Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 121-150).

Prevalence of HIV-associated cognitive impairment is rising, the worst form of which is HIV-associated dementia (HAD). The disease is fuiled by a chronic innate type pro-inflammatory response in the brain which is highly dependent upon the activation of microglia. We first created an in vitro model of HAD composed of cultured microglial cells synergistically activated by the addition of IFN-gamma and the HIV-1 coat glycoprotein, gp120. This activation, as measured by TNF-alpha and NO release, is synergistically attenuated through the alpha7nAChR and p44/42 MAPK system by pretreatment with nicotine, and the cholinesterase inhibitor, galantamine. As these medications have been FDA approved, and over time, have shown only minor improvement in neurodegenerative disease for a limited period, we next sought to explore natural compounds that may attenuate HAD mediated inflammation and related pathology. This inflammation is a key moderator of A-#914; plaque deposition in the brain. Indeed it is likely a contributing factor as epidemiological data suggests significant numbers of HIV survivors are at elevated risk of developing Alzheimer's disease (AD). HIV-1 Tat-induced A-beta deposition, tau phosphorylation, and subsequent neuronal death could be risk factors for subsequent AD and/or HAD. Recent reports suggest green tea-derived (-)-epigallocatechin-3-gallate (EGCG) can attenuate neuronal damage mediated by conditions such as brain ischemia. In order to investigate the therapeutic potential of EGCG to mitigate the neuronal damage characteristic of HAD, IFN- gamma was evaluated for its ability to enhance well-known neurotoxic properties of HIV-1 proteins gp120 and Tat in primary neurons and mice. Indeed, IFN-gamma enhanced the neurotoxicity of gp120 and Tat via increased JAK/STAT signaling. Additionally, primary neurons pretreated with a JAK1 inhibitor, or those from STAT1-deficient mice, were largely resistant to the IFN- gamma-enhanced neurotoxicity of gp120 and Tat. Moreover, EGCG treatment of primary neurons from normal mice reduced IFN-gamma-enhanced neurotoxicity of gp120 and Tat by inhibiting JAK/STAT1 pathway activation. EGCG was also found to mitigate the neurotoxic properties of HIV-1 proteins in the presence of IFN-#947; in vivo. To explore the mechanism by which HIV may augment AD-like pathology, we found HIV-1 Tat protein inhibits microglial uptake of A-beta-1-42 peptide, a process enhanced by IFN-#947; and rescued by EGCG. To mimic the HAD clinical condition, we generated mice with HIV-1 Tat-induced AD-like pathology by cross-breeding HIV-1 Tat expressing mice (expressed under control of GFAP, Doxycline inducible promoter) with the PSAPP mouse model of AD. To simulate chronic Tat secretion over we used an optimized dose of 54 mg/kg/day on a biweekly basis over three months Tat significantly induced neuron degeneration and tau phosphorylation in Tat transgenic mice, dox dependently (P<0.001). Similar effects at the chronic 54 mg/kg/day dose were observed in PSAPP/Tat mice induced with dox. These mice also showed significantly more A-beta deposition (P < 0.05), neurodegeneration, neuronal apoptotic signaling, and phospho-tau than PSAPP mice (P < 0.05). In conclusion, HIV-1 Tat significantly promotes AD-like pathology in PSAPP/Tat mice. This model may provide a framework in which to identify new mechanisms involved in cognitive impairment in the HIV infected population, and possible treatments. Additional works will be needed to fully characterize the mechanism(s) of HIV- induced amyloid deposition, and to uncover viral mechanisms promoting AD-like pathology in general.

