Browsing by Subject "HIV-1"

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APOBEC3 Transcriptional Regulation and HIV-1 Restriction in T Lymphocytes
(2017-08) Anderson, Brett
Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), has caused one of the most widespread and devastating pandemics in human history, and continues to persist as a substantial burden on global healthcare, social and economic systems, despite significant advances in modern anti-retroviral therapies. HIV-1 primarily infects CD4+ T lymphocytes, and to a lesser degree, macrophages, monocytes and dendritic cells. In the absence of therapeutic intervention, HIV-1 infection results in the gradual depletion of CD4+ T cells, leading to a severely compromised immune response and increased susceptibility to a wide range of opportunistic infections and malignancies. The innate immune response to HIV-1 infection in CD4+ T cells is mediated in part by members of the APOBEC3 family of DNA cytosine deaminases. In the absence of the viral Vif protein, multiple APOBEC3 enzymes can package into virions budding from an infected cell. Following virus entry into a new target cell, the APOBEC3 enzymes catalyze the deamination of cytosines to uracils in viral reverse transcription intermediates, resulting in mutations that can render viral gene products non-functional. The HIV-1 Vif protein counteracts the antiviral activity of the APOBEC3 enzymes by commandeering a cellular ubiquitin ligase comprised of CBF-β, ELOB, ELOC, CUL5 and RBX2, to polyubiquitylate the APOBEC3 enzymes and target them for proteasomal degradation. Thus, viral progeny are mostly protected from APOBEC3 mutagenesis. Despite significant advances in understanding the mechanisms that govern APOBEC3-dependent HIV-1 restriction, as well as Vif-dependent counteraction of this iii host defense, little is known about how these innate antiviral enzymes are regulated at the transcriptional level. The first part of this thesis identifies the CBF-β/RUNX transcription complex as a critical regulator of APOBEC3 gene expression in CD4+ T cells (APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H, but not APOBEC3A or APOBEC3B). This unexpected discovery suggests that HIV-1 Vif may employ a secondary mechanism in counteracting the host APOBEC3 defense by hijacking CBF-β from RUNX-associated transcription complexes to downregulate transcription of the APOBEC3 genes themselves. Thus, Vif may disarm the host APOBEC3 response by targeting these enzymes for proteasomal degradation, while simultaneously interfering with their ongoing expression at the transcriptional level. The seven membered APOBEC3 gene family is highly polymorphic within the human population, and several common genetic variations manifest as clear biochemical phenotypes. The second part of this thesis focuses on a rare variant of APOBEC3C (S188I), which confers enhanced HIV-1 restriction activity in comparison to the predominant S188 variant, which has been largely disregarded as playing a role in innate immunity to HIV-1 in T cells. The studies within characterize the antiviral activity of this APOBEC3C variant in multiple CD4+ T cell lines, and ultimately demonstrate that the S188I polymorphism renders APOBEC3C capable of protecting cells against Vif- deficient virus replication. These findings provide an additional example of meaningful variation within the human APOBEC3 repertoire that may impact virus replication and transmission in vivo, and will likely be the subject of follow up in several large ongoing HIV-1 infected patient cohort studies.
