Browsing by Subject "HIV"

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    Cloning and characterization of MRI, a modulator of retroviral infection.
    (2009-12) Agarwal, Sumit
    Although significant progress has been made in elucidating the life cycle of HIV and identifying host cell proteins which interact with HIV, a much greater understanding of these processes is necessary in order to eradicate this disease. The studies presented in this dissertation further examine these processes and identifies a new host cell protein, MRI, which appears to be involved in the poorly understood step of viral uncoating.The first part of my project involved the creation, characterization, and utilization of a novel toxic retroviral vector encoding barnase, a non-specific protease derived from Bacillus amyloliquefaciens. In conjunction with an inhibitor of barnase (barstar), I showed that barnase-based retroviral vectors were capable of inducing cell death in a wide variety of cell types and that the presence of barstar was essential for the creation of high titer retroviral vectors. The second part of my project was to was to create a heavily mutagenized hamster cell line, V79-4, and repeatedly challenge this cell line with the toxic retroviral vector in order to isolate clonal populations of cells resistant to retrovirally mediated transduction. Ultimately, I isolated two cell lines, 31-2 and 67-1, which we characterized further. 31-2 was determined to be least five fold resistant to both MLV- and lenti-viral at a stage in viral replication post reverse transcription. 67-1 was minimally ten fold resistant to both MLV- and lenti-viral infection at a stage between viral entry and uncoating. We demonstrated that the mutation in 67-1 implicated the proteasome and identified a gene we designated MRI that reversed the inhibition of infection in the mutated 67-1 line. The implication of MRI in the retroviral lifecycle identifies a new protein that could facilitate the creation of novel host cell based therapy for HIV.
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    Correlates of Annual Testing for Sexually Transmitted Infections (STIs) in an Online Sample of Men Who Have Sex with Men (MSM): Study Sample Validity, Measure Reliability, and Behavioral Typologies
    (2013-07) Grey, Jeremy
    Objective: Testing for STIs has been prioritized as part of a comprehensive HIV/AIDS prevention plan. Internet-based studies of STI testing among men who have sex with men (MSM) are efficient methods of recruiting non-clinic samples from diverse geographic areas. However, online survey methods raise unique concerns regarding threats to the validity of study samples and unknown measurement properties. Thus, this dissertation had two aims. The first was to examine methods related to online survey research by evaluating a protocol to detect invalid survey entries and determining the test-retest reliability of online measures of sexual behavior and STI testing. The second aim was to use the validated sample and reliable measures to examine correlates of STI testing in the year prior to the survey. Methods: In Manuscript 1, survey submissions were classified as valid and invalid according to a de-duplication and cross-validation protocol. Logistic regression models were used to determine associations between invalidity and key demographic and behavioral variables. In Manuscript 2, test-retest reliability over one week was evaluated using intraclass correlation coefficients (ICCs) and kappa statistics for measures of sexual behavior, HIV status, HIV testing, and STI diagnoses. Finally, in Manuscript 3, the valid sample from Manuscript 1 and measures that were evaluated in Manuscript 2 were used to examine the clustering and correlates of STI testing behaviors. Results: In Manuscript 1, three components of the protocol for detecting invalid submissions were responsible for identifying the most invalid survey submissions: duplicate IP address, changed eligibility responses, and duplicate payment name. A total of 146 (11.6%) of the submissions were identified as invalid. Invalid submissions had lower odds of reporting HIV testing in the past year. Hispanic/Latino identity, age, and HIV status were also significantly associated with invalidity. In Manuscript 2, counts of sexual partners (three months), HIV status, HIV testing, and STI diagnoses were found to have substantial (0.61-0.80) to almost perfect (0.81-1.00) seven-day test-retest reliabilities, according to commonly used cutpoints. Partner-specific data, however, were only fairly or moderately reliable (0.21-0.60). Finally, in Manuscript 3, a latent class analysis indicated five STI testing classes: no STIs, all STIs, bacterial STIs and hepatitis, bacterial STIs only, and hepatitis only. The largest class was no STIs, indicating that 45.8% of the validated sample had not been tested for STIs in the past year. Predictors of being in a testing class versus no STI testing included age, education, outness about having sex with men, HIV status, and having a sexual partner in the last three months. Conclusions: This dissertation served two primary aims. The first was to evaluate sample validity and measure reliability in an online study of MSM. The second was to apply the information from those analyses to examine the presence and correlates of a latent variable of STI testing. Across all three manuscripts, online survey research appears to be a viable method of studying STI testing in Internet-based samples of MSM.
