Biomedical subjects

Dana Gabuzda

Publications and source records attributed to Dana Gabuzda.

At least 19 recordsLinked to original sources

PubMed2006

Macrophage entry mediated by HIV Envs from brain and lymphoid tissues is determined by the capacity to use low CD4 levels and overall efficiency of fusion.

HIV infects macrophages and microglia in the central nervous system (CNS), which express lower levels of CD4 than CD4+ T cells in peripheral blood. To investigate mechanisms of HIV neurotropism, full-length env genes were cloned from autopsy brain and lymphoid tissues from 4 AIDS patients with HIV-associated dementia (HAD). Characterization of 55 functional Env clones demonstrated that Envs with reduced dependence on CD4 for fusion and viral entry are more frequent in brain compared to lymphoid tissue. Envs that mediated efficient entry into macrophages were frequent in brain but were also present in lymphoid tissue. For most Envs, entry into macrophages correlated with overall fusion activity at all levels of CD4 and CCR5. gp160 nucleotide sequences were compartmentalized in brain versus lymphoid tissue within each patient. Proline at position 308 in the V3 loop of gp120 was associated with brain compartmentalization in 3 patients, but mutagenesis studies suggested that P308 alone does not contribute to reduced CD4 dependence or macrophage-tropism. These results suggest that HIV adaptation to replicate in the CNS selects for Envs with reduced CD4 dependence and increased fusion activity. Macrophage-tropic Envs are frequent in brain but are also present in lymphoid tissues of AIDS patients with HAD, and entry into macrophages in the CNS and other tissues is dependent on the ability to use low receptor levels and overall efficiency of fusion.

AIDS Dementia Complex↗

PubMed2006

The HIV Env variant N283 enhances macrophage tropism and is associated with brain infection and dementia.

HIV infects tissue macrophages and brain microglia, which express lower levels of CD4 and CCR5 than CD4+ T cells in peripheral blood. Mechanisms that enhance HIV tropism for macrophages in the CNS and other tissues are not well understood. Here, we identify an HIV envelope glycoprotein (Env) variant in the CD4-binding site of gp120, Asn 283 (N283), that is present at a high frequency in brain tissues from AIDS patients with HIV-associated dementia (HAD). N283 increases gp120 affinity for CD4 by decreasing the gp120-CD4 dissociation rate, enhancing the capacity of HIV Envs to use low levels of CD4 for virus entry and increasing viral replication in macrophages and microglia. Structural modeling suggests that the enhanced ability of Envs with N283 to use low levels of CD4 is due to a hydrogen bond formed with Gln 40 of CD4. N283 is significantly more frequent in brain-derived Envs from HAD patients (41%; n=330) compared with non-HAD patients (8%; n=151; P<0.001). These findings suggest that the macrophage-tropic HIV Env variant N283 is associated with brain infection and dementia in vivo, representing an example of a HIV variant associated with a specific AIDS-related complication.

AIDS Dementia Complex↗

PubMed2006

CD4 and MHC-I downregulation are conserved in primary HIV-1 Nef alleles from brain and lymphoid tissues, but Pak2 activation is highly variable.

HIV-1 compartmentalization in the CNS has been demonstrated for gag, pol, and env genes. However, little is known about tissue compartmentalization of nef genes and their functional characteristics in brain. We have cloned 97 nef genes and characterized 10 Nef proteins from autopsy brain and lymphoid tissues from 2 patients with AIDS and HIV-1-associated dementia. Distinct compartmentalization of brain versus lymphoid nef genes was demonstrated within each patient. CD4 and MHC-I downregulation were conserved in all tissue-derived Nefs. However, MHC-I downregulation by brain-derived Nefs was weaker than downregulation by lymphoid-derived Nefs. The motifs KEEE- or EKEE- at the PACS-1 binding site represented brain-specific signature patterns in these 2 patients and contributed to the reduced MHC-I downregulation activity of brain-derived Nefs from these patients. Pak2 association was highly variable in Nefs from both patients. Three of 10 tissue-derived Nefs coimmunoprecipitated activated Pak2, with strong association demonstrated for only 2 Nefs. The ability of Nef to associate with activated Pak2 did not correlate with brain or lymphoid tissue origin. Nef genes from viruses isolated from brain by coculture with PBMC were not closely related to sequences amplified directly from brain tissue, suggesting that viral selection or adaptation occurred during coculture. This study of tissue-derived HIV-1 Nefs demonstrates that CD4 and MHC-I downregulation are highly conserved Nef functions, while Pak2 association is variable in late stage AIDS patients.

