Browsing by keyword "Gene Products"
Now showing items 1-4 of 4
-
Characterization of the Nef-TCR Zeta Interaction and Its Role in Modulation of Src Family Kinase Activity: A DissertationOne of the hallmarks of an infection with pathogenic HIV-1 is the elevated level of immune activation that leads to rapid progression to AIDS. Surprisingly, nonhuman primates naturally infected with SIV do not exhibit an augmented activation phenotype nor severe immunodeficiency. One of the viral components implicated in determining the state of immune activation is the accessory protein Nef which has been demonstrated to affect T cell signaling pathways from within the intracellular compartment and for Nef from SIV, to downregulate TCR surface expression. Recently, Nef from HIV-1 and SIV have been demonstrated to bind the ζ chain of the TCR which functions as the primary signaling subunit of the receptor. However, the molecular details of the Nef-TCRζ interaction as well as the role of complex formation in modulation of immune activation remain largely unknown. This thesis describes work directed at elucidating the biochemical and structural features of the Nef-TCRζ interaction and the functional consequences of complex formation relevant to T cell activation. Chapter I provides a brief introduction on HIV/SIV classification and pathogenesis with an emphasis on Nef and its pleiotropic function in T cells. Chapter II describes the biochemical characterization of the interaction of the conserved core domain of Nef proteins from HIV-1, HIV-2 and SIV with the cytoplasmic domain of TCRζ. The core domains of HIV-2 ST and SIVmac239 are demonstrated to bind the cytoplasmic domain of TCRζ at two distinct regions and with different affinities. In contrast, the core domain of HIV-1 isolate ELI Nef only binds to one region and with the weakest calculated affinity among the HIV-1, HIV-2 and SIV Nef proteins studied. In addition, both the N-terminal domain and the strong TCRζ-binding core domain of SIVmac239 Nef each are demonstrated to be necessary but not sufficient for downregulation of TCR surface expression. Chapter III describes the crystallization and structure determination methods used to solve the crystal structures of the core domain of SIVmac239 Nef in complex with two overlapping TCRζ polypeptides. Crystals of Nef in complex with the longer TCRζDP1 (L51-D93) polypeptide grew in a tetragonal space group but only diffracted to low resolution. In contrast, crystals of the Nefcore-TCRζA63-R80 complex grew in an orthorhombic space group and diffracted to high resolution but were nearly perfectly pseudo-merohedrally twinned thus complicating structure determination. Following identification of the twin law relating the twin domains, the structure of the Nefcore-TCRζA63-R80 complex was determined using refinement procedures that accounted for crystal twinning to 2.05 Å. The structure of the Nefcore-TCRζDP1 complex was solved to 3.7 Å from a single non-twinned crystal. The altered crystal packing induced by the shorter TCRζA63-R80polypeptide is postulated to have led to a reduction in crystal symmetry and increase in proneness to crystal twinning. Chapter IV provides a detailed analysis of the structure of the Nefcore-TCRζA63-R80 complex and demonstrates its effect on modulation of Src family kinase activity. The TCRζ polypeptide adopts an alpha helical conformation and occupies a hydrophobic crevice on Nef not shared by any of Nef’s reported interaction partners. The interaction of Nefcore with TCRζ is mediated primarily by the burial of hydrophobic residues on TCRζ (L75, L77) in a hydrophobic pocket on Nef and a salt bridge between a glutamic acid (E74) on TCRζ and a basic patch on Nef consisting of two conserved arginines (R105, R106). The TCRζ polypeptide additionally orders the N-terminus of Nefcore into a polyproline type II helix that has been described to bind the SH3 domain of Src family kinases. We demonstrate that in vitro phosphorylation of TCRζcyt by Fyn and Src is specifically augmented by HIV-1 and SIV Nefcoreand suggest that Nef-TCRζ complex formation cooperatively enhances kinase activity. Chapter V contains overall conclusions, future directions and a model illustrating the proposed role of the Nef-TCRζ interaction in immune activation modulation. The Appendices contain sequences of the proteins, gene constructs and primers used in this work.