Determination of the endogenous HIV-restrictive APOBEC3 repertoire
(2014-08) Refsland, Eric William
Despite over 3 decades of intense research, an estimated 35.3 million people today are living with HIV-1 (Human Immunodeficiency Virus 1), the etiological agent of AIDS (Acquired Immunodeficiency Syndrome). The good news is that deaths are declining, in large part to combinations of highly active antiretroviral drugs. However, all of the current treatment options do not amount to a cure and a vaccine to prevent future infections is not yet available. With nearly 2 new infections for every 1 patient beginning an antiretroviral treatment regimen, the need is as great as ever to explore every avenue for new therapeutic interventions. One of these avenues is harnessing proteins endogenous to the very cells targeted for infection by HIV-1. Collectively referred to as host restriction factors, these innate immune proteins share a number of characteristics. First, they decrease virus replication. Second, expression of host restriction factors is often inducible and coupled to the immediate innate immune response to a foreign attack. Third, under the immense pressure of adapting to a pathogenic agent, host restriction factors often exhibit high degrees of rapid evolutionary change. Finally, if a restriction factor is potent enough, the virus will have evolved a counter-restriction mechanism. My thesis research has focused on the APOBEC3 family of host restriction factors that constitute an important arm of innate immunity. These enzymes, armed with the capacity to trigger cytosine to uracil mutagenesis in single-stranded DNA, function to defend the genome against attacks from foreign DNA elements including the retrovirus HIV-1. A more comprehensive understanding of the entire APOBEC3 family, its cellular functions, and how HIV-1 counteracts these activities with its accessory protein Vif, will all be essential before the design of therapeutic approaches incorporating endogenous APOBEC3 proteins in the fight against HIV/AIDS can be achieved. All 7 APOBEC3 proteins have been implicated in restricting HIV-1. The overall objective of my thesis research, detailed in the following chapters, was to define which of the 7 APOBEC3 proteins are capable of restricting the replication of HIV-1 in a T cell and which are potentially the source of the high levels of G-to-A mutagenesis observed in patient-derived HIV-1 sequences. To begin to narrow down which proteins are involved in vivo, we solved a long-standing problem in the APOBEC3 field. Quantifying expression levels had been impeded by the degree of homology between APOBEC3s, both at the nucleic acid and protein levels. My colleagues and I developed and characterized specific and efficient expression assays for each APOBEC3 family member. Using these assays we demonstrate that multiple APOBEC3s were expressed in relevant cell types and tissues including the major target of HIV-1, CD4+ T lymphocytes. To interrogate the contribution of individual APOBEC3s to HIV-1 restriction, we took a genetic approach and performed targeted deletion and knockdown experiments in a cell line that expresses multiple APOBEC3s. This approach allowed for a direct and definitive test of our hypothesis that multiple endogenous APOBEC3s restrict Vif-deficient HIV-1 replication. Based on HIV-1 replication kinetics and the levels of viral genome mutation, we concluded that 4 APOBEC3s are involved in HIV-1 restriction and that any future strategies employing the restrictive APOBEC3s will benefit from liberating all four proteins from Vif counteraction rather than any single one alone.Finally, to explore the consequences of genetic variation in the APOBEC3 locus on the HIV-1 restriction capacity of these proteins, we analyzed the expression levels and activity of the 7 haplotypes of the most genetically diverse APOBEC3, APOBEC3H. Through a series of primary cell experiments and HIV-1 spreading infections, we found that a subset of APOBEC3H haplotypes produce proteins that are stably expressed and capable of restricting naturally occurring HIV-1 variants that haven't evolved or have lost the capacity to neutralize this APOBEC3. The ramifications of this variability in a human restriction factor coinciding with diversity in HIV-1 variants able to counteract it will be an exciting area of future research. Overall, my research demonstrated that HIV-1 must contend with 4 APOBEC3 proteins to efficiently replicate in T cells. Expression of the APOBEC3s in human cells is widespread and inducible, and these host restriction factors combine to mutagenize the viral genome in the absence of HIV-1 Vif or, in the case of APOBEC3H, with Vif-proficient HIV-1 variants that are unable to mount an effective counter-defense. Consequently, unleashing all of these potent antiviral agents and allowing them to directly attack the virus' genetic code may lead to the first targeted innate immune therapy against HIV-1.
Establishment of Human Retrovirus Particle Assembly Sites
(2022-12) Schilling, Daniel
Human immunodeficiency virus (HIV) currently infects about 37 million people globally and has led to over 35 million deaths since its emergence in the human population by causing acquired immunodeficiency syndrome (AIDS). HIV type 1 (HIV-1) is responsible for the AIDS pandemic). HIV-1 spreads by intravenous drug use, sexual contact, blood transfusions, breastfeeding). Virus particle assembly is a foundationally important aspect in all modes of infectious spread and virus transmission. Comparative analyses with closely related human retroviruses can be particularly informative when studying areas including virus particle assembly and host-cell interactions that influence this process – which represents an important knowledge gap in the field. In preliminary studies, our collaborative research team has observed that the actin cortex can act as physical barrier for HIV-1 Gag recruitment to the plasma membrane (PM), particularly retroviral Gag-genomic RNA (gRNA) complexes. The actin cortex is functioning as a non-specific barrier that limits potential virus assembly sites on the PM. Here, in this fellowship application, experiments are proposed to gain further insights into the fundamental aspects of how virus-host cell interactions establish virus particle assembly sites. First, studies are proposed to investigate virus assembly sites in the context of the actin cortex. Multiple experimental approaches with be used to test the hypothesis that the actin cortex acts as a physical barrier for Gag-gRNA complexes, including comparative analyses in the context of cell-cell contact and using closely related human retroviruses. Second, I propose to investigate the role of the tumor suppressor adenomatous polyposis coli (APC) protein enrichment at virus particle assembly sites, testing the hypothesis that APCs interaction with the Gag protein helps to stabilize the Gag lattice and enhance the efficiency of Gag puncta biogenesis and gRNA recruitment. Comparative analyses will be leveraged to understand how APC may rely on interactions with actin filaments and play a more significant role during virus particle assembly at cell-cell contacts. Together, these studies will provide new insights into HIV-1 particle assembly, and advance knowledge in this crucial aspect of virus replication.
