Interactions between HIV-1 gp120, chemokines, and cultured adult microglial cells.

HIV dementia (HIVD), a disease that is apparently mediated by neurotoxins and viral proteins secreted by HIV infected microglia, is characterized neuropathologically by an increased number of activated microglia in the brains of affected individuals. Consequently, the rational design of potential therapeutic strategies should take into account the mechanisms that lead to microglial activation and to their increased prominence in the adult brain. In this regard, one leading hypothesis proposes that microglia are recruited to specific sites in the central nervous system (CNS) as a result of interactions between microglial chemokine receptors and chemokines, or even the viral glycoprotein gp120, which binds chemokine receptors in the process of cellular entry. Adult microglia express the functional chemokine receptors CCR5 and CXCR4 molecules that mediate chemotaxis in these and other cell types. We determined that purified adult microglial cultures contain a heterogeneous population with respect to their ability to respond to the alpha- and beta-chemokines, SDF1alpha, and MIP-1beta. A mean of 14.6% of the microglia assayed responded to both alpha- and beta-chemokines (CCR5(+)CXCR4(+) phenotype); 45.4% of microglia were phenotyped as CCR5(+)CXCR4(-); 12.9% of the microglia were CXCR4(+)CCR5(-); and 27.0% of microglia did not respond to either chemokine. No increase in intracellular calcium levels was seen in the vast majority of microglia exposed to the soluble HIV envelope protein, gp120, or to HIV envelope (gp120/gp41) expressed on MLV virus pseudotypes. However, exposure of microglia to soluble fractalkine or to other chemokines resulted in an intracellular calcium flux. Our results raise the possibility of microglial heterogeneity with respect to their response to chemokines, and indicate that any effects due to gp120 are likely to be considerably less robust than the response of microglia to the natural ligands of their chemokine receptors, for example SDF1alpha and MIP-1beta.

CXCR3 expression in human central nervous system diseases.

The CXCR3 chemokine receptor, expressed on activated T lymphocytes, is seen within the central nervous system (CNS) in inflammatory conditions where a T-cell response is prominent. However, the distribution of CXCR3 in parenchymal CNS cells is unknown. Using a monoclonal antibody against CXCR3 and post-mortem tissue of patients with and without CNS pathology, we have determined its expression pattern. CXCR3 was found in subpopulations of cells morphologically consistent with astrocytes, particularly reactive astrocytes, and in cerebellar Purkinje cells. It was also detected in arterial endothelial and smooth muscle cells, particularly in areas associated with atherosclerotic plaques. CXCR3-positive astrocytes were particularly prominent in the CNS of HIV-positive patients, in patients with Multiple Sclerosis (MS), in ischaemic infarcts and in astrocytic neoplasms. Immunofluorescence studies of mixed adult primary glial cultures and fetal glial cultures also showed expression of CXCR3 in astrocytes. CXCR3 mRNA was detected in Purkinje cells by in situ hybridization with a CXCR3-specific probe. Thus, the predominant expression of CXCR3 in reactive astrocytes may indicate that it plays a role in the development of reactive gliosis in a variety of infectious, inflammatory, vascular and neoplastic processes in the CNS. The relationship between CXCR3 expression in astrocytes to its expression in Purkinje cells, endothelial cells and smooth muscle cells is yet to be determined.

HIV-associated neuropathies: role of HIV-1, CMV, and other viruses.

The role of the human immunodeficiency virus (HIV) and other viruses in the development of neuropathies associated with HIV infection is controversial. Distal symmetric polyneuropathy (DSP), the most common subtype of HIV-associated neuropathy, is characterized by an abundance of reactive macrophages within the peripheral nerve, but HIV replication is limited to a small percentage of the macrophages. Thus, the pathological destruction may be mediated by pro-inflammatory signals amplified by activated glial elements within the nerve, similar to the proposed mechanism of damage caused by HIV within the central nervous system. In contrast, in mononeuropathy multiplex (MM) and progressive polyneuropathy (PP), cytomegalovirus (CMV) replication in the peripheral nerve is consistently demonstrable, and this replication likely results in direct damage to the infected cells (neurons and glia). The rarest form of HIV-associated neuropathy, the diffuse infiltrative lymphocytosis syndrome (DILS), is characterized by an intense CD8+ T lymphocyte infiltration into the nerve and abundant HIV infection of macrophages. Finally, while other viruses (varicella zoster, herpes simplex) are associated with myelitis in HIV-infected individuals, there is little support for a role for these viruses in HIV-associated neuropathy.