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    Correlates of cytotoxic T lymphocyte vaccine-induced control against Simian immunodeficiency virus (SIV)
    (2018-10) Mohammad, Hadia
    There is an urgent need to develop a successful HIV vaccine. Despite continuous efforts, development of a protective HIV-1 vaccine remains a big challenge. HIV-1/SIV-specific CD8+ T cells play a pivotal role in the control of virus replication. During chronic HIV-1 and SIV infections, virus replication is most concentrated within lymphoid B cell follicles, whereas virus-specific CD8+T cells concentrate in extrafollicular areas of secondary lymphoid tissues and tend to be excluded from follicular areas. My thesis focuses on identifying immune correlates of control associated with successful CD8+T cell-based SIV vaccines to help understand what is required to develop a successful HIV vaccine. My central hypothesis is that vaccine-induced control will be associated with induction of an early SIV-specific CD8+T cell response at the portal of viral entry and in lymphoid tissues and also with induction of high levels of virus-specific CD8+T cells in follicular areas of lymphoid tissues. I determined the location, abundance, and phenotype of virus-specific CD8+T cells and the abundance of virus-infected cells in vaccinated and unvaccinated rhesus macaques. Freshly collected unfixed tissue samples were analyzed using in situ tetramer staining combined with immunohistochemistry, in situ hybridization, confocal imaging, and quantitative image analysis. I found that vaccine-induced control was associated with induction of virus-specific CD8+T cells before challenge with pathogenic SIV. Virus-specific CD8+T cells expanded rapidly after challenge and resulted in high effector (SIV-specific CD8+T cells) to target (SIV-infected cells) ratios at the portal of viral entry and in lymph nodes. Additionally, vaccine-induced control correlated with the induction of high follicular: extrafollicular ratio of virus-specific CD8+T cells in lymphoid tissues. Moreover, this control correlated with induction of SIV-specific CD8+T cells that expressed little perforin and with SIV-specific CD8+ T cells in which perforin was exclusively localized to the cell membrane in lymphoid tissues. These cells are likely effector memory T cells with an immediate killing ability. These findings provide a better understanding of the immune correlates of CD8+T cell-based vaccine-induced control against SIV and provide understating of what is needed to create an effective HIV vaccine.
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    Disruption of Gut Homeostasis by Opioids in the Early Stages of HIV Infection
    (2014-12) Sindberg, Gregory
    Opioids are a common comorbidity with HIV, with the use of opioids being present in up to 40% of the HIV infected population in some countries. Opioids have been shown to worsen HIV pathogenesis, including increased viral replication and faster progression to AIDS. HIV pathogenesis has been shown to be important in the gastrointestinal tract, where early loss of CD4+ T-cells has been observed in SIV infection and infection with either HIV or SIV show evidence of systemic bacterial translocation which is believed to drive HIV replication. Opioids are believed to worsen this effect and have been shown to increase bacterial translocation in HIV patients. The second chapter study was performed to understand the underlying disruption of gut homeostasis that contributes to bacterial translocation. HIV models were validated to show bacterial translocation, and then look at gut morphology, tight junction localization on gut epithelium, and immune function within the gut at early time points of exposure to HIV infection. Overall, based on the measures examined, opioids enhanced the pathogenesis of HIV in the gut at early infection which likely contributes to the greater replication and faster development of AIDS. While the loss of gut homeostasis is strongly believed to occur at least in part through changes in the host defenses in the gut, namely on immune populations and epithelial barrier integrity, recent evidence suggests that the microbiome of late stage HIV infected individuals is altered and may contribute to the observed disruption. The third chapter investigated the microbiome in early HIV infection to see if dysbiosis occurs, and whether opioids are associated with earlier changes. Using two animals models of infectious HIV, microbial dysbiosis was not observed at early time points of infection in either model. However, this study shows for the first time that morphine induced strong changes in the microbiome, which likely occurs via a combination of constipation and immune mediated effects. Altogether, these findings suggests another mechanism for morphine influencing HIV pathogenesis at early stages of disease. Combined, these studies show the wide ranging effects that opioids and HIV have on gut defenses, including epithelial barrier, immune function, and dysbiosis from the normal microbiome. While mostly descriptive in nature, the results give potential therapeutic opportunities, including potential oral administration of TLR2 and TLR4 antagonists, opioid antagonist naloxone, and bile acids in order to supplement deficiencies in metabolites observed.