-
Cytoplasmic Localization of HIV-1 Vif Is Necessary for Apobec3G Neutralization and Viral Replication: A DissertationThe binding of HIV-1 Vif to the cellular cytidine deaminase Apobec3G and subsequent prevention of Apobec3G virion incorporation have recently been identified as critical steps for the successful completion of the HIV-1 viral life cycle. This interaction occurs in the cytoplasm where Vif complexes with Apobec3G and directs its degradation via the proteasome pathway or sequesters it away from the assembling virion, thereby preventing viral packaging of Apobec3G. While many recent studies have focused on several aspects of Vif interaction with Apobec3G, the subcellular localization of Vif and Apobec3G during the viral life cycle have not been fully considered. Inhibition of Apobec3G requires direct interaction of Vif with Apobec3G, which can only be achieved when both proteins are present in the same subcellular compartment. In this thesis, a unique approach was utilized to study the impact of Vif subcellular localization on Vif function. The question of whether localization could influence function was brought about during the course of studying a severely attenuated viral isolate from a long-term non-progressor who displayed a remarkable disease course. Initial observations indicated that this highly attenuated virus contained a mutant Vif protein that inhibited growth and replication. Upon further investigation, it was found that the Vif defect was atypical in that the mutant was fully functional in in vitro assays, but that it was aberrantly localized to the nucleus in the cell. This provided the basis for the study of Vif localization and its contribution to Vif function. In addition to the unique Vif mutant that was employed, while determining the localization and replication phenotypes of the differentially localized Vif proteins, a novel pathway for Vif function was defined. Copious publications have recently defined the mechanism for Vif inhibition of Apobec3G. Vif is able to recruit Apobec3G into a complex that is targeted for degradation by the proteasome. However, this directed degradation model did not fully explain the complete neutralization of Apobec3G observed in cell culture. Other recent works have proposed the existence of a second, complementary pathway for Vif function. This pathway is defined here as formation of an aggresome that prevents Apobec3G packaging by binding and sequestering Apobec3G in a perinuclear aggregate. This second mechanism is believed to work in parallel with the already defined directed degradation pathway to promote complete exclusion of Apobec3G from the virion. The data presented here provide insight into two areas of HIV research. First, the work on the naturally occurring Vif mutant isolated from a long-term non-progress or confirms the importance of Vif in in vivo pathogenesis and points to Vif as a potentially useful gene for manipulation in vaccine or therapy design due to its critical contributions to in vivo virus replication. Additionally, the work done to address the subcellular localization of Vif led to the proposal of a second pathway for Vif function. This could have implications in the field of basic Vif research in terms of completely understanding and defining the functions of Vif. Again, a more complete knowledge about Vif can help in the development of novel therapies aimed at disrupting Vif function and abrogating HIV-1 replication.
-
HIV-1 Gene Expression: Transcriptional Regulation and RNA Interference Studies: a DissertationGene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression. To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies. To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.
-
Identification and Characterization of SNAPIN as a Novel Antagonist of HIV-1 Egress: A DissertationVpu has been shown to possess two distinct roles in the pathogenesis of HIV. First, Vpu has been shown to down-regulate the expression of CD4 molecules at the plasma membrane of infected cells by targeting CD4 molecules for degradation in the endoplasmic reticulum. Second, Vpu promotes viral egress in specific cell lines termed non-permissive cells by mechanism that remain relatively unclear. Therefore, experiments were conducted in order to identify cellular factors involved in the Vpu-dependent phenotype. Using full-length Vpu as bait in yeast two-hybrid experiments, several candidate cellular factors were identified. One protein, SNAPIN, was identified as a cellular factor putatively involved in the Vpu-dependent phenotype. Further experiments determined that not only do SNAPIN and Vpu interact, but that Vpu also leads to the degradation of SNAPIN by both proteasomal and lysosomal degradation pathways. Over-expression of SNAPIN in cell lines that do not normally require Vpu expression for viral production resulted in a Vpu-dependent phenotype. While over-expression of SNAPIN in otherwise permissive cell lines significantly reduced Vpu-deficient virus production, wild type levels remained relatively constant. Importantly, no defective viral structural protein production was observed; however, intracellular p24/p55 did not accumulate suggesting that in SNAPIN expressing cells, Gag is also targeted for degradation. In addition, the reduction of SNAPIN expression in non-permissive cell lines significantly increased viral titers in supernatants. Of particular interest, even in cells expressing Bst-2 (a previously identified cellular factor involved in the Vpu-phenotype), siRNA mediated knockdown of SNAPIN led to increased viral titers. In addition, the co-transfection of siRNAs targeting both SNAPIN and Bst-2 resulted in an additive effect, in which Vpu-deficient viral titers were nearly equivalent to wild-type titers. Surprisingly, siRNA-mediated knockdown of SNAPIN in Jurkat cells was sufficient to overcome any restriction in viral egress imposed by the deletion of Vpu. Conversely, siRNA targeting Bst-2 had little or no effect on viral titers in Jurkat cells regardless of whether it was transfected alone or in combination with siRNAs targeting SNAPIN. These experiments provide evidence of an alternate cellular restriction mechanism involved in viral egress that is countered by the HIV-1 accessory protein, Vpu. In addition, this research may provide further insight into the complex cellular networks involved in the trafficking of Gag through cellular endosomal pathways.