High-throughput Screening and Chemical Synthesis for the Discovery of APOBEC3 DNA Cytosine Deaminase Inhibitors
(2015-09) Olson, Margaret
APOBEC3 (APOBEC3A-APOBEC3H) enzymes catalyze single-stranded (ss)DNA cytosine-to-uracil (C-to-U) deamination as a function of innate immune defense against foreign DNA. Host cellular protection results from APOBEC3-catalyzed lethal mutagenesis of the offending genome. When misregulated, however, APOBEC3 enzymes have been demonstrated to drive the genetic evolution of numerous cancers and HIV-1. Specifically, sub-lethal levels of APOBEC3D/F/G/H-catalyzed mutation can enable HIV-1 escape from immune defense and antiretroviral therapies. Moreover, APOBEC3B over-expression in breast, bladder, cervical, lung, and head/neck cancers generates high levels of C-to-U mutation, which drives tumor formation, metastasis, and chemotherapeutic resistance. Thus, small molecule inhibition of APOBEC3-catalyzed deamination may provide a novel strategy for HIV-1 and cancer drug development. This thesis highlights efforts to discover small molecule inhibitors of the APOBEC3s through high-throughput screening (HTS) and chemical synthesis. Chapter 2 discusses an HTS of 168,192 compounds against APOBEC3B and APOBEC3G. In this effort, MN23 was discovered as a potent inhibitor of APOBEC3B (IC50 = 150 nM) and APOBEC3G (IC50 = 5.5 µM). Chapter 2 also reports a novel synthesis of MN23, and preliminary efforts to elucidate its mechanism of inhibition. Chapter 3 presents a class of covalent APOBEC3G-specific inhibitors based on a 1,2,4-triazole-3-thiol substructure. This compound class is predicted to inhibit APOBEC3G by covalently binding C321, forcing an inhibitory conformational change within the enzyme active site. Chapter 4 reports the discovery of a novel APOBEC3G inhibitory chemotype, which was discovered from the deconvolution of an impure HTS hit. In this effort, we also identified a previously unreported Pan Assay Interference Scaffold (PAINS), and characterized the mechanism by which compounds of this class undergo oxidative decomposition. Finally, Chapter 5 describes how benzthiazolinone-based APOBEC3 inhibitors are being developed into probes of APOBEC3 structure and function. Brief descriptions of two unrelated projects performed concurrent with these studies are also detailed in the Appendices.
