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The ability to link a particular phenotype to its causative genotype is one of the most challenging objectives for biological research. Although the genetic code provides an explicit formula for determining the sequence of amino acid phenotypes produced by a given nucleotide sequence, identifying specific residues that are functionally important remains problematic. Many computational approaches have been developed that use patterns observed in DNA sequences to identify these critical sites. However, very few research studies have used empirical data to test whether these approaches are truly able to identify sites of interest.In most empirical studies, the actual protein function and selective pressures are unknown; thus it is difficult to assess whether computational approaches are correctly identifying critical sites. Here I present two studies that utilize well-characterized empirical systems to evaluate and compare the performance of several computational approaches. In both cases, the proteins under study have specific amino acid substitutions that are confirmed to alter protein function and expected to be constrained by natural selection. In chapter 2, I examine functional variants in angiopoietin-like protein 4 (ANGPTL4), a protein involved in regulating plasma triglyceride levels; loss-of-function variants in this gene are believed to decrease the risk of cardiovascular disease. I apply several computational approaches to identify functional variants, including phylogenetic approaches for detecting positive selection. In chapter 3, I investigate the emergence of drug-resistance in HIV-1 during the course of antiretroviral drug therapy. I compare the performance of eight selection detection methods in identifying drug-resistant mutations in 109 intrapatient datasets with HIV-1 sequences isolated at multiple timepoints throughout drug treatment.It is critical that we develop methods to detect positively selected sites. The ability to detect these sites in silico, without the need for expensive and time consuming assays, would be invaluable to researchers in evolutionary biology, human genetics, and medicine. Through the research presented in this thesis, I hope to provide insight into the strengths and weaknesses of current approaches, thereby facilitating future research towards the development and improvement of evolutionary models.

Vpr is a 14 kDa accessory protein conserved amongst extant primate lentiviruses that is required for virus replication in vivo. Although many functions have been attributed to Vpr, its primary role, and the function under selective pressure in vivo, remains elusive. The minimal importance of Vpr in infection of activated CD4+ T cells in vitro suggests that its major importance lies in overcoming restriction to virus replication in quiescent CD4+ T cells and non-cycling myeloid cell populations, such as macrophages and dendritic cells. Previous studies from our laboratory demonstrated that HIV-1 replication is attenuated in the absence of Vpr in monocyte-derived dendritic cells (MDDCs) and macrophages, which is correlated with the ability of HIV-1 Vpr to overcome a post-integration transcriptional defect in these cells. In contrast to HIV-1 Vpr-mediated transcriptional enhancement of the viral LTR, here I describe a role for HIV-2 and SIVmac Vpr homologs in the suppression of innate immune sensing of primate lentiviral infection in monocyte-derived dendritic cells (MDDCs). Specifically, the Vpr proteins of HIV-2 and SIVmac, but not that of HIV-1, suppress innate immune detection and induction of type I and type III IFN at two distinct stages of the viral life cycle: prior to and during integration. We posit that HIV-2/SIVmac-lineage Vpr homologs gained this function upon the acquisition of Vpx, a Vpr paralog in the lentiviral genome, that targets the retroviral restriction factor SAMHD1 for proteasomal degradation. Mutational analysis shows that suppression of pre-integration innate immune sensing by HIV-2/SIVmac Vpr homologs is tied to their interaction with the DNA damage response protein human Uracil DNA glycosylase hUNG. Interestingly, the HIV-1 Vpr degrades hUNG, whilst the HIV-2/SIVmac Vpr homologs do not. This difference correlates with the inability of HIV-1 Vpr to suppress type I and III IFN responses in SIVmac Vpx supplemented infections of MDDCs. These results highlight how divergent lentiviruses have tailored interactions of their Vpr proteins with members of the DNA damage response to promote replication in diverse cellular contexts. This work also describes the conserved role of primate lentiviral Vpr homologs in the transcription of extrachromosomal or unintegrated viral DNA. This function is dependent on Vpr engagement with the host E3-ubiquitin ligase complex Cul4-DDB1-DCAF1 (DCAFCRL4) and ability to activate the DNA damage response. These findings give insight into the mechanisms driving transcription from an underappreciated and long-lived source of viral antigen, and further the field of non-integrating lentiviral vectors, a frequently used tool for the genetic modification of non-dividing cells. Together, both studies shed light on the way Vpr proteins from diverse primate lentiviruses converge in their manipulation of the DNA damage response to facilitate multiple stages of the virus lifecycle. / 2024-01-24T00:00:00Z

In this study, a Monte Carlo simulation was designed to observe the self-assembly of the HIV-1 capsid protein. The simulation allowed a coarse grained model of the capsid protein with defined interaction sites to move freely in three dimensions using the Metropolis criterion. Observations were made as to which parameters affected the assembly the process. The ways in which the assembly were affected were also noted. It was found that proper dimerization of the capsid protein was necessary in order for the lattice to form properly. It was also found that a strong trimeric interface could be responsible for double-layered assemblies. Further studies may be conducted by further varying of parameters or reworking the dynamics of the simulation. The possible causes of curvature within the assembly still need to be researched further.

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