AIDS Dementia Complex↗

PubMed2006

Elicitation from virus-naive individuals of cytotoxic T lymphocytes directed against conserved HIV-1 epitopes.

Cytotoxic T lymphocytes (CTL) protect against viruses including HIV-1. To avoid viral escape mutants that thwart immunity, we chose 25 CTL epitopes defined in the context of natural infection with functional and/or structural constraints that maintain sequence conservation. By combining HLA binding predictions with knowledge concerning HLA allele frequencies, a metric estimating population protection coverage (PPC) was computed and epitope pools assembled. Strikingly, only a minority of immunocompetent HIV-1 infected individuals responds to pools with PPC >95%. In contrast, virus-naive individuals uniformly expand IFNgamma producing cells and mount anti-HIV-1 cytolytic activity. This disparity suggests a vaccine design paradigm shift from infected to normal subjects.

Journal Article↗

PubMed2006

CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands.

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.

Antigens, CD↗

PubMed2006

A zinc-binding region in Vif binds Cul5 and determines cullin selection.

Human immunodeficiency virus-1 (HIV-1) Vif overcomes the anti-viral activity of APOBEC3G by targeting it for ubiquitination via a Cullin 5-ElonginB-ElonginC (Cul5-EloBC) E3 ligase. Vif associates with Cul5-EloBC through a BC-box motif that binds EloC, but the mechanism by which Vif selectively recruits Cul5 is poorly understood. Here we report that a region of Vif (residues 100-142) upstream of the BC-box binds selectively to Cul5 in the absence of EloC. This region contains a zinc coordination site HX5CX17-18CX3-5H (HCCH), with His/Cys residues at positions 108, 114, 133, and 139 coordinating one zinc ion. The HCCH zinc coordination site, which is conserved among primate lentivirus Vif proteins, does not correspond to any known class of zinc-binding motif. Mutations of His/Cys residues in the HCCH motif impair zinc coordination, Cul5 binding, and APOBEC3G degradation. Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cul5 and inhibit APOBEC3G degradation. The Vif binding site maps to the first cullin repeat in the N terminus of Cul5. These data suggest that the zinc-binding region in Vif is a novel cullin interaction domain that mediates selective binding to Cul5. We propose that the HCCH zinc-binding motif facilitates Vif-Cul5 binding by playing a structural role in positioning hydrophobic residues for direct contact with Cul5.

APOBEC Deaminases↗

PubMed2006

Use of laser capture microdissection to detect integrated HIV-1 DNA in macrophages and astrocytes from autopsy brain tissues.

The importance of astrocytes as a reservoir of human immunodeficiency virus type 1 (HIV-1) in the brain remains elusive. By combining immunohistochemistry, laser capture microdissection, and triple-nested Alu-PCR, we demonstrate integrated HIV-1 in astrocytes and macrophages isolated directly from autopsy brain tissues of HIV-1-infected subjects. The ability of HIV-1 to integrate in terminally differentiated astrocytes suggests a permanent reservoir of provirus in brain that will impact the development and likely success of strategies aimed at eradicating HIV-1.

AIDS Dementia Complex↗

PubMed2006

Reconstitution of human immunodeficiency virus-induced neurodegeneration using isolated populations of human neurons, astrocytes, and microglia and neuroprotection mediated by insulin-like growth factors.

