Browsing by Subject "Vif"

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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.
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    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.
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    The Dynamic Interplay Between Lentiviral Vif and Human APOBEC3 Proteins
    (2019-07) Wang, Jiayi
    Four members of the APOBEC3 (A3) family of DNA cytosine deaminases are capable of inhibiting HIV-1 replication by deaminating viral cDNA cytosines and interfering with reverse transcription. HIV-1 counteracts restriction with its Vif protein, which nucleates a ubiquitin ligase complex that directly binds A3 enzymes and targets them for proteasomal degradation. My thesis research aims at understanding the dynamic interplay between lentiviral Vif and human A3 enzymes, from the molecular determinants of this interaction to its implications in HIV-1 transmission within a large patient cohort. This has been addressed through three separate studies. The primate A3 repertoires show considerable variations likely due to positive selection during evolution. Lentiviral Vif proteins have rapidly evolved to counteract this immune pressure, resulting in present-day host-pathogen interactions that are largely species specific. However, a simian immunodeficiency virus (SIV) Vif exhibits cross-species degradation capability against multiple human A3 enzymes. We used mutagenesis coupled with functional assays to determine the residues involved in the interaction between the SIV Vif and A3B, and demonstrated that it resembles the HIV-1 Vif-human A3G interaction. This may be a molecular remnant of an ancestral Vif activity or result of molecular mimicry between human A3B and A3G. A3H is unique among family members by dimerizing through cellular and viral duplex RNA species. RNA binding is required for proper localization of A3H to the cytoplasmic compartment, for efficient packaging into nascent HIV-1 particles, and ultimately for effective virus restriction activity. To investigate the role of RNA in HIV-1 Vif-mediated degradation of A3H, we used structural and cell biology approaches to study RNA binding mutants and their sensitivity to Vif-mediated degradation. We found that RNA is not strictly required for Vif-mediated degradation of A3H and that RNA and Vif bind the enzyme on largely distinct surfaces, but the degradation process may be affected by changes in subcellular localization/mobility and/or differences in the constellation of A3H interaction partners. In humans, A3H is the most polymorphic member of the family and includes seven haplotypes with three encoding for stable proteins and the rest unstable. Stable A3H proteins contribute to HIV-1 restriction and can only be counteracted by fully functional Vif variants (dictated by amino acids at key positions). We tested the hypothesis that stable A3H enzymes provide a transmission barrier to HIV-1 isolates harboring less-than-fully functional Vif alleles. We have determined the A3H and viral Vif genotypes of a large cohort of African HIV-1 serodiscordant couples and have shown stable A3H is unlikely to be a general protective factor in HIV-1 acquisition. However, stable A3H enzymes may still serve positive roles in slowing virus spread and disease progression. Overall, my thesis research contributes to the growing knowledge of the A3-Vif interaction, particularly interactions between the Vif protein of pandemic HIV-1 and the contemporary restriction factor A3H. These studies will help guide future efforts to disrupt this interaction as an antiviral therapy.
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    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.
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    HIV-1 Vif requires core binding factor Beta to degrade the APOBEC3 restriction factors and facilitate viral replication
    (2012-12) Hultquist, Judd F.
    While there are a number of antiretroviral drugs for the treatment of Human Immunodeficiency Virus (HIV), they are all expensive, invasive, susceptible to resistance, and are not curative. One potential future drug target is the interaction between the human antiviral APOBEC3 proteins and the HIV counterdefense protein, Vif. Vif binds to and neutralizes the DNA-mutating APOBEC3 proteins by recruitment of an E3 ubiquitin ligase complex that targets them for degradation. Design of small molecule therapeutics to disrupt this interaction and free the antiviral APOBEC3 proteins has been hampered by an incomplete understanding of the Vif E3 ubiquitin ligase complex and conflicting reports as to which of the seven different APOBEC3 proteins contribute to HIV restriction in vivo. To determine which APOBEC3 proteins contribute to HIV restriction, we performed a comprehensive analysis of both human and rhesus macaque APOBEC3 families in T cells. Based on six criteria (expression, virion incorporation, HIV restriction, viral genome mutation, neutralization by Vif, and conservation), we found that four APOBEC3 proteins have the potential to restrict HIV replication. To better understand the Vif E3 ligase complex responsible for neutralizing these proteins, we performed extensive purification experiments with HIV Vif and discovered that Vif interacts with the cellular transcription factor Core Binding Factor Beta (CBFB). We discovered that CBFB not only allows for reconstitution of the Vif E3 ligase complex in vitro, but also stabilizes Vif in vivo, subsequently facilitating ligase assembly and allowing for APOBEC3 degradation. This functional interaction is highly conserved, being required to enhance the steady-state levels of Vif proteins from all tested HIV subtypes and required for the degradation of all restrictive human and rhesus APOBEC3 proteins by their respective lentiviral Vif proteins. Mutagenesis screening revealed that CBFB interacts with Vif and its normal RUNX transcription partners on genetically separable interfaces, indicating this essential virus-host interaction may serve as a viable drug target with minimal off-target effects. Disruption of this newly identified and highly conserved CBFB-Vif interaction would release the entire multitude of restrictive APOBEC3 proteins and significantly inhibit HIV infection, making this interaction a promising new target for small molecule therapeutics.
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    Human immunodeficiency virus evasion of APOBEC3 restriction factors
    (2012-10) Albin, John Squire
    The human immunodeficiency virus accessory protein Vif protects the viral genome from the mutational activity of APOBEC3 subfamily DNA cytosine deaminases by facilitating their proteasomal degradation, thereby preserving viral infectivity. A comprehensive understanding of the components of the Vif-APOBEC3 interaction is therefore important for consideration of the potential for novel antiretroviral approaches aimed at modulating this critical host-pathogen interaction. Here, we establish APOBEC3F among the seven subfamily members as a valid model for the study of the APOBEC3-Vif interaction. By utilizing this model as a starting point, we further define the APOBEC3-Vif interaction sites in each protein and the downstream ubiquitin acceptor sites modified en route to APOBEC3 degradation, in the process deriving broader insights into the nature of the interactions between different APOBEC3 proteins and Vif. In contrast with the diversiform APOBEC3-Vif interactions proposed in the extant literature, we find that the interaction of Vif with different APOBEC3 proteins likely proceeds through a conserved helix-helix interaction. Even if one were to successfully block this interaction for therapeutic purposes, however, the virus may develop accessory mechanisms of APOBEC3 evasion to bypass the intervention. While we find that this can occur, present evidence suggests that such alternatives may be insufficient to circumvent restriction in cells that naturally express multiple APOBEC3 proteins. Thus, it may be possible to potentiate the action of multiple endogenous antiretroviral proteins to counteract human immunodeficiency virus infection by targeting a conserved interaction motif as described herein.