymatic properties. Yet, the details underlying the deamination-independent restriction by the proteins remain unclear. Through my work, I have advanced our current understanding of this elusive process by defining the essential role for RNA-binding in the inhibition of the early steps of infection by APOBEC3G (A3G). I also demonstrate that the ability to bind RNA is important for the selection of DNA dinucleotides targeted for deamination by A3 enzymes. Based on the premise that the DNA context for deamination may alter viral fitness in various ways, I then investigated the gene inactivation potency of different A3 based on their preferred DNA substrate. My experiments showed that mutations introduced in a 5'CC context by A3G are much more lethal for the virus because of the high frequency of termination codons that are generated. I therefore clearly established that deamination target specificity has a strong influence on the overall restriction potency of A3 proteins and demonstrated that such specificity was linked to the ability of A3 proteins to bind RNA. Finally, in addition to retroviruses, mobile elements such as retrotransposons can also lead to genomic instability if not properly controlled. The A3 protein family has been shown to play a crucial role in the restriction of these elements through a mechanism that is not believed to require the enzymatic activity of the proteins, although the details of the restriction mechanism are not yet understood. Here, I provide molecular insights on the potential mechanism of retrotransposon restriction by showing that the RNA-binding properties of the enzymes are not involved in the restriction of L1 retrotransposition. A complete elucidation of the modes of restriction employed by the A3 could lead to the development of a new generation of antiretroviral drugs. Overall, my research has led to the design of a new research tool to detect and quantify A3-induced mutations in retroviruses, but more importantly, it has enabled a better understanding of how the RNA-binding abilities of A3 proteins play an essential role in the overall restriction potency of retroviruses and retrotransposons.

Masters of Science / The viral Integrase (IN) protein is an essential enzyme of all known retroviruses, including HIV-1. It is responsible for the insertion of viral DNA into the human genome. It is known that HIV-1 is highly diverse with a high mutation rate as evidenced by the presence of a large number of subtypes and even strains that have become resistant to antiretroviral drugs. It remains inconclusive what effect this diversity in the form of naturally occurring polymorphisms/variants exert on IN in terms of its function, structure and susceptibility to IN inhibitory antiretroviral drugs. South Africa is home to the largest HIV-1 infected population, with (group M) subtype C being the most prevalent subtype. An investigation into IN is therefore pertinent, even more so with the introduction of the IN strand-transfer inhibitor (INSTI) Dolutegravir (DTG). This study makes use of computational methods to determine any structural and DTG drug binding differences between the South African subtype C IN protein and the subtype B IN protein. The methods employed included homology modelling to predict a three-dimensional model for HIV-1C IN, calculating the change in protein stability after variant introduction and molecular dynamics simulation analysis to understand protein dynamics. Here we compared subtype C and B IN complexes without DTG and with DTG.

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Retroviruses are capable of infecting diverse vertebrates, and successful infection requires intimate interaction between virus and the host cell. During an infection, retroviral particles must bind specifically to cell surface receptors on the target cell, cross the plasma membrane, reverse-transcribe their RNA genome into double stranded DNA, find their way to the nucleus, enter the nucleus and integrate its DNA into host chromosomes. Following integration, expression of viral mRNA ensues, followed by viral mRNA export into the cytoplasm, translation of viral mRNA into proteins, and assembly of new virions that will egress from the host cell. We now appreciate that at many steps of this complex process, the virus must hijack the cellular machinery to replicate. At the same time, the host cell mobilizes a variety of cellular defense mechanisms to suppress viral infection. This thesis investigates various aspects of virus-host interactions. I will first describe the involvement of cellular transcriptional repressor protein ErbB3 binding protein 1 (EBP1) in facilitating transcriptional shutdown of Moloney murine leukemia virus (MLV) gene expression in mouse embryonic cells. Next, I describe a novel means of regulating the activity of Yin Yang 1 (YY1), a cellular transcription factor regulating retroviral gene expression, through post-translational modifications. I show that YY1 is a target of tyrosine phosphorylation by Src family kinases. Phosphorylation of YY1 impairs its ability to bind DNA and RNA, thereby downregulating its activity as a transcription factor on retroviral and cellular promoters. Apart from studying retroviral gene expression, I have also investigated intrinsic cellular defenses against retroviral infection. This is exemplified by our finding that mouse cells are intrinsically resistant to infection by betaretroviruses such as Mason-Pfizer monkey virus (M-PMV). The block against M-PMV occurs after reverse transcription and prior to viral nuclear entry. Finally, I will present ongoing work examining the fate of viral DNAs following infection, focusing on the kinetics of its association with cellular core histones and viral structural proteins. Together, this work provides critical insights into numerous aspects of the virus-host interactions.

Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends (the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed substrates to probe the processing reaction and a disintegration substrate to determine the number of protomers in a functional multimeric complex. In single-turnover studies, a novel "splicing" reaction was observed that revealed complications with accurate quantification of enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity synapsed substrates were designed that were not susceptible to the splicing reaction and that allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the processing reaction. A disintegration substrate was used in presteady-state active site titrations to determine a reaction stoichiometry of four integrase protomers per one substrate molecule for the disintegration reaction. A tetrameric active complex was then confirmed using atomic force microscopy to image integrase-DNA complexes during the first catalytic turnover. The observed increase of the tetramer population in the presence of substrate DNA demonstrates that the binding of the disintegration substrate induces assembly of the active tetramer and suggests that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003

Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized. Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining. Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints. Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.

<p>Nevirapine (NVP) is a cheap anti-retroviral drug used in poor countries worldwide, administered to pregnant women at the onset of labour to inhibit HIV enzyme reverse transcriptase. Viruses which may get transmitted to newborns are deficient in this enzyme, and HIV-1 infection cannot be established, thereby preventing mother to child transmission (MTCT). In some cases, babies get infected and positive selection for viruses resistant to nevirapine may be inferred. Positive selection can be inferred from sequence data, when the rate of nonsynonymous substitutions is significantly greater than the rate of synonymous substitutions.</p> <p>Unfortunately, it is found that available positive selection methods should not be used to analyse before- and after- NVP treatment sequence pairs associated with MTCT. Methods which use phylogenetic trees to infer positive selection trace synonymous and nonsynonymous substitutions further back in time than the short time duration during which selection for NVP occurred. The other group of methods for inferring positive selection, the pairwise methods, do not have appreciable power, because they average susbtituion rates over all codons in a sequence pair and not just at single codons. We introduce a simple counting method which we call the Pairwise Homologous Codons (PHoCs) method with which we have inferred positive selection resulting from NVP treatment in longitudinal HIV-1 reverse transcriptase sequences. The PHoCs method estimates rates of substitutions between before- and after- NVP treatment codons, using a simple pairwise method.</p>

Clonally derived liver tumours and an ovarian cyst developed in mice following EIAV and FIV delivery in utero. LAM PCR and 454 sequencing was used to retrieve proviral insertion sites. TaqMan analysis revealed gene expression changes in lentiviral infected tumours. STRING and IPA networks identified links between genes flanking the lentivirus provirus and oncogenic pathways supporting the role of insertional mutagenesis in Hepatocellular Carcinoma (HCC). Global methylation analysis demonstrated increased relative methylation levels in lentivirus (EIAV, FIV, and HIV) infected normal and tumour samples. This provided strong evidence for host defence against lentivirus infection by epigenetic means. Microarray data showed altered expression of Dnmt1 and Dnmt3b and TaqMan analysis revealed specific changes in Dnmts levels when compared to uninfected liver. The evidence found for involvement of DNA methylation associated with lentivirus infection and possibly tumour development required that this study be repeated in vitro. DNA methylation was investigated at early time points after lentivirus and retrovirus infection in HepG2 cells. Results revealed sharp increases in global methylation and Dnmt levels at 24 and 30hrs post infection. E2F targets play a key role in the regulation of gene expression and aberrations result in the development of cancer. Of the 94 E2F target genes analysed 77.7% were involved in DNA damage and repair mechanisms, 21.3% were known oncogenes or shown to exert oncogenic activity and 80.9% were categorised as HCC target genes. The fact that all lentiviral/retroviral vectors used in this study were found to cause changes in methylation and gene expression in vivo and in vitro suggests that these vectors, at least in the mouse, are genotoxic. Findings here support the use of the fetal animal model to identify vector genotoxicity and the mechanisms of lentiviral vector-induced tumorigenesis. This model may be a valuable tool to evaluate the safety of lentiviruses for gene therapy.