Differential CD4/CCR5 utilization, gp120 conformation, and neutralization sensitivity between envelopes from a microglia-adapted human immunodeficiency virus type 1 and its parental isolate.

Human immunodeficiency virus type 1 (HIV-1) infects and induces syncytium formation in microglial cells from the central nervous system (CNS). A primary isolate (HIV-1(BORI)) was sequentially passaged in cultured microglia, and the isolate recovered (HIV-1(BORI-15)) showed high levels of fusion and replicated more efficiently in microglia (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. González-Scarano, J. Virol. 70:7654-7662, 1996). The parent and adapted viruses used CCR5 as coreceptor. Recombinant viruses demonstrated that the syncytium-inducing phenotype was associated with four amino acid differences in the V1/V2 region of the viral gp120 (J. T. C. Shieh, J. Martin, G. Baltuch, M. H. Malim, and F. González-Scarano, J. Virol. 74:693-701, 2000). We produced luciferase-reporter, env-pseudotyped viruses using plasmids containing env sequences from HIV-1(BORI), HIV-1(BORI-15), and the V1/V2 region of HIV-1(BORI-15) in the context of HIV-1(BORI) env (named rBORI, rB15, and rV1V2, respectively). The pseudotypes were used to infect cells expressing various amounts of CD4 and CCR5 on the surface. In contrast to the parent recombinant, the rB15 and rV1V2 pseudotypes retained their infectability in cells expressing low levels of CD4 independent of the levels of CCR5, and they infected cells expressing CD4 with a chimeric coreceptor containing the third extracellular loop of CCR2b in the context of CCR5 or a CCR5 Delta4 amino-terminal deletion mutant. The VH-rB15 and VH-rV1V2 recombinant viruses were more sensitive to neutralization by a panel of HIV-positive sera than was VH-rBORI. Interestingly, the CD4-induced 17b epitope on gp120 was more accessible in the rB15 and rV1V2 pseudotypes than in rBORI, even before CD4 binding, and concomitantly, the rB15 and rV1V2 pseudotypes were more sensitive to neutralization with the human 17b monoclonal antibody. Adaptation to growth in microglia--cells that have reduced expression of CD4 in comparison with other cell types--appears to be associated with changes in gp120 that modify its ability to utilize CD4 and CCR5. Changes in the availability of the 17b epitope indicate that these affect conformation. These results imply that the process of adaptation to certain tissue types such as the CNS directly affects the interaction of HIV-1 envelope glycoproteins with cell surface components and with humoral immune responses.

Expression pattern of CXCR3, CXCR4, and CCR3 chemokine receptors in the developing human brain.

Chemokine receptors are essential components of the immune and central nervous systems, but little is known about their distribution during development. We evaluated the distribution of 3 chemokine receptors: CXCR3, CXCR4, and CCR3 in the human developing brain. Of these, CXCR3 was the only receptor expressed in fetal brain at 26 wk of gestation and its expression was restricted to glial cells, endothelial cells, and the choroid plexus. Neuronal staining was only seen at term in the Purkinje cells of the cerebellum. CCR3 appeared only at term in both neurons and glial cells. The expression pattern of these 2 receptors in the late gestation and term resembled that of adults. CXCR4 could not be detected in the fetal brain on neurons nor on glial cells. By examining pediatric cases, we determined that CXCR4 expression commences sometimes between 3.5 and 4.5 yr. Two of the chemokine receptors examined, CCR3 and CXCR4, can be used as co-receptor together with CD4 for HIV entry, but neither was expressed during the second trimester of pregnancy. Our findings suggest that it is unlikely that CCR3 or CXCR4 play a major role in HIV-1 transmission in the fetal brain before 37 wk of gestation.

Immunohistochemical analysis of CCR2, CCR3, CCR5, and CXCR4 in the human brain: potential mechanisms for HIV dementia.