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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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    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.
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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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    HIV-associated Cryptococcal Meningitis in sub-Saharan Africa: Factors affecting short and long-term survival
    (2013-09) Rolfes, Melissa
    Cryptococcal meningitis (CM) is a wide-spread, yet under-recognized, fungal opportunistic infection occurring primarily among people living with advanced HIV/AIDS. While vast advances in understanding the pathogenesis and treatment options for CM have reduced mortality, major gaps remain in understanding factors that contribute to mortality rates of 12-20% in the first two weeks. The intent of this dissertation was to contribute towards the efforts to address these gaps and provide evidence that could further improve short and long-term recovery from HIV-associated CM in sub-Saharan Africa. The first paper was focused on mortality in the days after CM diagnosis and understanding whether lumbar punctures (LPs) to reduce intracranial pressure also reduce mortality. Raised intracranial pressure is common in CM patients and contributes to many of the disease's signs and symptoms. Two hundred forty-eight HIV-positive, CM patients from Uganda and South Africa were evaluated for the occurrence of therapeutic LPs and mortality within 11 days of CM diagnosis. Analysis was conducted using a marginal structure model, with time-varying exposure. The results suggest that undergoing at least one repeat LP reduced 11-day mortality by 69% (95% CI: 18% to 88%), adjusted for heart rate, CSF fungal burden and altered mental status. This beneficial effect was independent of the baseline CSF opening pressure, demonstrating that increases in intracranial pressure may be common among all CM patients and that all patients may benefit from an additional LP during initial therapy. The second paper was aimed at investigating baseline demographic and clinical features predictive of CM treatment success. The recommended initial treatment regimen for CM rapidly reduces infection; however, nearly 50% of CM patients will continue to have viable fungus in their central nervous system at the end of therapy. Being able to predict patientutcomes has many advantages including optimizing treatment for each patient and reducing drug toxicities. One hundred seventy-seven HIV-positive, CM patients undergoing 2 weeks of antifungal treatment, including amphotericin B and fluconazole, were evaluated for sterility of a CSF culture by the end of therapy. The baseline CSF quantitative fungal burden was a strong and practical predictor of achieving CSF sterility, along with the rate of fungal clearance over the first week of treatment, and the baseline hemoglobin. Information on the baseline burden of infection could be used to tailor the duration of treatment, thus avoiding unnecessary toxicity and treatment costs for individuals with a lower burden of infection and potentially shifting resources to allow for more aggressive treatment of high-risk patients. The third paper aimed to understand the consequence of residual fungal infection at the end of initial antifungal therapy in terms of mortality in the first weeks and months after 2-week therapy ends. Among 154 HIV-positive individuals surviving the initial 2-week phase of therapy, there was no evidence that either the presence or the amount of residual cryptococcal infection in the CSF had an association with mortality in the following 3 weeks or 6 months. It is possible that a higher dose of fluconazole used at the end of amphotericin therapy in the present cohort may have contributed to the lack of association. The objective of this dissertation was to expand the understanding of CM treatment and recovery in resource-limited settings. The results suggest that additional benefits could be gained from the use of therapeutic lumbar punctures during the acute phase of treatment, the possibility of customizing therapy to further reduce treatment toxicities, and, finally, describing the relationship between residual infection and CM mortality with indirect support for higher doses of fluconazole during subsequent phases of CM therapy.