Human immunodeficiency virus type 1 (HIV-1) is the cause of acquired immune deficiency syndrome (AIDS). HIV-1 is a worldwide epidemic that currently affects over 35 million people worldwide, and continues to spread at an appalling rate. A universal HIV-1 preventive vaccine is considered to be the optimal solution in achieving the ultimate goal of AIDS eradication. Regretfully, most endeavors thus far of developing a prophylactic vaccine have been largely disappointing. Highly Active Anti-Retroviral Therapy (HAART) has been shown to reduce the plasma HIV-1 RNA level to below the detection limit of clinical assays (50 copies/ml); it combines three or more antiretroviral drugs which belong to at least two different classes – targeting distinct steps in the viral life cycle, and inhibiting viral replication. However, unless the infection is eradicated, strict adherence to a lifelong treatment regimen is required. HAART is limited by its high cost, drug availability, complicated administration schedules, serious side effects, and the potential that the virus will ultimately develop drug resistance. A more plausible approach lies in therapeutic vaccines that provide immunity to partially control viral replication postinfection – delaying or minimizing ART, and offering “drug holidays”. The primary goal of a therapeutic vaccine is to effectively induce HIV-1 specific cytotoxic T lymphocyte (CTL) responses, which plays a critical role in control of viral proliferation. Dendritic cells (DCs)-based therapeutic vaccines have been showing the most promising results. However, the therapeutic efficacy of DCs based vaccines is limited. This is partially due to the fact that DCs induced CD8+ T cell responses are largely CD4+ T cell dependent, while HIV-1 infection usually renders the immune system very “helpless” from CD4+ T cells. In addition, infection, impaired function, and physical depletion of DCs are often reported during the early stage. Furthermore, DCs are often found to be inflammatory and immunosuppressive, which is mainly mediated by the interaction between HIV-1 Env gp120 and DC receptors. Thus, the search for a novel therapeutic vaccine strategy is warranted. Using T-APC (T cells-antigen-presenting cells) as a novel T cell-based vaccine has emerged as a potential candidate for a HIV-1 therapeutic vaccine, which aims at boosting HIV-specific CTL responses. Our previous work demonstrated that CD4+ and CD8+ T cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice via co-culture with OVA-pulsed DCs (DCOVA) can be activated, acquiring pMHC I, pMHC II, and costimulatory molecules, thus act as CD4+ T helper-antigen-presenting cells (Th-APCs) and CD8+ cytotoxic T-antigen-presenting cells (Tc-APCs). We also elucidated that DC-derived exosomes (EXO), which are 50- to 90-nm diameter vesicles containing antigen-presenting, tetraspan, adhesion, and costimulatory molecules, can transfer the antigen-presenting activity of DCs to activated CD4+T cells through EXO uptake. EXOOVA-targeted activated CD4+T (aTexo) cells can (1) stimulate more efficient central memory CD8+ CTL responses and T cell memory than EXOOVA or DCOVA, (2) activate CD8+ CTL responses independent of CD4+Th cells, and (3) counteract CD4+25+regulatory T (Tr) cell-mediated immune suppression. These results formed the new concept of novel EXO-targeted CD4+ T cell vaccines. In this study, we tailored EXO-targeted T cells vaccine by using polyclonal activated CD8+ T cells instead of CD4+ T cells, as CD4+ T cells served as the primary target for HIV-1 infection. We showed that (1) OVA-specific exosome-targeted CD8+ T cell-based vaccine (OVA-Texo) can stimulate efficient OVA-specific CD8+ CTL and memory responses, inducing sufficient antitumor immunity against OVA-expressing tumor cells in mouse models. (2) This exosome-targeted CD8+ T cell-based vaccine strategy could be applied to HIV-1-Gag protein, provoking effective Gag-specific CD8+ CTL, T cell memory, and antitumor immunity against Gag-expressing tumor cells. (3) Engineering Gag-Texo with up-regulated 4-1BBL (APC derived costimulatory molecule) expression could improve the performance of Gag-Texo vaccine. (4) OVA-Texo is able to evoke a successful immune response in bystander chronic infection, converting CD8+ T cell exhaustion, restoring effector functions of exhausted CD8+ T cells. Moreover, combination of OVA-Texo vaccine with PD-L1 blockage in a dual treatment could result in a synergistic effect in rescuing CTLs exhaustion in chronic infection. Those desired features make EXO-targeted CD8+ T cells vaccine an appealing novel strategy in HIV-1 infection. The EXO-targeted CD8+ T cells vaccine may be applicable to therapeutic HIV treatment through the use of autologous T cells with uptake of EXOs derived from engineered DCs.