The CXC chemokine receptor CXCR4 was the first molecule identified as a coreceptor working in conjunction with CD4 to mediate cellular entry for the human immunodeficiency virus (HIV-1). Since that original discovery, 11 other seven-mtransmembrane domain molecules, many of which are chemokine receptors, have been shown to facilitate HIV entry into cells. These include CCR5, CCR3, CCR2, CCR1, CCR8, CX3CR1, STRL33 (BONZO), GPR15 (BOB), GPR1, US28, and APJ. In studies done by this and other labs, CCR3, CCR5, and CXCR4 have been identified in CNS microglia and several laboratories, including ours, have shown that CXCR4 is expressed in neurons. Neuronal expression of CCR2, CCR3, and CCR5 has been less consistent. We performed a semiquantitative immunohistochemical analysis of the expression of CCR2, CCR3, CCR5, and CXCR4 in 23 regions of the brain and in two sections of the spinal cord. Hippocampal neurons were positive for CCR2, CCR3, and CXCR4, but not for CCR5. In other regions of the brain, neurons, and glial cells reacted with anti-CCR2, anti-CCR3, and anti-CXCR4 antibodies, whereas only glial cells (primarily microglia) were positive for CCR5. The areas of highest expression, however, seem to be subcortical regions and the limbic system. The limbic system plays a key role in memory, and the presence of CXCR4-which can bind the viral envelope protein gp120-min a subset of neurons from this system may play a role in the development of HIV-related dementia.

Efavirenz is a potent nonnucleoside reverse transcriptase inhibitor of HIV type 1 replication in microglia in vitro.

The objective of this study was to determine whether reverse transcriptase inhibitors (RTIs) could decrease viral replication in microglia. Human microglia obtained from individuals undergoing temporal lobectomy were cultured and infected with HIV-1 isolates from the central nervous system (CNS) as previously described (Strizki JM, et al. J Virol 1996;70:7654-7662). These microglial cultures were treated with one of three nucleoside RTIs (NRTIs) or with efavirenz, a nonnucleoside RTI (NNRTI), at various time points before and during HIV-1 infection. The drug levels sufficient to provide > 90% inhibition of microglial HIV replication (IC90) were determined by comparison of p24(gag) release in the cultures among treated and untreated microglia. Infectious virus released from the infected cultures was also measured with U373-MAGI-CCR5 cells. Efavirenz, an NNRTI, blocked HIV-1(DS-br) infection of microglia with an IC(90) of 0.7-7 nM. This value is similar to the efavirenz IC(90) values for inhibition of laboratory and clinical isolates in lymphocytes, is 2-3 logs lower than the IC90 values of AZT and d4T, and is 1-2 logs lower than that of ddC in microglia. Efavirenz also inhibited infection with other neurotropic isolates, and with viruses isolated from other compartments that also replicated well in microglia. Thus, efavirenz is a potent inhibitor of HIV-1 infection in microglia. Furthermore, efavirenz IC(90) drug levels are present in the cerebrospinal fluid (CSF) of patients taking this once daily NNRTI.

Characterization of cultured microglia that can be infected by HIV-1.

Parenchymal microglia are targets of HIV infection. We, as well as others, have used in vitro microglia culture systems to study the tropism and replication of HIV. Characterization of perivascular and parenchymal microglia surface markers in vivo, in vitro, and ex vivo, has led to the understanding that these cell populations are different, and data from both the HIV and SIV models support the hypothesis that they may play different roles in infection of the CNS. We determined that human adult parenchymal microglia cultured from temporal lobe tissue for use in HIV replication studies, were CD11c+, CD45+, CD68+, CD14- when cultured with standard serum/cytokine-supplemented media. To determine the influence of serum and cytokines on HIV replication in microglia, we designed a new protocol for culturing microglia, and compared the results obtained with this protocol with the standard approach previously described. Microglia cultured in the presence of a 'feeder' layer of glial cells and in the absence of serum and cytokines expressed the same surface markers as pure microglia (>95%) cultured in supplemented media. However, pure microglia cultured in the absence of both serum/cytokines supplements and other glial cells, did not have characteristic microglial morphology and did not support HIV replication to as high a level. Lastly, we determined that unlike monocytes, ex vivo parenchymal microglia were capable of supporting HIV replication.

Determinants of syncytium formation in microglia by human immunodeficiency virus type 1: role of the V1/V2 domains.

