Paul R. Gorry

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity.

ABSTRACT The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.

Increased CCR5 Affinity and Reduced CCR5/CD4 Dependence of a Neurovirulent Primary Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT 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.

Genetic and Functional Analysis of Full-Length Human Immunodeficiency Virus Type 1 env Genes Derived from Brain and Blood of Patients with AIDS

ABSTRACT 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.

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.

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.

Persistence of dual-tropic HIV-1 in an individual homozygous for the CCR5Δ32 allele

Summary 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 (CCR5Δ32). 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.

Viral Phenotypes and Antibody Responses in Long-Term Survivors Infected with Attenuated Human Immunodeficiency Virus Type 1 Containing Deletions in the nef and Long Terminal Repeat Regions

ABSTRACT The Sydney Blood Bank Cohort (SBBC) consists of eight blood transfusion recipients infected with nef-attenuated human immunodeficiency virus type 1 (HIV-1) acquired from a single donor. Here, we show that viral phenotypes and antibody responses differ considerably between individual cohort members, despite the single source of infection. Replication of isolated virus varied from barely detectable to similar to that of the wild-type virus, and virus isolated from five SBBC members showed coreceptor usage signatures unique to each individual. Higher viral loads and stronger neutralizing antibody responses were associated with better-replicating viral strains, and detectable viral replication was essential for the development of strong and sustained humoral immune responses. Despite the presence of strong neutralizing antibodies in a number of SBBC members, disease progression was not prevented, and each cohort member studied displayed a unique outcome of infection with nef-attenuated HIV-1.

Chapter 5.3 – HIV-1-Associated Dementia

Publisher Summary nThis chapter discusses the role of chemokine receptors and their ligands in the disease mechanisms. HIV-1-associated dementia (HAD) results from complex interactions between viral and host factors. Four chemokine receptors and their ligands (CXCR4/SDF-1, CCR5/MIP-1α and β, RANTES, CCR2/MCP-1, and CX3CR1/fractalkine) are of particular importance in HAD. HIV-1 infection of macrophages and microglia in the CNS is primarily mediated by CCR5. A subset of strains can infect macrophages and microglia via CXCR4. Increased monocyte trafficking into the CNS of patients with HAD is likely to result from increased expression of MCP-1, in addition to CCR5 chemokines. Neurodegenerative mechanisms in HAD may involve interactions between the HIV-1 envelope glycoproteins (Env) and CXCR4, or possibly other chemokine receptors on neurons, and possibly other CNS cell types. CCR5 and CXCR4 are promising targets for the prevention and treatment of HAD. Several compounds that selectively block virus entry without affecting physiological functions of these receptors are being developed, and some are in early clinical trials. Understanding the role of CCR5, CXCR4 and other chemokine receptors and their ligands in HIV-1 neuropathogenesis will be important for advancing the development of new therapeutic strategies for the prevention and treatment of HAD and other neurological complications of HIV-1 infection.