Regulatory T cells represent a specialized subpopulation of T lymphocytes that may modulate spontaneous HIV-1 disease progression by suppressing immune activation or inhibiting antiviral T cell immune responses. While effects of classical \(CD25^{hi}FoxP3^+CD4^+\) regulatory T cells during HIV-1 infection have been analyzed in a series of recent investigations, very little is known about the role of non-classical regulatory T cells that do not express intracellular FoxP3. Here I evaluated two groups of non-classical Treg cells. One is phenotypically identified by the surface expression of HLA-G, an HLA class Ib molecule. The other Treg cell population is characterized by the surface expression of latency-associated peptide (LAP), a membrane-bound form of \(TGF-\beta\). Both HLA-G and LAP-expressing T cells are present in small proportions in peripheral blood of healthy individuals. I performed a systematic study on the phenotypic and functional profile of HLAG- and LAP- expressing regulatory T (Treg) cells in patients with different stages of HIV-1 infection. I found that HLA-G-expressing Treg cells were highly susceptible to HIV-1 infection, and were significantly reduced in individuals with progressive HIV-1 disease courses. Moreover, the proportion of \(HLA-G^+\) CD4 and CD8 T cells was positively correlated with CD4 T cell count and inversely correlated with markers of HIV-1 associated immune activation. Mechanistically, this correlation corresponded to a substantially increased ability of \(HLA-G^+\) Treg cells to inhibit bystander immune activation, while only minimally affecting functional properties of HIV-1-specific T cells. In contrast, no significant change in \(LAP^+\) Treg cell frequencies was found in progressive HIV-1 infection, and these frequencies were not correlated with immune activation. This observation was consistent with functional analysis, which indicated that \(LAP^+\) Treg cells did not suppress bystander activation. These investigations indicate an important role of \(HLA-G^+\) Treg cells for balancing bystander immune activation and anti-viral immune activity in HIV-1 infection, and suggest that the loss of these cells during advanced HIV-1 infection may contribute to immune dysregulation and HIV-1 disease progression. In the meantime, \(LAP^+\) Treg cells do not appear to play an important role in determining HIV-1 disease outcome.

An effective vaccine to protect against HIV-1/AIDS remains elusive due to the extensive mechanisms employed by the HIV-1 virus to evade immune attack. Highly potent broadly neutralizing antibodies isolated from chronically infected individuals, however, show that such relevant antibodies can be naturally produced, implying that their elicitation through vaccination is a realistic possibility. These broadly neutralizing antibodies target different regions on the trimeric spikes formed by three protomers of the envelope (Env) protein. Each Env protein is comprised of the gp120 surface subunit in non-covalent association with the gp41 transmembrane subunit. Four regions have been identified: the CD4 binding site, the V1/V2 segment and the V3/glycan area all on the gp120 subunit as well as the MPER segment on the gp41 subunit. This dissertation focuses on the gp41 MPER segment given its highly conserved amino acid sequence among all HIV-1 clades and viral strain isolates and essential function in Env-mediated fusion and HIV entry. Of note, the MPER segment contains several adjacent epitopes targeted by broadly neutralizing antibodies, suggesting that the immune system is capable of producing neutralizing antibodies against this specific region. Analysis of both clade B and C MPER segments shows them to be L-shaped, consisting of two &#61537; helices separated by a hinge. We have found that the hinge region of the MPER segment provides the conformational flexibility necessary for the Env-mediated hemifusion and fusion processes. A significant reduction in virus infectivity is observed when the hinge region is disrupted by introduction of two amino acid mutations that eliminate &#61537;-helical capping residues and the tandem hinge joints. The importance of the hinge region of the MPER segment is further supported by the action of four MPER-specific neutralizing antibodies 2F5, 4E10, 10E8 and Z13E1. These neutralizing antibodies block virus infection by disrupting MPER hinge-related function.

Multivalent interactions are common in nature, such as influenza virus infecting epithelial cells, clearance of pathogens by antibody-mediated attachment to macrophages, etc. To mimic nature, we utilized a bottom-up approach to develop various multivalent self-assembling systems based on leucine-zipper peptides. We tethered several pairs of leucine-zipper peptides to PAMAM dendrimers to form leucine-zipper dendrimers (LZDs). We conjugated Fos/Jun to the dendrimer to make D0Fos4 and D0Jun4, and studied the interactions between these LZDs and their cognate peptide target, either Jun or Fos. Our experiments showed that the D0Fos4 can non-covalently assemble four copies of Jun, and this approach can be further used for the rapid non-covalently assembling of multimeric ligands. We also pursued the multivalent target of GPCRs with a Fos/Jun assembly, and found the complex can potentially be used as a molecular switch to target GPCRs with controlled ligand activity. In a related project for bio-material design based on self-assembly of LZDs, we synthesized a different pair of LZDs, D-Ez4 and D-Kz4, and established that they can assemble at neutral pH to form helical fibrils which display higher order self-organized structures, providing a new methodology for bio-material design. In another effort for studying multivalent interactions, we conjugated three copies of the F23, mini-protein that binds the HIV-1 capsid protein, to a trimesic acid and obtained a trivalent inhibitor, Tri-F23. Tri-F23 showed enhanced binding in ELISA against gp120, but was not significantly more effective preventing HIV entry. This methodology provides a new strategy for developing multivalent inhibitors for preventing HIV-1 infection at the entry level. In a related area, we are developing imaging agents based on quantum dots that can detect GPCRs on whole cells and at the single molecule level. To this end, a new method was developed for biocompatible amphphilic polymers to coat quantum dots. This amphiphilic polymer facilitates rapid quantum dot conjugation to any ligand with a free thiol or engineered cysteine. Several GPCR targeted peptides have been utilized for imaging receptors on whole cells and as single molecules. These efforts will guide the rational design of multivalent ligands for targeting GPCRs and other cell surface proteins.