HIV-1 Counteraction Mechanisms Versus APOBEC3-mediated Restriction
(2018-07) Richards, Christopher
Human Immunodeficiency Virus type 1 (HIV-1) is responsible for the etiology of Acquired Immunodeficiency Syndrome (AIDS). Almost 40 years’ worth of intensive HIV-1 research have not yet led to a cure, nor is an efficacious vaccine available. The number of deaths caused by HIV-1/AIDs is declining due to effective, though non-curative, combinations of highly active antiretroviral therapy (HAART) regimens. Given that in 2016: 1.8 million newly infected people were infected with HIV-1 in 2016, 36.7 million people globally were living with HIV-, and 1 million people died from AIDS-related (UNAIDS Fact Sheet – 2018), every avenue to discovering a cure should be sought out. HIV-1’s life cycle is characterized by eight steps. Step 1 is known as attachment, where HIV-1 binds to the receptors of a CD4+ cells. Step 2 is fusion, where the viral envelope fuses with the cellular membrane, granting the virus entry to the host cell. Step 3 is reverse transcription, where the viral RNA that is now inside the host cells is converted into viral DNA. Step 4 is integration, where the HIV-1 DNA is shuttled into the nucleus, as part of a high molecular weight pre-integration complex, by co-opting host nuclear import machinery, followed by HIV-1 integrase catalyzes the insertion of the viral DNA into the host’s genome making a provirus. Step 5 is replication, where the integrated viral DNA hijacks host machinery to make viral RNA copies as well as viral structural proteins that are used as building blocks for HIV-1 particle formation. Step 6 is assembly, where newly synthesized HIV-1 proteins and viral RNA are trafficked and used to assemble an immature (noninfectious) particle at the cell membrane. Step 7 is known as budding, where the immature particle undergoes membrane scission with the host cell releasing the particle into extracellular space. Step 8 is maturation, where HIV-1 protease is activated in the newly released particle and it begins to cleave the structural proteins of the virus, releasing intra-virion proteins that are required to make the particle infectious (mature). APOBEC3 (A3) enzymes are packaged into budding virions from a cell already infected with HIV-1 (Steps 6-7). After a virion containing A3 enzymes enters a target cell (Steps 1-2), the A3s can restrict HIV-1 via deaminase-dependent and -independent mechanisms during reverse transcription (Step 3), which is described in more detail below. However, HIV-1 encodes virion infectivity factor (Vif), which allows the virus to retain high levels of infectivity via proteasomal degradation of cellular A3 restriction factors in cells producing virus. Restrictive A3 enzymes capacity to incapacitate HIV-1 is such that no appreciable infectivity is observed in Vif-null systems, thereby suggesting that modulation of the A3-Vif axis in the host’s favor could be a potentially curative antiretroviral approach. In this thesis, three separate projects combine to advance our understanding of the A3-mediated restriction mechanism and the Vif-mediated counteraction mechanism. Chapter 2 uses human APOBEC3F (A3F) to adapt HIV-1 and create a genetic and structural map of the Vif interaction surface. Chapter 3 compares the HIV-1 restriction activity of splice variants human APOBEC3H (A3H) and reports differential antiviral activities and a novel viral protease-dependent counteraction mechanism. Chapter 4 explores potential antiviral strategies using synthetic peptides derived from Vif. Collectively, these studies increase our overall understanding of how HIV-1 counteracts A3 restriction factors. Ultimately, this work informs the next generation of approaches directed at discovering ways to modulate these interactions in potentially curative ways.
Host Cell Factors in HIV-1 Replication Cycle
(2014-06) Boso, Guney
Despite the development of more than 20 antiretrovirals in the last 30 years, HIV-1 continues to be one of the major infectious causes of mortality in the world. Due to invasive side effects and the high cost of antiretrovirals novel treatment strategies are in high demand. One of the strategies for targeting viral infection with drug intervention takes advantage of the fact that HIV-1 requires host cell machinery to complete its life cycle. Identifying host cell factors that are involved in HIV-1 life cycle as well as elucidating the roles they play in viral infection can lead to discovery of potential drug targets. In this dissertation my goal is to understand the role of some host cell processes in the replication cycle of HIV-1. I approached this in two ways: In the first two parts I studied the role of a ubiquitous cellular pathway called the N-end rule pathway in the early phase of the retroviral life cycle. In the third part I have characterized a human cell line that was found to be non-permissive to infection with retroviruses and retroviral vectors.
The mechanism of HIV-1 Tat-induced changes in NMDA receptor function
(2014-08) Krogh, Kelly A.
