The influence of T cell receptor signaling on human immunodeficiency virus infection

A significant barrier to curing Acquired Immunodeficiency Syndrome (AIDS) is the presence of latent reservoirs of cells infected with an integrated but transcriptionally silent provirus that is unaffected by the immune response or by antiretroviral drugs. Therefore, while antiretroviral therapy is able to suppress patient viral loads to clinically undetectable levels, upon cessation of treatment, viral loads rebound rapidly as virus is released from the latent reservoir. An understanding of the Human Immunodeficiency Virus (HIV) transcriptional regulatory events that contribute to viral latency is an important step towards eradicating the latent reservoir. Of particular interest are the mechanisms early in HIV infection that direct the cell towards productive or latent infection. The current study aims to determine whether the strength of T cell receptor (TCR) signaling at the time of HIV infection is correlated with the level of HIV transcription and to characterize the T cell receptor signaling pathways that regulate HIV transcription. We hypothesize that the strength of signaling at the time of infection determines the magnitude of early HIV transcription. Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing the C6.5 chimeric antigen receptor shows a 1.5-fold increase in transcription compared to unstimulated controls while Jurkat E6.1 T cells expressing the B1D2 chimeric antigen receptor shows a nearly 3-fold increase in transcription compared to unstimulated controls. These results suggest that the strength of signaling through the TCR at the time of infection determines the magnitude of HIV transcription early in infection and may contribute to the establishment of productive or latent infection within the cell. Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing the C6.5 or B1D2 chimeric antigen receptors in the presence of select inhibitors of T cell receptor signaling molecules indicate that TCR signaling through PI3 kinase and protein kinase C pathways may negatively and positively regulate HIV transcription, respectively. Understanding the specific TCR signaling pathways that lead to the initial establishment of latency within newly infected cells may lead to the discovery of novel therapeutic targets aimed at eliminating the latent reservoir

Studies on Cellular Host Factors Involved in the HIV-1 Life Cycle: A Dissertation

Human Immunodeficiency Virus Type 1 (HIV-1) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), currently the leading cause of death from infectious diseases. Since HIV-1 co-opts the host cellular machinery, the study of cellular factors involved is a rational approach in discovering novel therapeutic targets for AIDS drug development. In this thesis, we present studies on two such proteins. APOBEC3G is from the family of cytidine deaminases known to keep endogenous retroviruses and retrotransposons at bay to maintain stability of the human genome. APOBEC3G targets Vif-deficient HIV-1 particles and renders them noninfectious, partially through deaminase-dependent hypermutation of the provirus during reverse transcription. APOBEC3G largely localizes in mRNA processing (P) bodies, cytoplasmic structures involved in RNA metabolism. Here we explore the significance of APOBEC3G localization in P bodies. We found that disrupting P bodies does not affect virion incorporation of endogenous APOBEC3G, implying that the APOBEC3G fraction in P bodies is not directly involved in the production of nascent, non-infectious particles. We also study UPF1, another host protein encapsidated by HIV-1. It is an essential protein mainly studied for its role in nonsense-mediated decay (NMD) pathway and belongs to the same helicase superfamily as MOV10, a recently identified antiviral factor. We found that UPF1 is incorporated in HIV-1 virions in a nucleocapsid-dependent manner and is required for single-cycle infectivity at an early, post-entry step of the viral life cycle. This novel function of UPF1 most likely does not involve NMD since depletion of UPF2 does not affect viral infectivity

Investigating the feasibility of incorporating Vpx into lentiviral gene therapy vectors: at a global and targeted scale

Lentiviral gene therapy has the potential to target HIV-1 reservoirs. However, a major obstacle in effectively targeting the HIV-1 latent reservoir is the lack of efficient gene delivery that occurs within cells. The viral protein Vpx from the SIVSM/HIV-2 lineage is capable of increasing lentiviral reverse transcription (RTn); by antagonizing the restriction factor SAMHD1. This study aimed to investigate the feasibility of incorporating Vpx into lentiviral vectors. Primary macrophages and dendritic cells were utilised as resting cell models. Firstly, we determined the short and long-term effects post-Vpx exposure. Results revealed Vpx exposure led to increased permissiveness of cells to HIV-1 infection over a period that exceeded two weeks and was associated with lower potency of HIV antiretrovirals that target HIV RT or integration. Secondly, we incorporated Vpx with lentiviral gene therapy approaches that target HIV pre- and post-RTn and determined whether the inclusion of Vpx increased their effectiveness. When Vpx was incorporated into anti-HIV gene therapy constructs, transduction levels were significantly increased. However, increase in gene delivery only translated into an increase in HIV protection with constructs that inhibit pre-RTn phases of the virus life cycle. Thirdly, we utilised proteomics to determine any hidden phenotypes, in macrophages and dendritic cells, post-Vpx exposure, which could affect the usage of Vpx in gene therapy protocols. Significant protein changes were further investigated by generating a panel of THP1 knockdown/overexpression cell lines and examining whether individual changes, similar to SAMHD1 depletion, could increase HIV-1 infection. No detrimental hidden phenotypes were revealed in both macrophages and dendritic cells. Importantly, two knockdown cell lines, CALR and FKBP5, were shown to enhance HIV-1, in the absence of Vpx. In conclusion, this study supports the use of Vpx with gene therapy vectors as a way to overcome the major genetic delivery obstacle presented in resting cells. This effect was the greatest when incorporating anti-HIV constructs that target HIV-1 prior to reverse transcription. Importantly this approach can target cells of the existing HIV-1 latent reservoir or in resting cells that require HIV-1 protection