Human immunodeficiency virus (HIV) is the causing agent of the acquired immune deficiency syndrome (AIDS). Like all retroviruses, HIV carries two copies of viral genomic RNA in each virion. HIV genome encodes three structural genes, including gag, pol and env, as well as two regulatory genes (rev and tat) and four accessory genes (vif, vpr, vpu and nef). It is noted that none of these nine viral proteins bears the helicase activity. Helicases are able to unwind RNA duplex and remodel the structure of RNA-protein (RNP) complexes using energy derived from hydrolysis of nucleotide triphosphates (NTPs). They are involved in every step of cellular RNA metabolisms. It is conceivable that HIV needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. / In this study, we found that a DEAD-box protein named DDX24 associates with HIV-1 Gag in an RNA-dependent manner but is not found within virus particles. Knockdown of DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.

No description available.

Human Immunodeficiency Virus type I genome consists of two identical RNA molecules that are non-covalently linked to form a dimer. HIV-1 immature and mature genomic RNA (gRNA) dimers were found in protease defective (PR -) and wild type virions, respectively, and the 5'untranslated region (5' UTR) was shown to play key roles during the genome dimerization process; but the dimerization mechanism still remains to be clarified My research project is to characterize the dimerization process and the role of 5' UTR in genome dimerization in virions produced by tissue culture cells. I'll firstly show the dimer maturation processes of HIV-1 gRNA isolated from newly released to grown-up (&ge;10h old) wild type, PR-, and SL1 defective (DeltaIDS) virions respectively. The results showed that HIV-1 gRNA dimer maturation process was protease-dependent and involved multiple steps: from low to high dimerization level and dimer thermostability, and from low dimer mobility to intermediate and high mobility. PR- virions did not freeze gRNA conformation in the primordial nascent state and gRNA changed from monomeric in newly released virions to half dimeric in grown-up virions, which showed that genome was packaged in the form of monomeric RNA or fragile dimers, more thermolabile than immature dimers in grown-up PR- virions. DeltaDIS inhibited gRNA dimerization by about 50% in newly released virions, though grown-up DeltaDIS gRNA was fully dimeric, which indicated that the DIS played the initiation role in gRNA dimerization in HIV-1 virions. The gRNA dimerization rate in PR- or DeltaDIS virions was much slower than that in wild type virions. These results show for the first time the whole process of dimer maturation after virion release, the gRNA conformation rearrangement in PR- virions, and the initiation role of the DIS in HIV-1 virions. Next, I'll provide a rather systematic search for the contribution of different regions in 5' UTR to HIV-1 gRNA dimerization by studying selected mutations singly or together with defective SL1. The results showed that the 5'trans-activation response element (5'TAR) was directly involved in gRNA dimerization, and a long distance base-pairing interaction between a sequence in U5 region (nts105-1l5) and another around the initiation codon of the gag gene (nts334-344) was structurally contributive to gRNA dimerization. Deletions of sequences around the 3'end of Primer Binding Site (PBS) stem-loop moderately decreased gRNA dimerization level. Other sequences in 5' UTR except DIS/SL1, which was previously known to play important roles, didn't show any systematic role. Here the results suggested that the absence of inhibition on gRNA dimerization level with defective DIS might be the compensation of the direct role of 5'TAR; and wild type-like dimerization level of DeltaTAR must be the direct contribution of the DIS.