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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.
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    Identifying medication-related needs of HIV patients: foundation for community pharmacist-based services
    (University of Minnesota, College of Pharmacy, 2014) Kauffman, Yardlee; Barkowitz, Elana; Cerussi, Nicole; Pringle, Jan; McGivney, Melissa
    Background: Patients living with HIV/AIDS have complex medication regimens. Pharmacists within community pharmacy settings can have a role managing patients living with HIV/AIDS. Patients’ perspectives surrounding implementation about community pharmacist-based services is needed as limited information is available. Objective: To identify medication-related needs of HIV-infected patients who receive prescriptions from a community pharmacy. To determine patient perspectives and knowledge of community pharmacist-based services. Methods: A qualitative research study involving in-depth, semi-structured interviews with patients was conducted. Inclusion criteria included: HIV positive men and women at least 18 years of age who receive care at a HIV clinic, currently take medication(s) and use a community pharmacy for all prescription fills. Patients were recruited from one urban and one rural health center. Patients answered questions about their perceptions and knowledge about the role and value of pharmacy services and completed a demographic survey. The recordings of the interviews were transcribed verbatim and were analyzed using principles of Grounded Theory. Results: Twenty-nine interviews were conducted: 15 participants from the urban site and 14 from the rural site. Five main themes emerged including: patients experience ongoing and varying medication-related needs; patients desire a pharmacist who is caring, knowledgeable and integrated with health care providers; patients expect ready access to drug therapy; patients value an individualized patient encounter, and patients need to be informed that a pharmacist-service exists. Conclusion: Patients with HIV value individualized and personal encounters with pharmacists at time intervals that are convenient for the patient. Patients felt that a one-on-one encounter with a pharmacist would be most valuable when initiating or modifying medication therapy. These patient perspectives can be useful for pharmacists and pharmacies interested in providing advanced care to patients with HIV.
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    Lymphoid tissue damage in HIV and SIV infections depletes naive T cells and limits immune reconstitution after anti-retroviral therapy.
    (2012-06) Zeng, Ming
    HIV infection causes the Acquired Immunodeficiency Syndrome (AIDS), a condition in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. HIV primarily infects and depletes CD4+ T cells, a cell type that is central to the maintenance of host's immunocompetency. The slow depletion of CD4+ T cells has been attributed to direct mechanisms such as viral killing and indirect mechanisms such as increased apoptosis accompanying the chronic immune activation associated with HIV infections. Highly active anti-retroviral therapy (HAART) can potently suppress HIV viral replication and largely normalize associated immune activation thereby significantly increasing the CD4+ T cells. Yet more than a decade after the introduction of HAART, it is clear that relatively few individuals will achieve normal levels of peripheral blood CD4+ T cells, and up to 20% of the patients have little or no increase despite the impact of HAART on the viral replication. This limited immune reconstitution is most prevalent in patients starting HAART in the chronic stage of infection and strongly correlates with significantly higher morbidity and mortality compared to uninfected populations. This suggests that there is a persistent defect in the homeostasis of CD4+ T cell population that is not fully corrected by HAART. However, the underlying mechanisms of this limited immune reconstitution after HAART remain elusive. More recently, there is increasing evidence showing that there is progressive fibrotic damage during HIV infection to the structure of lymphoid tissue (LTs) and the extent of this damage strongly correlates with the extent of depletion of naïve CD4+ T cells before HAART and the extent of immune reconstitution after HAART. Since LT structure is critical for the homeostasis of naïve T cells, we hypothesized that the LT damage during HIV infection contributes to CD4+ T cell depletion and limits immune reconstitution after HAART. In this dissertation, I tested this hypothesis by providing experimental data to show the mechanisms of LT damage and how LT damage contributes to CD4+ T cell depletion and limited immune reconstitution. The understanding of these mechanisms points to novel avenues to improve immune reconstitution in HIV-infected patients.
