Human Immunodeficiency Virus 1 (HIV-1) is a lentivirus that forms persistent latently infected reservoirs that are the remaining major hurdle for current HIV-1 treatments. APOBEC3 (A3) proteins are intrinsic retroviral restriction factors that introduce GA mutations during reverse transcription, while Reverse Transcriptase (RT) introduces on average 2-3 mutations every reverse transcription cycle due to a lack of proofreading ability. The goal of this research is to characterize the infectivity and activation of mutated HIV-1 viruses that display reduced transcription upon infection, viruses that we term latency prone viruses (LPVs). We hypothesize that GA transition mutations in the HIV-1 Long Terminal Repeat (LTR) region of the LPVs introduced through Reverse Transcriptase and low levels of A3 protein activity can create HIV-1 sequences that display a reversible, latency-like phenotype. Variable levels of transcription and promoter activation were seen among the LPVs when tested against four classes of Latency Reversing Agents (LRAs). Subsequently, three tested LPVs demonstrated an initial latency-like phenotype before rebounding in infectivity. This project demonstrates for the first time that HIV-1 latency is not simply a byproduct of the infection timing and cellular conditions, but that replication-competent HIV-1 latent viruses can also be created through sublethal mutagenesis of their viral promoter sequence introduced through A3 and RT exposure. The characterization of the complete mechanism of HIV-1 latency induction, maintenance, and reversal is critical in the development of sterilizing and functional cures for HIV-1 infection.

Murine leukemia virus (MLV), classified as a gammaretrovirus, has been studied extensively to enhance our understanding of the biology and replication of retroviral infection. Typically referred to as a simple retrovirus, its usefulness as a model is highlighted owing to its minimal genome. The genetic material for MLV was thought to only code the basic and essential defining features of a retrovirus. Through the understanding developed from the use of simple retroviruses, the clinical and research communities were immeasurably more prepared to combat the more complex and decidedly infamous human immunodeficiency virus (HIV). Interestingly, a scenario of convergent evolution has directed MLV to encode an accessory protein, termed Glycosylated Gag (gGag), that shares functionality reminiscent of several HIV proteins. Herein, I present a dissection of a novel function of this enigmatic protein, paired with an improved understanding of the biology of MLV that was revealed by the development of small particle flow cytometry performed on viruses, also known as flow virometry. Initially, we elucidated that gGag is responsible for the resistance of MLV towards the restriction factor murine APOBEC3 (mA3). I showed that even endogenous mA3 from primary cells exhibited an enhanced enzymatic activity towards MLV with mutant gGag proteins which have lost glycosylation sites. In our following study, I illustrated that these mutants displayed a reduced viral core stability, the severity of which was correlated directly with susceptibility to mA3. These results are in line with the hypothesis that viral core stability and APOBEC3-susceptibility are directly linked. Furthermore, I showed for the first time that unprocessed gGag was associated with viral particles released from producer cells in the orientation of a type I membrane protein, with the structural regions directed within the viral core. This may be the direct evidence of how gGag improves capsid stability, a mechanism which is still unresolved. On the flip side, gGag as a type II membrane protein was observed exclusively on virus-like particles devoid of detectable envelope glycoprotein (Env). This marks a potential new function for gGag in the context of infection. Given the ubiquitous necessity of an optimized core stability for any virus, combined with the overlapping function of gGag with HIV accessory proteins, continuation of this work represents an as of yet clinically unexplored avenue for the development of HIV therapeutics. At the same time, in order to characterize individual viral particles, I played an instrumental role in developing the technique of flow virometry within our core facility. I illustrated that the Env of MLV does not significantly accumulate on extracellular vesicles (EVs) and acts as an effective marker for viral particles. With this evidence in hand, the enumeration of MLV virions was made possible. By correlating this information with an absolute viral genome determination, I was able to estimate the packaging efficiency for MLV in a quantitative manner. This information suggests that roughly 80-85% of MLV particles are missing their essential genetic information. These findings may implicate the disease progression of MLV infection may be enhanced by the use of defective-interfering particles, a theory that has been suggested for HIV. This work highlighted the fact that flow virometry is uniquely capable to discriminate viral particles from other cell-derived membraned vesicles in a highly sensitive manner. Overall, my work has unveiled new complexities of a simple retrovirus, while laying the groundwork towards both diagnostics and therapeutics for the ongoing battle with HIV.