Primary human neuron cultures are an important in vitro model system for studies on mechanisms involved in human immunodeficiency virus (HIV)-associated dementia (HAD) and other neurological disorders. Here, more than 80 cell surface antigens were screened to identify a marker that could readily distinguish between neurons and astrocytes and found that neurons lack CD44 surface expression, whereas astrocytes and other cell types in brain are CD44+. Neurons and astrocytes were isolated from human fetal brain based on differential expression of CD44. Using purified neurons cocultured with astrocytes and/or microglia, it was demonstrated that HIV infection of microglia induces cellular activation and production of soluble factors that activate uninfected microglia and astrocytes and induce neuronal cell death. Activated astrocytes promoted HIV replication in microglia, thereby amplifying HIV-induced neurotoxicity. A screen for 120 cytokine/proteins detected upregulation of insulin-like growth factor (IGF)-binding protein (IGFBP)-2, interleukin (IL)-6, and CCL8/MCP-2 (monocyte chemoattractant protein 2) in supernatants of HIV-infected brain cell cultures. IGF-1 and -2 increased neuronal survival in HIV-infected brain cell cultures, whereas IGFBP-2 inhibited prosurvival effects of these growth factors. These findings identify CD44 as a marker that can be used to sort neurons from other cell types in brain, suggest the importance of microglia-astrocyte interactions in neurodegenerative mechanisms associated with HIV infection, and indicate a role for insulin-like growth factors in neuroprotection from HIV-induced neurodegeneration. The ability to reconstitute brain cultures using isolated populations of neurons, astrocytes, and microglia will be valuable for studies on pathogenic mechanisms in HAD and other neurological disorders, and will also facilitate neuroactive drug discovery.

AIDS Dementia Complex↗

PubMed2006

A hydrophobic binding surface on the human immunodeficiency virus type 1 Nef core is critical for association with p21-activated kinase 2.

The interaction of human immunodeficiency virus type 1 (HIV-1) Nef with p21-activated kinase 2 (Pak2) has been proposed to play an important role in T-cell activation and disease progression during viral infection. However, the mechanism by which Nef activates Pak2 is poorly understood. Mutations in most Nef motifs previously reported to be required for Pak2 activation (G2, PxxP72, and RR105) also affect other Nef functions, such as CD4 or major histocompatibility complex class I (MHC-I) downregulation. To better understand Nef interactions with Pak2, we performed mutational analysis of three primary HIV-1 Nef clones that exhibited similar capacities for downregulation of CD4 and MHC-I but variable abilities to associate with activated Pak2. Our results demonstrate that Nef amino acids at positions 85, 89, 187, 188, and 191 (L, H, S, R, and F in the clade B consensus, respectively) are critical for Pak2 association. Mutation of these Nef residues dramatically altered association with Pak2 without affecting Nef expression levels or CD4 and MHC-I downregulation. Furthermore, compensation occurred at positions 89 and 191 when both amino acids were substituted. Since residues 85, 89, 187, 188, and 191 cluster on the surface of the Nef core domain in a region distinct from the dimerization and SH3-binding domains, we propose that these Nef residues form part of a unique binding surface specifically involved in association with Pak2. This binding surface includes exposed and recessed hydrophobic residues and may participate in an as-yet-unidentified protein-protein interaction to facilitate Pak2 activation.

Amino Acid Sequence↗

PubMed2006

Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele.

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5delta32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5delta32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.

Alleles↗

PubMed2006

CD16+ monocytes produce IL-6, CCL2, and matrix metalloproteinase-9 upon interaction with CX3CL1-expressing endothelial cells.

The CD16+ subset of peripheral blood monocytes (Mo) is expanded dramatically during inflammatory conditions including sepsis, HIV-1 infection, and cancer. CD16+ express high levels of CX3CR1, which mediates arrest onto CX3CL1-expressing endothelial cells (EC) under flow conditions. In contrast, attachment of CD16- Mo onto cytokine-activated EC is independent of CX3CL1. Here, we investigate the ability of CD16+ and CD16- Mo to produce proinflammatory cytokines upon interaction with CX3CL1-expressing HUVEC. We demonstrate that CD16+ but not CD16- Mo produce high levels of IL-6, CCL2, and matrix metalloproteinase (MMP)-9 when cocultured with TNF/IFN-gamma-activated HUVEC or nonactivated HUVEC expressing CX3CL1. Furthermore, supernatants from Mo cocultured with cytokine-activated HUVEC induce neuronal death in vitro. These results suggest that membrane-bound CX3CL1 stimulates production of IL-6, CCL2, and MMP-9 by CD16+ Mo, likely via engagement of CX3CR1. Thus, expansion of CD16+ Mo and their accumulation onto CX3CL1-expressing EC may result in recruitment of Mo and T cell subsets at sites of inflammation in response to CCL2, IL-6-induced cell activation and/or differentiation, and MMP-9-mediated vascular and tissue injury.