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    Mechanisms of HIV-associated neurotoxicity: Tat-induced synapse loss and recovery
    (2013-05) Brunner, Angela Haijung
    HIV infection is a worldwide pandemic. A debilitating neurological consequence of HIV infection is progressive cognitive decline, known as HIV-associated neurocognitive disorders (HAND). HAND afflicts up to 50% of all HIV patients to varying degrees, and as survival of HIV patients improves with current antiretroviral therapies, the prevalence of HAND is also increasing. This, coupled with the lack of current effective HAND therapies, creates a dire need to understand the mechanisms underlying the cognitive decline associated with HIV. HAND symptoms correlate closely with processes of neuronal injury, which are early events that precede overt neuronal death. One such injurious process is synapse loss. The HIV protein transactivator of transcription (Tat) is a neurotoxic viral protein released from infected cells into the central nervous system. Tat contributes to the pathologies seen in HAND patients, and induces loss of excitatory synapses between rat hippocampal neurons in culture. Using an innovative live cell imaging assay, our laboratory has previously shown that Tat induces reversible synapse loss via a pathway that is distinct from cell death. In this dissertation, I outline three studies that stem from the current knowledge involving Tat-induced synapse loss. These studies elucidated important information regarding the mechanisms by which HIV Tat exerts its neurotoxic effects, emphasizing the importance of subunit composition when determining toxic or beneficial effects of NMDA receptor activation as well as unmasking the importance of the postsynaptic density as the central target of Tat's effects. Furthermore, these studies highlight the reversibility of synapse loss and uncover a new role for the canonical NO/cGMP/PKG pathway in modulating synapse recovery downstream of GluN2B-containing NMDA receptors. Tat-induced synapse loss and subsequent recovery can correlate to symptoms of cognitive decline seen in HAND. Targeting these mechanisms can shed new light on therapeutic strategies to treat HAND patients.
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    Modulation of the Endocannabinoid System By Hiv Tat Protein
    (2020-06) Wu, Mariah
    Despite the success of combined antiretroviral therapy (cART) in extending the lifespan and enhancing the quality of life for those infected with HIV, nearly half of the 37.9 million HIV-positive individuals experience some degree of neurocognitive impairment. These impairments are collectively termed HIV-associated neurocognitive disorders (HAND). HAND symptoms range from subtle difficulties in the tasks of daily living to severe functional impairment and dementia. cART effectively suppresses viral load and improves patient survival; however, while the severity is reduced, the prevalence of HAND remains high and may be increasing due to the prolonged lifespan of HIV patients. Currently, there are no effective treatments for HAND. HIV neurotoxicity is mainly mediated by inflammatory and excitotoxic agents released from infected macrophages and microglia. The HIV trans-activator protein Tat is one such agent that plays a major role in the neuropathogenesis of HAND. While cART suppresses viral replication, it does not halt the production of Tat once HIV has integrated into the host genome. Numerous studies have demonstrated that Tat causes excitotoxicity, which leads to a loss of spine density and altered network function; this synaptic dysfunction predicts marked impairments in learning and memory paradigms in various in vitro and in vivo models of HAND. Thus, it is essential to elucidate the mechanisms that cause and exacerbate neurotoxicity, in order to find effective therapeutic targets to ultimately treat HAND symptoms. A growing body of literature indicates that components of the endocannabinoid (eCB) system may be viable therapeutic targets. Across many models of neurological disorders that share a common pathophysiological pathway including excitotoxicity and neuroinflammation, activation of the eCB system attenuates detrimental cellular processes. Recent reports show that activating the eCB system protects against HIV-induced neurotoxicity, indicating that modulation of the eCB system may be a viable therapeutic approach to suppress the neuronal damage that underlies HAND. However, one important determinant of clinical outcome is the degree to which the target biological system is functional. Exposure to excitotoxic stimuli alters the eCB system; whether eCB signaling is altered in the presence of HIV is unknown. In this dissertation, I explore the effects of HIV Tat protein on eCB signaling in vitro. Using an electrophysiological approach, I determined that Tat impaires cannabinoid type 1 receptor (CB1R) function in a subset of neurons without affecting other components of the eCB system. Taken together, these results indicate that Tat specifically impairs CB1R-mediated presynaptic inhibition of glutamate release. This observation suggests that eCB-mediated neuroprotection may be reduced in vivo, possibly exacerbating synaptodendritic injury caused by HIV. Thus, this dissertation provides new insight into processes that further contribute to HIV-induced neurotoxicity and suggests that protecting or enhancing the neuroprotective eCB system may attenuate the symptoms of HAND.
