Biomedical subjects

Dana Gabuzda

Publications and source records attributed to Dana Gabuzda.

32 records · Page 2Linked to original sources

PubMed2004

Camelized rabbit-derived VH single-domain intrabodies against Vif strongly neutralize HIV-1 infectivity.

We recently developed a specific single-chain antibody from immunized rabbits to HIV-1 Vif protein that was expressed intracellularly and inhibited reverse transcription and viral replication. The Vif of HIV-1 overcomes the innate antiviral activity of a cytidine deaminase Apobec3G (CEM15) that induces G to A hypermutation in the viral genome, resulting in enhancement of viral replication infectivity. Here, we have developed a minimal scaffold VH fragment with intrabody properties derived from anti-Vif single-chain antibody that was engineered to mimic camelid antibody domains. Non-specific binding of VH by its interface for the light chain variable domain (VL) was prevented through amino acid mutations in framework 2 and 4 (Val37F, G44E, L45R, W47G and W103R). Our results demonstrate that all constructed anti-Vif VH single-domains preserve the antigen-binding activity and specificity in the absence of the parent VL domain. However, only the most highly camelized domains had high levels of intracellular expression. The expression in eukaryotic cells showed that VH single-domains could correctly fold as soluble proteins in the reducing environment. The results demonstrated an excellent correlation between improvements in protein solubility with gradually increasing camelization. Camelized single-domains efficiently bound Vif protein and neutralized its infectivity enhancing function, by reducing late reverse transcripts and proviral integration. The activity of the anti-Vif single-domains was shown to be cell-specific, with inhibitory effects only in cells non-permissive that require Vif for HIV-1 replication. Moreover, cell specificity of anti-Vif intrabodies was correlated with an increase of Apobec3G, which potentiates viral inhibition. The present study strongly suggests that camelization of rabbit VH domains is a potentially useful approach for engineering intrabodies for gene therapy.

Amino Acid Sequence↗

PubMed2004

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins.

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.

Asparagine↗

PubMed2004

Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions.

CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a consequence of their massive accumulation onto inflamed brain vascular endothelial cells expressing FKN and other adhesion molecules.

Brain↗

PubMed2004

Contribution of proteoglycans to human immunodeficiency virus type 1 brain invasion.

As a neurotropic virus, human immunodeficiency virus type 1 (HIV-1) invades the brain and causes severe neuronal, astrocyte, and myelin damage in AIDS patients. To gain access to the brain, HIV-1 must migrate through brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB). Given that BMECs lack the entry receptor CD4, HIV-1 must use receptors distinct from CD4 to enter these cells. We previously reported that cell surface proteoglycans serve as major HIV-1 receptors on primary human endothelial cells. In this study, we examined whether proteoglycans also impact cell-free HIV-1 invasion of the brain. Using an artificial BBB transmigration assay, we found that both heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are abundantly expressed on primary BMECs and promote HIV-1 attachment and entry. In contrast, the classical entry receptors, CXCR4 and CCR5, only moderately enhanced these processes. HSPGs and CSPGs captured HIV-1 in a gp120-dependent manner. However, no correlation between coreceptor usage and transmigration was identified. Furthermore, brain-derived viruses did not transmigrate more efficiently than lymphoid-derived viruses, suggesting that the ability of HIV-1 to replicate in the brain does not correlate with its capacity to migrate through the BBB as cell-free virus. Given that HIV-1-proteoglycan interactions are based on electrostatic contacts between basic residues in gp120 and sulfate groups in proteoglycans, HIV-1 may exploit these interactions to rapidly enter and migrate through the BBB to invade the brain.

Blood-Brain Barrier↗

PubMed2004

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure.

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.

Animals↗

PubMed2003

Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway.

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses.

APOBEC-3G Deaminase↗

PubMed2003

Fractalkine preferentially mediates arrest and migration of CD16+ monocytes.

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Antibodies, Monoclonal↗

PubMed2003

CD2 engagement induces dendritic cell activation: implications for immune surveillance and T-cell activation.

We have shown previously that primary dendritic cells and monocytes express equal levels of CD14 but are distinguishable by the presence of CD2 on dendritic cells. CD2 is known to mediate the activation of T and natural killer (NK) cells through its interaction with CD58. CD2 epitopes recognized by anti-T111, -T112, and -T113 monoclonal antibodies (mAbs) are present on dendritic cells. Here we show that CD2 engagement significantly increases class II, costimulatory (CD40, CD80, CD86), adhesion (CD54, CD58), and CCR7 molecule expression on primary dendritic cells. Conversely, minimal or no change in the expression of the above antigens occurs on monocyte-derived dendritic cells, because these molecules are already maximally expressed. However, both kinds of dendritic cells release interleukin-1beta (IL-1beta) and IL-12 after CD2 engagement. Lastly, interference with dendritic cell CD2-T-cell CD58 engagement decreases naive CD4+CD45RA+ T-cell proliferation. Collectively, our results suggest another role of the CD2-CD58 pathway that allows nonimmune and immune cells to interact directly with dendritic cells and initiate innate and adaptive immune responses.