Cell Communication↗

PubMed2006

Mechanisms of HIV-1 neurotropism.

Human immunodeficiency virus type 1 (HIV) infects macrophages and microglia in the CNS and frequently causes neurocognitive impairment. Although antiviral therapy generally reduces the viral load in the CNS and improves HIV-associated neurological dysfunction, most current antiviral drugs have poor CNS penetrance and cannot completely suppress viral replication. Furthermore, drug-resistance mutations can evolve independently in the CNS. Thus, a long-lived viral reservoir persists in macrophages and microglia in the brain despite antiviral therapy. This review discusses mechanisms underlying the neurotropism of HIV, focusing on the role of the HIV envelope glycoproteins and their interactions with CD4 and the chemokine receptors CCR5 and CXCR4. We review data from studies of neurotropic HIV derived from the brains of patients with HIV-associated neurocognitive impairment as well as studies of nonhuman primate models. Understanding mechanisms that underlie HIV neurotropism and neurovirulence is critical for development of therapeutics to inhibit CNS infection and preventing neurological injury in HIV-infected individuals.

Animals↗

PubMed2005

CD16+ monocytes exposed to HIV promote highly efficient viral replication upon differentiation into macrophages and interaction with T cells.

The CD16+ subset of monocytes is dramatically expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unknown. Here, we demonstrate that CD16+ but not CD16- monocytes promote high levels of HIV replication upon differentiation into macrophages and interaction with T cells. Conjugates formed between CD16+ monocyte-derived macrophages and T cells are major sites of viral replication. Furthermore, similar monocyte-T cell conjugates detected in peripheral blood of HIV-infected patients harbor HIV DNA. Thus, expansion of CD16+ monocytes during HIV infection and their subsequent recruitment into tissues such as lymph nodes, brain, and intestine may contribute to HIV dissemination and establishment of productive infection in T cells.

CD4 Antigens↗

PubMed2005

HIV-1 envelope pseudotyped viral vectors and infectious molecular clones expressing the same envelope glycoprotein have a similar neutralization phenotype, but culture in peripheral blood mononuclear cells is associated with decreased neutralization sensitivity.

Recombinant lentiviral vectors pseudotyped with heterologous HIV-1 envelope glycoproteins allow rapid and accurate measurement of antibody-mediated HIV-1 neutralization. However, the neutralization phenotypes of envelope pseudoviruses have not been directly compared to isogenic replication competent HIV-1. We produced pseudoviruses expressing three different HIV-1 envelope glycoproteins and subcloned the same three env genes into a replication competent NL4-3 molecular clone. For each of the antibodies tested, the neutralization dose-response curves of pseudoviruses and corresponding replication competent viruses were similar. Thus, envelope pseudoviruses can be used to study the anti-HIV-1 neutralizing antibody response. A single passage of replication competent virus derived from 293T cells through peripheral blood mononuclear cells (PBMC) caused a substantial decrease in sensitivity to neutralizing antibodies. This was associated with an increase in average virion envelope glycoprotein content of the PBMC-derived virus. Replication competent HIV-1 and isogenic envelope pseudoviruses have similar neutralization characteristics, but passage into PBMC is associated with decreased sensitivity to neutralization.

HIV Antibodies↗

PubMed2005

Nuclear localization of HIV type 1 Vif isolated from a long-term asymptomatic individual and potential role in virus attenuation.