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    Neural adaptations following exposure to HIV proteins
    (2018-07) Green, Matthew
    HIV associated neurocognitive disorders (HAND) affect nearly half of the population infected with HIV. Symptoms range from subclinical cognitive deficits to severe dementia which can affect daily living. The neurotoxicity produced is likely indirect as the virus does not infect neurons but infects other cells in the brain such as microglia and astrocytes which can release viral proteins and inflammatory cytokines that then affect neurons and lead to synaptodendritic damage. Thus, understanding how these toxic viral factors affect neurons and the mechanisms underlying these changes will aid in the discovery of therapies for HAND. In this dissertation, I report viral proteins altering two types of receptors involved in regulating neuronal excitability and synaptic transmission, the excitatory NMDA receptor (NMDAR) and the inhibitory GABAAR. NMDARs initially become overactivated but then downregulate in hippocampal cultures exposed to the HIV protein transactivator of transcription (Tat). This downregulation was mediated by GluN2A-containing NMDARs signaling to the kinase Akt and the E3 ubiquitin ligase, Mdm2. This novel mechanism delineates a signaling pathway activated by viral proteins that regulates NMDAR-dependent loss of excitatory synapses. Second, I determined how the viral protein gp120 affects GABAergic inhibition. In hippocampal cultures, gp120 caused an increase in tonic GABAAR currents mediated by extrasynaptic GABAARs and an increase in the number of inhibitory synapses. Both increases in inhibition were dependent on microglial activation and release of interleukin-1β which activated interleukin-1 receptors. The increase in inhibitory synapses was dependent on IL-1R-mediated Src activation and subsequent potentiation of GluN2A-containing NMDARs and protein synthesis. On the other hand, the increase in tonic inhibition was dependent on IL-1R-mediated p38 MAPK activation and selective upregulation of α5-containing GABARs. The increases in inhibition likely dampen neuronal excitability and network function and may contribute to the cognitive deficits in HAND. These studies elucidate changes in two types of receptors affected by viral proteins and identify novel signaling pathways that may lead to therapeutic targets for HAND.
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    Pathologies of Care: HIV Treatment and Prevention in Turkey
    (2023) Atuk, Tankut
    This dissertation investigates the ambivalent, elusive, and contradictory nature of care that produces abandonment and harm but dialectically gives rise to vital modes of belonging and carefulness. In Turkey, the number of HIV diagnoses has increased by 620%, and AIDS- related deaths have more than doubled over the last decade. Yet, formal regimes of HIV care block access to testing, condoms, and pre- and post-exposure prophylaxis; impose monogamy as a scientifically legitimate prevention method; deny (health)care to LGBTQI+s and people living with HIV; and refuse sexual health education. This dissertation draws on twenty months of embedded ethnography, public and private archives, medical records, newspaper articles, official government reports, and fifty in-depth interviews with governmental, nongovernmental, medical, and pharmaceutical sector workers. Through the concept of pathogenic care, this investigation uncovers how conservative and neoliberal ethics and mechanisms of public health aggravate the conditions of those living with HIV and increase the risk of transmission for others, especially the marginalized, who are socio- immunologically more vulnerable. In other words, this research demonstrates that HIV care has become pathological in Turkey by facilitating a joint epidemic of HIV and HIVfobi, i.e., status-based stigma and discrimination. By setting a sterile distance between themselves and the so-called “contagious others,” formal regimes of HIV care create zones of abandonment where particularly queer and trans communities are left vulnerable to HIV transmission and socio-medical discrimination. This study makes three central arguments with important theoretical and public health implications: (1) the Turkish HIV epidemic is not an inevitable result of dissident/terrorist sexualities, foreign lifestyles, or human immunodeficiency virus; (2) public health mechanisms, institutions, and actors in place to provide HIV treatment and prevention have become unexpected vectors of HIV transmission under neoliberal Islam; (3) the fear of medical and social contagion and the consequent impulse to immunize life against “risky” others lie at the center of Turkey’s failed public health response to the growing HIV epidemic.