La protéine Nef du VIH-1 joue un rôle important dans la pathogenèse du VIH-1 en modulant les voies de signalisation de la cellule hôte. La signalisation par le TcR est essentielle à la sélection positive pour générer les cellules simples positives (SP) CD4+ et simples positives (SP) CD8+, processus largement dépendant de l’activité de la Src kinase Lck et de son habileté à lier la queue cytoplasmique des corécepteurs CD4 et CD8. Nous avons précédemment trouvé que l’expression de Nef dans le VIH ou VIS peut induire une sévère déplétion des thymocytes et une baisse d’expression du corécepteur CD4 à la membrane. Nous avons également montré que Nef bloque la génération des thymocytes doubles positifs (DP) CD4+ CD8+ en plus d’altérer la transition des cellules DP vers CD4+ SP. Par contre, ce phénotype est récupérable par plusieurs approches dont le croisement d’une souris transgéniques exprimant Nef avec une souris exprimant la forme constitutivement active de Lck Y505F. Les résultats indiquent que la maturation des cellules CD4+ est altérée par le dysfonctionnement de la signalisation CD4-Lck. Toutefois, les mécanismes moléculaires par lesquels Nef contribue au bloc de la génération des cellules CD4+ dans le thymus demeurent très imprécis. Dans cette étude, en utilisant des approches biochimiques et de microscopie confocale, nous avons trouvé que les thymocytes transgéniques Nef+ expriment plus de Lck que les thymocytes Nef-. Malgré cette augmentation, une partie significative de Lck est incapable d’atteindre la membrane plasmique. Cette fraction était significativement accumulée dans un compartiment intracellulaire des thymocytes transgéniques exprimant Nef. Également, en utilisant la technique d’essai kinase in vitro, nous avons trouvé que l’activité kinase de Lck est significativement augmentée dans les thymocytes transgéniques mais demeure stable suite à une stimulation par un α-CD3ε + α-CD4. Également, comparativement aux thymocytes Nef-, la kinase Lck dans les thymocytes transgéniques était résistante à la dégradation suite à une stimulation. En examinant le statut de c-Cbl, le principal régulateur négatif de Lck, nous avons montré que c-Cbl colocalise faiblement avec Lck, malgré son hyperphosphorylation constitutive. Ceci pourrait expliquer l’échec de la dégradation de Lck. En plus, nous avons trouvé que suite à une stimulation par un α-CD3ε + α-CD4, la phosphorylation de Zap-70 en tyrosine 493 par Lck est diminuée, résultant d’une importante baisse de l’activité kinase de Zap-70 et d’un bloc des premiers évènements de la voie de signalisation par le TcR. Ces données indiquent que la signalisation CD4-Lck est interrompue par la présence de Nef. / HIV-1 Nef protein plays an essential role in the HIV-1 pathogenesis by modulating the host signaling transduction pathways. TcR signalling is important for the thymic selection process to CD4 and CD8 single positive T cells and is greatly dependent on the activity of Src kinase Lck and its ability to bind to CD4 and CD8 cytoplasmic tail. We previously found that expression of HIV or SIV Nef can induce severe thymocytes depletion and downregulation of CD4 expression in Nef+ mice. We also recently showed that Nef blocks generation of double positive thymocytes and impairs DP to CD4+ SP T cells transition. The reversal of this phenotype was accomplished by several approaches, among them by crossing Nef+ mice with mice expressing constitutively active Lck Y505F. These results imply that the maturation of CD4+ T cells is disrupted due to impairment of Lck-mediated CD4 receptor signaling. However, the molecular mechanisms by which Nef contributes to the impairment in thymic CD4 generation remains largely unclear. In this study, using confocal microscopy and biochemical approaches, we found that Nef+ thymocytes express more Lck than the Nef- control. Despite of this increase, a significant portion of Lck molecules were unable to reach to the plasma membrane. It was significantly accumulated in the intracellular endosomal compartment of the Nef+ thymocytes. Moreover, using IVKA we found that the activity of Lck is significantly increased in Nef+ thymocytes but was not further increased upon stimulation by α-CD3ε, α-CD4 or α-CD3ε + α-CD4. Moreover, compared to Nef- controls, Lck kinase in Nef+ thymocytes was resistant to degradation upon stimulation. Examining the status of c-Cbl, the main negative regulator of Lck, showed that c-Cbl localized with Lck poorly, despite his constitutive hyperphosphorylation. This explains the failure of Lck degradation. In addition, we found that upon stimulation, Zap-70 phosphorylation at tyrosine 493 by Lck is decreased, resulting by a decrease of Zap-70 kinase activity and TcR proximal event block. These data indicate that CD4-Lck signaling was interrupted by the presence of Nef.