Worldwide, more than 35 million people are currently infected with the human immunodeficiency virus (HIV). Approximately half of HIV-infected patients in the U.S. experience cognitive impairment despite effective control of viral load with combination anti-retroviral therapy (cART). The neurological complications that stem from an HIV infection are known as HIV-associated neurocognitive disorders (HAND). HAND ranges in severity from subtle difficulties with day-to-day tasks to severe functional impairment requiring assistance to survive. Although cART has improved patient survival by effectively managing viral load, it is ineffective at treating the majority of HAND. Consequently, the prevalence of HAND remains persistently high. The symptoms of HAND correlate with neuronal damage, such as synapse loss and dendritic beading. Such synaptodendritic damage results from HIV-infected cells within the central nervous system (CNS) shedding neurotoxic agents, such as the HIV-1 protein transactivator of transcription (Tat). Tat potentiates N-methyl D-aspartate (NMDA) receptor function allowing excessive Ca2+ influx leading to neurotoxicity. In this dissertation, two studies are outlined investigating the mechanisms of NMDA receptor (NMDAR) dysfunction following exposure to Tat. The graphical abstract summarizes these studies. First, the effect of Tat on NMDAR function was investigated. This study showed that Tat caused a time-dependent, biphasic change in NMDAR function. Initially, Tat potentiated NMDAR function via the low-density lipoprotein receptor-related protein (LRP) and activation of Src tyrosine kinase. Subsequently, NMDAR function adapted by gradually returning to basal levels following 24 h exposure to Tat and eventually falling below control responses by 48 h. Adaptation resulted from activation of a nitric oxide synthase (NOS), soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PKG) signaling pathway. Next, effectors downstream of PKG responsible for adaptation of NMDAR function were identified. Tat activated a signaling pathway including the small GTPase RhoA and Rho-associated protein kinase (ROCK). RhoA/ROCK activation caused remodeling of the actin cytoskeleton resulting in reduced NMDAR function. Taken together, these studies indicate that Tat causes a biphasic change in NMDAR function. Potentiation of NMDAR function is mediated by LRP-dependent activation of Src kinase; adaptation of NMDAR function occurs after activation of a NOS/sGC/PKG signaling pathway leading to RhoA/ROCK-mediated remodeling of the actin cytoskeleton. Adaptation of NMDAR function may be a neuroprotective mechanism to reduce excess Ca2+ influx and prevent neurotoxicity. These studies provide molecular and temporal detail of the dynamic changes in NMDAR function following exposure to Tat and offer insight into potential therapeutic targets for the treatment of HAND.
Structural studies of the deaminase domain of the human HIV-1 restriction protein APOBEC3G
(2010-09) Chen, Kuan-Ming
According to a report from the UNAIDS on the global AIDS epidemic in 2009, about 33.4 million people were living with the HIV virus, with a 2.7 million increase each year. HIV can lead to AIDS, which is a set of symptoms and infections resulting from the damage to the human immune system. However, due to the rapid mutability and productivity of the HIV, identifying treatments and therapeutic intervention remains challenging and has seen limited progress. In the past year, one novel innate defense against HIV infection was discovered, in which the human protein APOBEC3G (A3G) plays an important role. A3G was identified as a single-strand DNA deaminase that potently inhibited the replication of the HIV-1ΔVif virus. It produces the nonfunctional provirus by deaminating the cytosines to uracils on the minus-strand viral cDNA. Consequently, A3G can genetically inactivate HIV and recent studies have demonstrated that this activity is as potent as any current anti-retroviral drug. However, the exact model of this mechanism at atomic level has not yet been elucidated due to the low solubility of A3G which presents an obstacle for biochemical and structural studies. The research in this thesis took a structural approach to screen for a catalytically active and more soluble C-terminal deaminase of APOBEC3G (A3G-ctd) derivative, determine the NMR solution structure of this variant (A3G-2K3A) and characterize its interaction with single-strand DNA. Due to intrinsically low solubility of A3G-ctd, a strategy to design more soluble derivatives of the catalytic domain was performed prior to NMR structure determination. Two key methods: a solubility test and a E. coli-based mutation assay were used to test the solubility and catalytic activity of APOBEC3G variants. Deletion mutant analyses of APOBEC3G found the minimal catalytic region consisted of amino acids 198-384 (A3G198-384). Various alanine and lysine substitution variants based on this fragment were constructed and examined to screen for improved solubility and enhanced activity. One variant A3G198-384-2K3A (L234K-C243A-F310K-C321A-C356A) showed a significant improvement in both assays, and was purified as a monomer. The three-dimensional structure of A3G198-384-2K3A was then determined by triple resonance NMR spectroscopy. It consists of five β-strands that form a hydrophobic platform surrounded by five α-helices. Summarizing the DNA titration data, E. coli-based catalytic activity, conserved residues and computational modeling, the DNA binding mechanism of A3G was proposed in which a canyon formed by positively charged residues guides single-strand DNA binding and positions the target cytidine for deamination. Subsequently, a longer catalytic domain, A3G191-384-2K3A, was found to have higher activity than that of the A3G198-384-2K3A derivative. The longer domain has an additional α1-helix (residues 201-206) that was not observed in the shorter variant and part of the last α-helix (residues 191-194) of the N-terminal domain. The truncated model of the N-terminal domain was generated from the C-terminal NMR structures based on the sequence homology. Finally, a novel full-length A3G model was constructed by physically overlapping the α-helix (residues 191-194) of the N-terminal domain model and the C-terminal domain structure.
