Browsing by Subject "APOBEC3"

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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.
Counteraction of APOBEC3 Proteins by Herpesvirus Ribonucleotide Reductases
(2019-08) Cheng, Adam
The APOBEC3 family of DNA cytosine deaminases plays an important role in antiviral innate immunity. In this thesis, we describe the novel function of APOBEC3B as a physiologic restriction factor against herpesviruses such as Epstein-Barr virus and herpes simplex virus type 1. We additionally define the counteraction mechanism imparted by herpesviruses using the virus-encoded ribonucleotide reductase large subunit. These viral proteins directly bind A3B to inhibit enzymatic activity, relocalize it away from replicating viral DNA, and protect the virus from A3B-mediated hypermutation for preservation of the viral genome. These results have the potential to reveal new modes of antiviral therapy and have implications in the treatment of A3B-driven cancers.
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.
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.
Dynamics of the mammalian APOBEC3 locus and the relationship between mammalian APOBEC3 and Lentiviral Vif proteins.
(2010-08) LaRue, Rebecca St. Claire
The mammalian immune system must be dynamic in the face of a diverse and ever-changing array of pathogens. Successful pathogens have evolved to overcome host immunity. One of the most successful pathogens in the last century is the human immunodeficiency virus (HIV), which is the etiological agent responsible for the global acquired immune deficiency syndrome (AIDS) epidemic. Currently, one of the most studied host-pathogen interactions is between the cellular anti-retroviral APOBEC3 (A3) proteins and HIV viral infectivity factor (Vif) protein. A3 proteins are cytosine deaminases that primarily inhibit retroviruses and retrotransposons by mutating cytosines to uracils in retroviral DNA during reverse transcription. When HIV Vif is present, it can bind to certain A3 proteins and recruit cellular degradation complexes to target these anti-retroviral proteins for proteasomal degradation. HIV-like viruses (lentiviruses) infect other mammals and are the inspiration for studying A3 proteins in other mammals. The ultimate goal of this comparative study was to gain a better understanding of how the lentiviral Vif protein counteracts host A3 proteins. The first component of this dissertation is dedicated to the process of determining the complete A3 protein repertoires of cow and sheep (representatives of the artiodactyl lineage), which are both infected with a lentiviruses. The second component of this dissertation was to test if these artiodactyl A3 proteins were functional. Cow and sheep A3 proteins demonstrate intrinsic DNA cytosine deaminase activity and localize in cells similar to human A3 proteins. Furthermore, certain cow A3 proteins are capable of restricting HIV and are neutralized in the presence of cattle-specific lentiviral Vif (bovine immunodeficiency virus). The last component of this dissertation, a panel of conserved mammalian A3 proteins were tested to determine if each A3 protein interacts specifically with its species lentiviral Vif protein. It was shown that each representative host A3 protein is degraded in the presence of its species specific lentiviral Vif, suggesting a conserved interaction between mammalian A3 proteins and their species specific lentiviral Vif protein.
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.
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.
Studies of the mutagenesis and virus-host interactions of hepadnaviruses and retroviruses
(2021-11) McDaniel, Yumeng
Although viral infection is preventable by vaccination and antiviral treatments are available, up to 1 million people die every year from hepatitis B virus (HBV)-associated liver diseases. In line with this notion, there is an effective vaccine available for feline leukemia virus (FeLV), but FeLV remains a common infection among domesticated cat populations and accounts for many clinical syndromes that typically end with death within 1 to 3 years after the initial diagnosis. Therefore, the need for new antiviral agents for treating human and veterinary viral pathogens exists, even in instances where effective vaccines are available. Reverse transcriptase (RT) is an essential enzyme for the replication life cycle of both retroviruses (i.e., human immunodeficiency virus type 1, HIV-1) and hepadnaviruses (i.e., HBV). Previous studies have shown two types of small molecules possessing anti-HIV-1 activity, mainly through targeting reverse transcriptase (RT) and the reverse transcription pathway - namely viral mutagens (i.e., decitabine, 5-azacytidine and KP1212) and ribonucleotide reductase inhibitors (RNRIs) (i.e., gemcitabine and resveratrol). Our research group has shown that decitabine and gemcitabine can also inhibit FeLV and murine leukemia viruses (MuLV) replication, which are closely related gammaretroviruses. Based upon these observations, Part 1 of this dissertation sought to test following two hypotheses: 1) mutagen and RNRI or the combination of a mutagen and an RNRI will possess potent anti-HBV activity; and 2) distinct antiviral mechanisms can be elicited by a viral mutagen. A family of host proteins, i.e., human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (APOBEC3, A3) proteins, can induce viral mutagenesis, and therefore act as a cellular-based viral mutagen. APOBEC3 family member proteins can deaminate cytosines in single-strand (ss) DNA, which restricts HIV-1, retrotransposons, and other viruses such as HBV, but can cause a mutator phenotype in many cancers. Part 2 of this dissertation sought to characterize the deamination hotspots of APOBEC3 proteins, and to test the hypothesis that deamination hotspots among APOBEC3 family members are defined by both target site sequence and ssDNA structure. Although anti-HBV therapeutic discovery targeting host factors and screening for inhibitors are in progress, there is a significant knowledge gap regarding HBV-host cell interaction. This line of research investigation is critical to public health as HBV infection accounts for 54% of all hepatocellular carcinomas (HCCs), which is notable as it is the second highest cause of cancer-related mortality worldwide. An enhanced understanding of HBV-host cell interactions will help to improve general knowledge of HBV biology, identify potential targets for antiviral intervention to prevent HCC, and provide insights that could prove useful in the early diagnosis of HCC and the discovery of HCC-targeted therapeutics. Part 3 of this dissertation describes the results of an analysis of HBV-infected cells for testing the hypothesis that host genes involved in the cellular antiviral response and HCC development can be identified by transcriptome analysis. Together, the studies conducted in this dissertation serve to lay the foundation for future basic and translational research studies.