The Human Immunodeficiency virus-1 can stay undetected and unaffected by host immune surveillance and antiretroviral therapy. This phenomenon is called proviral latency and the cells harbouring such viruses are part of the latently infected cell reservoir. In this situation, the viral genome integrates into the host's genome upon infection, whereby infected cells exhibit either very low levels or no viral transcription, and hence no viral proteins or egress viruses are produced that can be detected by the immune system. However, viral transcription can be re-activated to produce infectious viruses under certain circumstances. Host-encoded retroviral restriction factors like APOBEC3 (A3) proteins are part of our intrinsic immune defences against retroviral infection, introducing mutations in viral replication intermediates. We hypothesize that low levels of G-to-A transition mutations in the HIV-1 LTR region, introduced by APOBEC3G/F, could lead to a latency-like phenotype. These latent viruses pose major hurdles for HIV-1 cure therapies. Our lab previously created a library of clones possessing mutations in the LTR introduced by A3G/F. Later, mutated LTRs were cloned into 3 types of plasmid backbones: 1) a pEGFP expression vector to study the transcriptional activity of the mutated promoter, 2) into non-replicative pNL4 ∆env ∆vif viral expression vector, and 3) into a replicative pNL4-CXCR4 viral vector to study infection and induction by latency reversal agent (LRA) treatment to better understand the mechanism of latency and transcriptional induction. Viruses produced from these plasmids carrying mutated promoters are referred to as latency-prone viruses or LPVs in this thesis. Characterizing the transcription, infection, and induction to PMA/I of the LPVs would essentially help in evaluating the role of A3 mutations in viral latency and further help in the development of new therapeutics.

APOBEC3s (A3s) are a family of human cytidine deaminases that play important roles in both innate immunity and cancer. A3s protect host cells against retroviruses and retrotransposons by deaminating cytosine to uracil on foreign pathogenic genomes. However, when mis-regulated, A3s can cause heterogeneities in host genome and thus promote cancer and the development of therapeutic resistance. The family consists of seven members with either one (A3A, A3C and A3H) or two zinc-binding domains (A3B, A3D, A3D and A3G). Despite overall similarity, A3 proteins have distinct deamination activity and substrate specificity. Over the past years, several crystal and NMR structures of apo A3s and DNA/RNA-bound A3s have been determined. These structures have suggested the importance of the loops around the active site for nucleotide specificity and binding. However, the structural mechanism underlying A3 activity and substrate specificity requires further examination. Using a combination of computational molecular modeling and parallel molecular dynamics (pMD) simulations followed by experimental verifications, I investigated the roles of active site residues and surrounding loops in determining the substrate specificity and RNA versus DNA binding among A3s. Starting with A3B, I revealed the structural basis and gatekeeper residue for DNA binding. I also identified a unique auto-inhibited conformation in A3B that restricts access to the active site and may underlie lower catalytic activity compared to the highly similar A3A. Besides, I investigated the structural mechanism of substrate specificity and ssDNA binding conformation in A3s. I found an interdependence between substrate conformation and specificity. Specifically, the linear DNA conformation helps accommodate CC dinucleotide motif while the U-shaped conformation prefers TC. I also identified the molecular mechanisms of substrate sequence specificity at -1’ and -2’ positions. Characterization of substrate binding to A3A revealed that intra-DNA interactions may be responsible for the specificity in A3A. Finally, I investigated the structural mechanism for exclusion of RNA from A3G catalytic activity using similar methods. Overall, the comprehensive analysis of A3s in this thesis shed light into the structural mechanism of substrate specificity and broaden the understanding of molecular interactions underlying the biological function of these enzymes. These results have implications for designing specific A3 inhibitors as well as base editing systems for gene therapy.

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate.

No description available.

APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.