Characterizing Novel Filovirus Proteins and 3-Deazaneplanocin A Derivatives as Antivirals against Non-Segmented Negative Sense RNA Viruses

Filoviruses belong to a family of RNA viruses that includes deadly emerging zoonotic pathogens such as Ebola and Marburg viruses. A concern of public health is whether recently discovered filoviruses have the potential to infect humans and cause disease. Filoviruses encode proteins that suppress innate immune signaling and this is postulated as a contributing determinant of virulence in animals. Měnglà virus (MLAV), a recently discovered bat filovirus, can infect human cells using a vesicular stomatitis virus (VSV)-MLAV GP pseudotype system. In Chapter 2, we characterize MLAV’s VP35, VP40 and VP24 proteins on their ability to regulate both human and bat type I IFN responses. Our assessment also includes MARV and EBOV protein homologs for points of comparison. Analogous to its filovirus equivalents, MLAV VP35 and VP40 proteins inhibited type I IFN responses. MLAV VP40 suppressed the IFNβ production pathway, and this is independent of its inhibition on the type I IFN signaling pathway. MLAV VP24 did not behave like either EBOV VP24, an inhibitor of type I IFN, or MARV VP24, an activator of the antioxidant response pathway. Another critical concern is the lack of approved pan-filovirus therapeutics. Broad-spectrum nucleoside analogs have demonstrated antiviral activity against filoviruses. 3-deazaneplanocin (DzNep) and its brominated derivates (CL123, CL4033 and CL4053) are adenosine analogs and exhibit inhibition of non-segmented negative sense (NNS) RNA viruses. The antiviral effect is through inhibition of the enzyme, S-adenosylhomocysteine hydrolase (SAHase), resulting in obstruction of viral methyltransferase activity and consequently impaired translation of viral mRNA. The D-like-CL4033 and L-like-CL4053 exert antiviral activity against NNS RNA viruses, however the L-isomer, CL4053, has approximately a 1000 fold higher 50 percent inhibitory concentration (IC50) relative to the D-isomer, CL4033, suggesting an alternative antiviral mechanism. In chapter 3 we have elucidated, using VSV as a model NNS RNA virus, mechanisms of how DzNep, CL123, CL4033 and CL4053 exert their antiviral activity in cell culture. Our data indicates that DzNep, CL123 and CL4033 inhibit VSV by preventing viral mRNA cap methylation. A virus selected for CL123-resistance demonstrates cross-resistance against all derivatives, suggesting L-like-CL4053 may function through a similar mechanism of inhibition as the D-like-CL4033

A novel method for the selective elimination of HIV infected cells: dexamethasone and procaine as a combination therapy prototype

It has been established that various combinations of three or more antiretroviral drugs will lead to durable inhibition of HIV replication in patients. Despite its success in controlling the infection, highly active antiretroviral therapy (HAART) does not eradicate the virus; systemic infection re-emerges upon treatment interruption in all but exceptional cases, necessitating a lifetime of expensive drug therapy with an ever-present risk of emerging drug resistance. Thus, there is a critical need for the development of new treatment modalities not based solely on virally-encoded targets, and with the potential to actually cure HIV infection. Using standard colorimetric and fluorometric live/dead cell labeling methods, we have developed a novel "selective cell death" assay, and identified a lead drug combination for elimination of infected cells, involving a patented combination of 2 generic FDA approved drugs or their metabolites: the glucocorticoid dexamethasone (Dex) and N,N-diethylaminoethanolamine (DEAE, a metabolite of procaine). Neither drug alone, but only the combination, is effective in cell killing, and only in HIV-infected cells (P<0.0001). Furthermore, prolonged exposure of cells to this drug combination leads to a decline in viral load; two weeks of treatment results in a decrease of more than 50% relative to untreated control cells. Flow cytometric methods indicate that the mechanism of infected cell death is via apoptosis. In addition, microray analysis has highlighted several host genes whose expression is altered by Dex/DEAE treatment, providing initial clues to understanding the pathways leading to apoptosis in infected cells