Studies on the basis of HIV-1 variation and its prposeful increase
(2012-12) Dapp, Michael J.
Unconventional measures are needed in an effort to outpace the HIV/AIDS pan- demic. Even with access to the six classes of FDA-approved antiretrovirals, adverse drug effects and evolution of drug resistance still pose obstacles, and represent issues that will inevitably arise in the developing world. Along with the absence of a proven vaccine strategy, these shortcomings necessitate the continual search for new therapeutic targets. One such novel approach is to target HIV-1’s low copying fidelity with a deliberate in- crease in viral mutational load using exogenous small molecule mutagens. However, in- asmuch as this concept of viral lethal mutagenesis has gained notoriety, it is also essential to understand fundamental enzymatic components (i.e., reverse transcriptase) that con- tribute to variation. The goal of this dissertation was to advance basic models to better understand the causes and consequences of HIV-1 variation. The components that influence variation within an HIV-1 population structure are critical to predict the emergence and direction of viral evolution (e.g., drug resistance). For these studies, the relationship between viral fitness and mutation rate was investigat-ed. A panel of 10 reverse transcriptase mutants – most having drug resistance pheno- types – were analyzed for their effects based on these two biological properties. Muta- tion rate differences were measured using single-cycle vector assays, while fitness differ- ences were identified using ex vivo head-to-head competition assays. As anticipated, vi- ral mutants possessing either higher or lower mutation rate had a corresponding loss in fitness. These observations provided the first description of an interrelationship between HIV-1 fitness and mutation rate and support the conclusion that mutator and antimutator phenotypes correlate with reduced viral fitness. A second focus of this dissertation was directed at studying novel mechanisms by which viral mutagens diminish HIV-1 infectivity. I have detailed the antiviral mecha- nisms of the ribonucleoside analog 5-azacytidine. It was demonstrated that the primary antiviral activity of 5-azacytidine can be attributed to its effect on the early phase of HIV- 1 replication mediated by reverse transcriptase. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants. Sequencing analysis showed enrichment in guanosine-to-cytidine (G-to-C) transversion mutations. These re- sults indicated that 5-azacytidine was incorporated into viral DNA following its 2’-OH reduction to 5-aza-2’-deoxycytidine. Incorporation into the viral DNA led to an increase in mutant frequency, which is consistent with lethal mutagenesis. Lastly, studies were directed at understanding concomitant exposure of two unre- lated mutagenic agents. Because the APOBEC3 proteins’ restrictive nature associated with cytosine deamination, I wanted to investigate its interplay with the small molecule cytosine analog, 5-azacytosine. Reduced viral infectivity and increased viral mutagenesis were observed with both the viral mutagen 5-azacytosine (i.e., G-to-C mutations) and the host restriction factor APOBEC3G (i.e., guanosine-to-adenosine (G-to-A) mutations); however, when combined, they had complex interactions. Nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-azacytosine and APOBEC3G result- ed in an increase in G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. Al- so, APOBEC3G catalytic activity was required for the diminution in G-to-C mutagenesis. These findings provided the first demonstration for potentiation of the mutagenic effect of a cytosine analog by APOBEC3G expression, resulting in concomitant HIV-1 lethal mutagenesis. In summary, the studies conducted in this dissertation 1) provide the first direct experimental evidence of an interrelationship between HIV-1 fitness and mutation rate, 2) demonstrate that the primary antiviral mechanism of 5-azacytidine can be attributed to its ability to increase the HIV-1 mutation frequency through viral DNA incorporation during reverse transcription, and 3) provide the first demonstration for potentiation of the muta- genic effect of a cytosine analog (i.e., 5-azacytosine) by APOBEC3G expression, result- ing in concomitant HIV-1 lethal mutagenesis.

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