Studies On The Antiretroviral Mechanism Of Action Of Clofarabine
(2014-07) Beach, Lauren
Since the beginning of the AIDS epidemic over thirty years ago, human immunodeficiency virus type 1 (HIV-1) has infected seventy-five million people and has claimed the lives of over thirty-six million people worldwide, making HIV/AIDS one of the most devastating global infectious disease epidemics in history. To date, no preventative vaccine or curative treatment exists for HIV-1 infection. The availability of drugs to treat HIV-1 infection has led to drug resistance, which limits the utility of antiviral therapy. This has provided the basis for the continual need for identifying new targets for antiviral drugs. This dissertation investigated the antiretroviral activity and mechanism of action for clofarabine, a purine nucleoside antimetabolite. Clofarabine was demonstrated to exert antiretroviral activity against both HIV-1 and human immunodeficiency virus type 2 (HIV-2). Studies directed at elucidating the antiretroviral mechanism of action support a model in which clofarabine acts as an inhibitor of ribonucleotide reductase, leading to imbalances in cellular dNTP pools, which reduces viral infectivity through an increase in the HIV-1 mutation rate.
Studies on the Assembly and Morphology of Human T-Cell Leukemia Virus Type 1
(2019-08) Maldonado-Ortiz, José
The group-specific antigen (Gag) polyprotein is an essential retrovirus structural protein required for the assembly and release of virus particles. Present knowledge of Gag biology has been limited to a few retroviruses. Furthermore, current understanding of the diversity in the nature of Gag structure and function in virus particle assembly is limited. Human T-cell leukemia virus type 1 (HTLV-1) is a deltaretrovirus that causes an adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated-myelopathy/tropical spastic paraparesis (HAM/TSP), and other neurotropic conditions. HTLV-1 has infected approximately 15 million individuals worldwide. A general knowledge gap exists regarding the details of HTLV-1 replication, including particle assembly. To address this, and to test the overarching hypothesis that HTLV-1 particle assembly is distinct from that of other retroviruses, this dissertation focused on investigating three key aspects of HTLV-1 immature and mature particle morphology. First, an analysis of the morphology and Gag stoichiometry of HTLV-1-like particles and authentic, mature HTLV-1 particles by using cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission electron microscopy (STEM) was conducted. HTLV-1-like particles mimicked the morphology of immature authentic HTLV-1 virions. Importantly, it was observed for the first time that the morphology of these virus-like particles (VLPs) has the unique local feature of a flat Gag lattice that does not follow the curvature of the viral membrane, resulting in an enlarged distance between the Gag lattice and the viral membrane. Measurement of the average size and mass of VLPs and authentic HTLV-1 particles suggested a consistent range of size and Gag copy numbers in these two groups of particles. The unique local flat Gag lattice morphological feature observed suggests that HTLV-1 Gag could be arranged in a lattice structure that is distinct from that of other retroviruses characterized to date. Second, the effects of Gag proteins labeled at the carboxy terminus with a fluorophore protein were analyzed for their influence on particle morphology. In particular, a HTLV-1 Gag expression construct with the yellow fluorescence protein (YFP) fused to the carboxy-terminus was used as a surrogate for the HTLV-1 Gag-Pro to assess the effects of co-packaging of Gag and a Gag-YFP on virus-like particle morphology and particles were analyzed by cryo-TEM. STEM and fluorescence fluctuation spectroscopy (FFS) were also used to determine the Gag stoichiometry. Ratios of 3:1 (Gag:Gag-YFP) or greater were found to result in a particle morphology indistinguishable from that of VLPs produced with the untagged HTLV-1 Gag, i.e., a mean diameter of ~113 nm and a mass of 220 MDa as determined by cryo-TEM and STEM, respectively. This information is useful for the quantitative analysis of Gag-Gag interactions that occur during virus particle assembly and in released immature particles. Third, cryo-electron tomography (cryo-ET) was used to analyze mature HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity. Taken together, the findings of this dissertation provide new insights into the nature of immature and mature HTLV-1 assembly and morphology and provide foundational knowledge towards an advanced understanding of the HTLV particle assembly pathway.