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    Regulation of APOBEC3B and the Restriction of HIV-1 in Myeloid Cells
    (2018-07) Molan, Amy
    The APOBEC3 (A3) DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several A3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) and APOBEC3G (A3G) show strong virus restriction activities in a variety of experimental systems. A3B and A3G are also subject to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity of A3B and HIV-1 Vif-mediated degradation of A3G. After observing several potential acetylations on A3B in a mass spectrometry screen, we asked whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells. HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes (if any) involved in HIV-1 restriction in macrophages has yet to be delineated. We show that multiple APOBEC3 genes are expressed in myeloid cell lines including THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus indicating the presence of at least one functional APOBEC3 enzyme. Proviral DNA resulting from such infections shows strong G-to-A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses selectively sensitive to APOBEC3G show Vif-null virus-like infectivity levels and similarly strong APOBEC3G-biased G-to-A mutation spectra. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells. Overall, my thesis research provides new insights into the post-translational regulation of A3B as well as uncovering a novel restriction mechanism in myeloid lineage cells. This research provides a great backbone to build on in understanding how the HIV-1 replication cycle works in myeloid cells as well as contributing to the understanding of the post-translational regulation of A3B.
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    The Role of Ambulatory Care Pharmacists in an HIV Multidisciplinary Team within a Free and Bilingual Clinic
    (University of Minnesota, College of Pharmacy, 2013) Vanmali, Radha S; Mayer, Sallie D; Fugit, Ann M
    Objective: Describe the role and integration of ambulatory care pharmacists in a Human Immunodeficiency Virus (HIV) clinic within a free and bilingual clinic with regards to types of interventions made during the patient-pharmacist visit. Design: Retrospective, single-centered, chart review. Setting: Free, bilingual clinic in Richmond, VA. Participants: Thirty-two adult patients with diagnosed HIV receiving care in the clinic between June 30, 2010 and January 26, 2011. Main Outcome Measure: Types of interventions documented during the patient-pharmacist visit, categorized as medication review, patient education, or adherence monitoring. Results: Total of 32 patients accounted for 55 patient-pharmacist visits and 296 interventions. The most common interventions were medication review (66.9%), patient education (23.3%), and adherence monitoring (9.8%). Post-hoc analysis suggests Hispanic patients are more likely to be diagnosed with Acquired Immune Deficiency Syndrome (AIDS) (P = 0.01), have current or history of opportunistic infection (OI) (P=0.01), and have current or history of OI prophylaxis (P = 0.03). Adherence monitoring was less common amongst the non-Hispanics (7.1%) compared to the Hispanic sub-population (16.5%), (P = 0.04). Conclusion: The role of ambulatory care pharmacists in a free and bilingual clinic goes beyond adherence monitoring. Pharmacists can be a valuable part of the patient care team by providing medication review and patient education for HIV and other co-morbidities within free clinics. Further research is warranted to assess outcomes and to further explore the underlying barriers to early HIV diagnosis and adherence within the Hispanic population.