Animals↗

PubMed2003

Genetic and functional analysis of full-length human immunodeficiency virus type 1 env genes derived from brain and blood of patients with AIDS.

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Functional neutralization of HIV-1 Vif protein by intracellular immunization inhibits reverse transcription and viral replication.

Human immunodeficiency virus type 1 (HIV-1)-encoded Vif protein is important for viral replication and infectivity. Vif is a cytoplasmic protein that acts during virus assembly by an unknown mechanism, enhancing viral infectivity. The action of Vif in producer cells is essential for the completion of proviral DNA synthesis following virus entry. Therefore, Vif is considered to be an important alternative therapeutic target for inhibition of viral infectivity at the level of viral assembly and reverse transcription. To gain insight into this process, we developed a Vif-specific single-chain antibody and expressed it intracellularly in the cytoplasm. This intrabody efficiently bound Vif protein and neutralized its infectivity-enhancing function. Intrabody-expressing cells were shown to be highly refractory to challenge with different strains of HIV-1 and HIV-1-infected cells. Inhibition of Vif by intrabody expression in the donor cell produced viral particles that do not complete reverse transcription in the recipient cell. The anti-Vif scFv was shown to be specific for Vif protein because its function was observed only in nonpermissive cells (H9, CEM, and U38). Moreover, transduction of peripheral blood mononuclear cells with an HIV-derived retroviral vector expressing Vif intrabody was shown to confer resistance to laboratory-adapted and primary HIV strains. This study provides biochemical evidence for the role of Vif in the HIV-1 lifecycle and validates Vif as a target for the control of HIV-1 infection.

Animals↗

PubMed2002

Persistence of dual-tropic HIV-1 in an individual homozygous for the CCR5 Delta 32 allele.

Entry of HIV-1 into a cell happens only after viral envelope glycoproteins have bound to CD4 and a chemokine receptor. Generally, macrophage-tropic strains use CCR5, and T cell-line-tropic strains use CXCR4 as coreceptors for virus entry. Dual-tropic viruses can use both CCR5 and CXCR4. About 1% of white people are homozygous for a non-functional CCR5 allele, containing a 32 base pair deletion (CCR5 Delta 32). We studied the persistence of dual-tropic HIV-1 in an individual homozygous for this deletion. Our results suggest that structural features of the HIV-1 envelope linked to CCR5 tropism could confer a selective advantage in vivo.

Adult↗

PubMed2002

Regulation of CC chemokine receptor 5 and CD4 expression and human immunodeficiency virus type 1 replication in human macrophages and microglia by T helper type 2 cytokines.

Macrophages, microglia, and other mononuclear phagocytes serve as cellular reservoirs for viral persistence in patients with acquired immunodeficiency syndrome. To understand host mechanisms that affect human immunodeficiency virus type 1 (HIV-1) pathogenesis by modulating expression of coreceptors, cytokine regulation of CC chemokine receptor 5 (CCR5) and CD4 expression on monocytes, monocyte-derived macrophages (MDMs), and microglia was investigated. Interleukin (IL)-4 and IL-10 enhanced the entry and replication of HIV-1 in microglia through up-regulation of CD4 and CCR5 expression, respectively. IL-4 stimulated HIV-1 replication in MDMs but down-regulated CD4 and CCR5 expression and inhibited virus entry, whereas IL-10 had the opposite effects. Thus, mechanisms independent of CCR5 and CD4 expression levels are involved in pathways that regulate HIV-1 replication in MDMs. CCR5 up-regulation by IL-10 was associated with increased migration of microglia in response to macrophage inflammatory protein-1beta. These findings suggest that increased production of T helper type 2 cytokines in the later stages of disease can enhance virus entry and replication in mononuclear phagocytes and facilitate chemotactic migration.

CD4 Antigens↗

PubMed2002

Human immunodeficiency virus type 1 neutralization measured by flow cytometric quantitation of single-round infection of primary human T cells.

There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24(+) lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8(-) T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines.

Cells, Cultured↗

PubMed2002

Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate.

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

AIDS Dementia Complex↗