Studies on the Molecular Determinants of Human Retrovirus Diversity
(2021-09) Meissner, Morgan
The human immunodeficiency viruses (HIVs) are two species (HIV type 1, HIV-1; HIV type 2, HIV-2) in the Lentivirus genus of the Retroviridae family of viruses and are the etiological agents of acquired immunodeficiency syndrome (AIDS). Nearly 75 million people have been infected with HIV-1 and HIV-2 since their emergence in the human population and they are responsible for the deaths of almost 32 million individuals to date. One of the key drivers of the HIV-1 pandemic is the extreme genetic diversity of the virus, which drives the development of antiviral drug resistance and frustrates vaccine development. Retroviruses also exhibit considerable structural diversity, which may have important implications for infectivity and replication. Human T-cell leukemia virus type 1 (HTLV-1), within the Deltaretrovirus genus, is also a major pathogenic human retrovirus. HIV-2 and HTLV-1 exhibit markedly reduced rates of transmissibility and potentially evolution compared with HIV-1 and are therefore understudied relative to their pandemic counterpart. The overarching goal of this thesis was to characterize the viral and cellular determinants of the molecular diversity of human retroviruses, with an emphasis on HTLV-1 and HIV-2. To this end, experiments were conducted which 1) characterized the structural diversity of authentic HTLV-1 particles derived from the chronically infected SP cell line, demonstrating that intact capsid cores are relatively rare among HTLV-1 particles; and 2) examined the contribution of host apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins to HIV-2 mutagenesis, which revealed that despite their role as potent mutagens of HIV-1, APOBEC3-mediated mutagenesis of HIV-2 is limited. Additionally, a user-friendly sequence analysis workflow was developed that enables the ultra-accurate detection of mutations within HIV-1 and HIV-2, which reduces the background error rate of traditional Illumina next-generation sequencing by approximately 100-fold. This workflow is already being employed to characterize the contributions of additional cellular proteins to retroviral mutagenesis, including the host protein SAM domain and HD domain- containing protein 1 (SAMHD1). Taken together, these studies provide new insights into the structural and genetic diversity of human retroviruses, particularly those which have historically been poorly characterized and underappreciated.
Studies on the origins of HIV-1 mutation and genetic diversity
(2013-12) Holtz, Colleen Mary
A fundamental biological property of retroviruses and RNA viruses is their ability to rapidly mutate and evolve. The ability of these viruses to generate high levels of genetic diversity during replication has clearly had a profound impact on their ability to maintain their niche in nature, and to rapidly adapt to changing environmental conditions or opportunities to expand their host range. Previous reports with HIV-1 have indicated that the cell type in which HIV-1 replicates does not have a profound impact on HIV-1 diversity. However, due to differences in dNTP pool levels and expression levels of HIV-1 DNA editing enzymes, the hypothesis that cell type does influence the diversity of HIV-1 populations was formulated. To test this, a panel of relevant cell types (i.e., CEM-GFP, U373-MAGI, 293T, and SupT1) was analyzed for their ability to influence HIV-1 mutant rate and spectra. No differences were observed in overall mutation rate, but intriguingly, cell type differences impacted HIV-1 mutation spectra. These observations represent the first description of significant differences in HIV-1 mutation spectra observed in different cell types in the absence of changes in the viral mutation rate and, imply that such differences could have a profound impact on HIV-1 pathogenesis, immune evasion, and drug resistance. The most common mutation type that arises during HIV-1 replication is transition mutations, particularly G-to-A mutations. Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) proteins create G-to-A mutations at specific cytosine dinucleotides. In order to better define the locations of APOBEC3G (A3G)-mediated G-to-A mutations, we tested the hypothesis that sequence context and DNA secondary structure influence the creation of A3G-mediated G-to-A mutations. Single-stranded DNA secondary structure as well as the bases directly 3'and 5' of the cytosine dinucleotide were found to be critically important for A3G recognition. These observations provide the first demonstration that A3G cytosine deamination hotspots are defined by both sequence context and the single-stranded DNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on the generation of HIV-1 diversity, ultimately influencing the biological properties of the progeny virus variants.

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