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    Structural and Mechanistic Insight into APOBEC3G DNA Binding and Deamination
    (2015-07) Solomon, William
    Humans express the APOBEC3 family of proteins to defend against endogenous and exogenous DNA pathogens. APOBEC3 proteins display significant activity towards HIV-1 through incorporation into budding viral particle and interacting with HIV's RNA genome. In order to stably integrate into the human genome, HIV reverse transcribes the single stranded RNA genome into a double stranded DNA genome through a single stranded DNA intermediate. Once bound to the transient single stranded viral DNA intermediate, APOBEC3 proteins deaminate cytidines to uridines. Transcription over the resulting mutations results in G to A transversions in the coding sequence of the virus and non-functional gene products. APOBEC3 proteins are highly active on the single stranded DNA intermediate but lack catalytic activity on cytidines present in the viral RNA. APOBEC3G is the most potent of these innate viral mutagens and selectively targets tri-cytidine hotspot motifs in viral genome intermediates. When I began my thesis research, the effects of A3G mutagenesis had been extensively studied but the structural interactions with single stranded DNA and the mechanism of RNA exclusion were unknown. My thesis research helped identify the amino acid responsible for pH dependent effects on substrate binding. The identification of this residue allowed for the development of a pH insensitive variant of A3G that provided indirect evidence for a reduced pH in the viral capsid during the initiation of reverse transcription. These results, along with the kinetic characterization of A3Gctd and determination of the substrate factors crucial for deamination, are described in Chapter 2. In chapter 3, I continued to explore the effects of this pH dependent increase in binding affinity. I utilized NMR spectroscopy to identify the structural interactions of catalytic substrate binding. By generating a structurally stable but catalytically inactive mutant, I was able to identify differences between substrate interactions. This mutant also allowed me to explore structural interactions involved in substrate recognition and RNA exclusion. This lead to the identification of a novel substrate and a ribose sugar pucker dependent mechanism for target discrimination. The role of APOBEC3 proteins in retroviral restriction as well as their connection to several types of cancer makes them a prime target for therapeutic interventions. My thesis research in trying to understand the structural and mechanistic components of the APOBEC3/DNA interaction provides information that may be useful in the development of treatments targeting this family of proteins.
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    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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    Studies On The Determinants Of HIV Mutagenesis And Strategies For Its Enhancement
    (2015-12) Rawson, Jonathan
    Human immunodeficiency virus type-1 (HIV-1) is one of the fastest evolving entities on earth, due in part to a mutation rate that is at least 10,000-fold higher than that of eukaryotic genomic DNA. The adaptability of HIV-1 prevents clearance of the virus by the immune system, promotes drug resistance, and has impeded development of effective vaccines. The viral mutation rate is a composite result of mutations that are contributed by multiple host and viral enzymes involved in viral replication. However, the precise determinants of viral mutagenesis in HIV-1 and the degree of their contribution to HIV-1 genetic diversity remain incompletely understood. Furthermore, differences in viral mutagenesis among different HIV types (e.g., HIV-1 and human immunodeficiency virus type 2, HIV-2) and subtypes have not been explored to date. Lastly, the elimination of HIV-1 infectivity by increasing the viral mutation rate has not been extensively investigated – in terms of mechanism of action and in preclinical and clinical evaluations. Based upon these general observations, this dissertation research was conducted to test the following hypotheses: 1) HIV-1 and HIV-2 have different mutation rates; 2) decitabine, a HIV-1 mutagen, creates G-to-C transversion mutations in the absence of mutational hotspots; 3) 5-azacytidine causes HIV-1 mutagenesis after reduction to 5-aza-2’-deoxycytidine (i.e. decitabine); 4) the anti-HIV-1 activity of 5-azacytidine is antagonized by inhibitors of ribonucleotide reductase; 5) the anti-HIV-1 activity of ribonucleotide reductase inhibitors are potentiated by 5,6-dihydro-5-aza-2’-deoxycytidine. The studies conducted in this dissertation advance current understanding of the determinants for retroviral mutagenesis, the mechanisms by which small molecules promote mutations in HIV-1, and approaches for using combinations of small molecules to reduce viral infectivity by enhancing the mutagenesis of HIV-1. Supplementary data (figures, tables, and spreadsheets) associated with this thesis are available online.