Recent reports have determined that HIV-1 Vif counteracts an innate antiviral cellular factor, Apobec3G. However, the function of Vif during HIV-1 pathogenesis remains poorly understood. To gain a better understanding of Vif function, the viral isolate from an HIV-1-infected long-term nonprogressor (LTNP) that displayed a Vif-mutant replication phenotype was studied. This LTNP has been infected since before 1983 and has no HIV-related disease in the absence of antiretroviral therapy. From separate samples, obtained on more than one study visit, virus grew in cocultures of LTNP cells with Vif-complementing T cell lines, but not the parental T cell lines. An unusual amino acid motif (KKRK) was found in the Vif sequence at positions 90 to 93. Since this motif commonly functions as a nuclear localization sequence, experiments were performed to determine the ability of this KKRK motif to mediate nuclear localization of Vif. Wild-type Vif displayed a predominantly cytoplasmic distribution. In contrast, the KKRK Vif showed a predominantly nuclear localization. The effect of the KKRK mutation on virus production and infectivity was also studied. The KKRK motif that mislocalizes Vif to the nucleus also reduces viral replication and infectivity in nonpermissive cells. Our data highlight the importance of Vif in HIV-1 pathogenesis and also provide a unique tool to investigate the interaction of Vif and Apobec3G.

Amino Acid Motifs↗

PubMed2005

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions.

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.

Apoptosis↗

PubMed2005

Pathogenesis of macrophage tropic HIV-1.

Despite numerous studies on the impact of viral diversity, human immunodeficiency virus type 1 (HIV-1)-specific immune responses and host factors on disease progression, we still do not have a firm understanding of the pathogenesis of HIV-1 infection. Rapid depletion of CD4+ T-lymphocytes has been associated with a switch in viral coreceptor usage from CCR5 to CXCR4 in approximately 40 to 50% of infected individuals. However, the majority of infected individuals who progress to AIDS harbor only CCR5-dependent (R5) viral strains. HIV-1 disease progression is associated with an enhanced tropism of R5 viral strains for cells of the monocyte/macrophage lineage (enhanced M-tropism). However, the underlying molecular mechanisms contributing to enhanced M-tropism by R5 HIV-1 strains, and how HIV-1 variants with enhanced M-tropism cause CD4+ T-cell depletion in vivo are unknown. This review examines the relationship between viral coreceptor usage, M-tropism, and pathogenicity of HIV-1. We highlight evidence supporting the hypothesis that enhanced M-tropism of R5 HIV-1 results from adaptive viral evolution, resulting in HIV-1 variants that have increased ability to utilize relatively low levels of CD4 and CCR5 expressed on macrophages. The evidence also suggests that these late-emerging, R5 viral strains have reduced sensitivity to entry inhibitors, and increased ability to cause CD4+ T-lymphocyte loss. These variants are likely to impact HIV-1 disease progression, particularly in patients who persistently harbor only R5 viral strains.

Disease Progression↗

PubMed2004

Phosphorylation of a novel SOCS-box regulates assembly of the HIV-1 Vif-Cul5 complex that promotes APOBEC3G degradation.

HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the DNA deaminase APOBEC3G by targeting it for proteasomal degradation. We report here that Vif targets APOBEC3G for degradation by forming an SCF-like E3 ubiquitin ligase containing Cullin 5 and Elongins B and C (Cul5-EloB-EloC) through a novel SOCS (suppressor of cytokine signaling)-box that binds EloC. Vif binding to EloC is negatively regulated by serine phosphorylation in the BC-box motif of the SOCS-box. Vif ubiquitination is promoted by Cul5 in vitro and in vivo, and requires an intact SOCS-box. Thus, autoubiquitination of Vif occurs within the assembled Vif-Cul5 complex, analogous to F-box proteins that are autoubiquitinated within their SCF (Skp1-Cullin-F-box) complex. These findings suggest mechanisms that regulate the assembly and activity of Cul5 E3 complexes through phosphorylation or autoubiquitination of the SOCS-box protein, and identify interactions between Vif and host cell proteins that may be therapeutic targets.

APOBEC-3G Deaminase↗