Mechanism and mediated mechanistic analysis of HIV Nef

The nef gene of HIV is required for complete viral pathogenicity. As an accessory gene with no known paradigm its principle function has been enigmatic. Nef is capable of a variety of in vitro effects including CD4 downregulation, MHC I down regulation, alteration of T cell signalling activity and enhancement of viral infectivity. The effect of Nef on host CD4 T cell signalling paths is intriguing as it is the earliest expressed HIV gene and because HIV replicates to high levels in activated host cells. An effect of Nef on T cell activation state could dictate host T cell fate tipping the balance between productive viral infection and post-integration latency. Although Nef can form in vitro interactions with a variety of T cell signalling proteins in vivo evidence for signalling path activation has been inconclusive mainly due to the limitations of the experimental systems used to address the question. An in vivo role for Nef in alteration of T cell activation state should lead to gene expression changes in the host cell. Large scale gene expression profiling was used to monitor T cell gene expression after controlled expression of Nef using the tetracycline inducible system. Nef induces a widespread transcriptional program in T cells. Comparison of this profile with reference T ceil activation states reveals a strong identity to the gene expression profile of Jurkat activated by anti-CD3 cross-linking. Gene expression profiling was used to explore the mechanism by which Nef mediates this effect. After cyclosporin treatment the profiie was attenuated. A similar effect was observed after Nef expression in Jurkat T cells genetically deficient in ZAP-70 and functionally deficient in TCRÇ. Expression of a mutant defective in a functional SH3 binding domain had a stronger effect on attenuation of gene induction. Thus gene expression profiling demonstrates definitively Nef is capable of activating T cells at the transcriptional level and that this effect is initiated high in the T cell signalling path. Within the Nef expression profile differential expression of multiple genes with the ability to affect virai replication was noted. In a few instances it was possible to demonstrate the Nef mediated changes in mRNA levels were functionally significant. Proteomic analysis of Nef effect on T cells was initiated. 2D gel analysis of T cell lysates after Nef expression demonstrated a differential regulation of proteins in T cell lysates. The mechanism by which Nef initiates T cell signalling was explored using a similar approach. The proteins interacting with Nef in lipid rafts were identified after immunoprécipitation of HA tagged Nef from this subcellular compartment. 2D gel analysis reveals the presence of Nef changes the content of lipid rafts and that Nef co-immunoprecipitates with several proteins in the raft fraction. The identity of these interactors obtained by peptide microsequencing should give insight into the molecular mechanism by which Nef induces a CD4 T cell transcriptional programme

Targeting the ATP-dependent formation of herpesvirus ribonucleoprotein particle assembly as an antiviral approach

Human herpesviruses are responsible for a range of debilitating acute and recurrent diseases, including a number of malignancies. Current treatments are limited to targeting the herpesvirus DNA polymerases, however with emerging viral resistance and little efficacy against the oncogenic herpesviruses, there is an urgent need for new antiviral strategies. Herein we describe a mechanism to inhibit the replication of the oncogenic herpesvirus Kaposi’s sarcoma associated herpesvirus (KSHV), by targeting the ATP-dependent formation of viral ribonucleoprotein particles (vRNPs). We demonstrate that small molecule inhibitors which selectively inhibit the ATPase activity of the cellular human transcription/export complex (hTREX) protein UAP56, result in effective inhibition of vRNP formation, viral lytic replication and infectious virion production. Strikingly, as all human herpesviruses utilize conserved mRNA processing pathways involving hTREX components, we demonstrate the feasibility of this approach for pan-herpesvirus inhibition

Allosteric Integrase Inhibitors Reveal a Role for Integrase During HIV-1 Maturation

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.Medical Science

SERINC5: Its Sensitivity to Nef and Restriction of HIV-1

The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef-resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5

Allosteric Integrase Inhibitors Reveal a Role for Integrase During HIV-1 Maturation

Integration of the DNA copy of the HIV-1 genome is an essential step for virus replication and is mediated by a homotetrameric complex of the viral protein integrase (IN) in association with the ends of linear viral DNA (vDNA). HIV-1 integrates into actively transcribed genes, a trait mediated by cellular host cofactor LEDGF/p75. LEDGF/p75 engages IN in a pocket formed by dimerization of the IN catalytic core domain, a region that has been validated as a drug target for allosteric IN inhibitors (ALLINIs). Previous in vitro work suggested that ALLINIs function through disruption of two integration-associated functions: IN-vDNA complex formation and the IN-LEDGF/p75 interaction. We now demonstrate that ALLINI potency is accounted for during the late phase of HIV-1 replication where the inhibitors block the formation of the viral core, converting the normally electron-dense conical core to an eccentric phenotype where the electron-density exists as a condensate situated between a translucent core and the viral membrane. We have further elucidated the eccentric condensates to represent non-packaged viral ribonucleoprotein (vRNP) complexes and that either genetic or pharmacological inhibition of IN can impair vRNP encapsidation. Supplying IN in trans as part of a Vpr-IN fusion protein partially restored the formation of conical cores with the internal electron density. Moreover the ability of ALLINIs to induce eccentric condensate formation required both IN and viral RNA. Based on these observations, we propose an active role for IN during HIV-1 maturation that involves initiating core morphogenesis and vRNP encapsidation.Medical Science