Microglia are the main reservoir for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS), and multinucleated giant cells, the result of fusion of HIV-1-infected microglia and brain macrophages, are the neuropathologic hallmark of HIV dementia. One potential explanation for the formation of syncytia is viral adaptation for these CD4(+) CNS cells. HIV-1(BORI-15), a virus adapted to growth in microglia by sequential passage in vitro, mediates high levels of fusion and replicates more efficiently in microglia and monocyte-derived-macrophages than its unpassaged parent (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. Gonzalez-Scarano, J. Virol. 70:7654-7662, 1996). Since the interaction between the viral envelope glycoprotein and CD4 and the chemokine receptor mediates fusion and plays a key role in tropism, we have analyzed the HIV-1(BORI-15) env as a fusogen and in recombinant and pseudotyped viruses. Its syncytium-forming phenotype is not the result of a switch in coreceptor use but rather of the HIV-1(BORI-15) envelope-mediated fusion of CD4(+)CCR5(+) cells with greater efficiency than that of its parental strain, either by itself or in the context of a recombinant virus. Genetic analysis indicated that the syncytium-forming phenotype was due to four discrete amino acid differences in V1/V2, with a single-amino-acid change between the parent and the adapted virus (E153G) responsible for the majority of the effect. Additionally, HIV-1(BORI-15) env-pseudotyped viruses were less sensitive to decreases in the levels of CD4 on transfected 293T cells, leading to the hypothesis that the differences in V1/V2 alter the interaction between this envelope and CD4 or CCR5, or both. In sum, the characterization of the envelope of HIV-1(BORI-15), a highly fusogenic glycoprotein with genetic determinants in V1/V2, may lead to a better understanding of the relationship between HIV replication and syncytium formation in the CNS and of the importance of this region of gp120 in the interaction with CD4 and CCR5.

Murine leukemia virus pseudotypes of La Crosse and Hantaan Bunyaviruses: a system for analysis of cell tropism.

We have developed a system for the preparation of La Crosse virus (LAC) and Hantaan virus (HTN) pseudotypes using a murine leukemia virus vector. After concentration, the pseudotypes were present in quantities sufficient to analyze cell tropism and neutralization. Cells resistant to LAC could not be infected with the MLV (LAC) pseudotypes, and the pseudotypes were sensitive to neutralizing monoclonal antibodies prepared against LAC glycoproteins, as well as to inhibition by a soluble form of the virus cell-attachment protein, G1. Perhaps because of lower expression of the HTN glycoproteins at the cell surface, MLV (HTN) pseudotypes were present at lower titers. However, they were also sensitive to appropriate neutralizing antibodies. This pseudotype system will be useful for analysis of the entry process of the Bunyaviridae, and for neutralization studies with some Bunyaviruses whose high virulence normally requires specialized containment facilities.

Polymerase chain reaction analysis of human herpesvirus-6 sequences in the sera and cerebrospinal fluid of patients with multiple sclerosis.

Several studies have suggested a possible association of human herpesvirus-6 (HHV-6) with multiple sclerosis (MS), a demyelinating disease with a variable course and progression. To determine whether HHV-6 could be detected in the sera of CSF of patients with different subtypes of MS, we performed nested polymerase chain reaction (PCR) on samples obtained from MS patients as well as samples from normal adults or individuals with other neurological diseases. Ninety-six serum samples from 24 patients with MS, including 13 individuals with relapsing remitting MS, one individual with primary progressive MS, seven individuals with secondary progressive MS and three individuals with an unspecified type were analyzed. Multiple serum samples were examined from individuals over varying periods of time and included samples obtained during exacerbations, remissions, and at different stages of progressive disease. HHV-6 DNA was detected only in one out of 15 serum samples that were collected over a number of years from one individual with secondary progressive MS. No HHV-6 DNA was detected in CSF from six patients with MS or 14 patients with other neurologic disease. These results indicate that the presence of HHV-6 DNA in the serum or CSF of patients with MS is not a common phenomenon, at least within the limits of the sensitivity of our assay.

Microglia as mediators of inflammatory and degenerative diseases.

Microglia are the principal immune cells in the central nervous system (CNS) and have a critical role in host defense against invading microorganisms and neoplastic cells. However, as with immune cells in other organs, microglia may play a dual role, amplifying the effects of inflammation and mediating cellular degeneration as well as protecting the CNS. In entities like human immunodeficiency virus (HIV) infection of the nervous system, microglia are also critical to viral persistence. In this review we discuss the role of microglia in three diseases in which their activity is at least partially deleterious: HIV, multiple sclerosis, and Alzheimer's disease.

Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates.

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1beta and SDF-1alpha, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.

The effect of human herpesvirus-6 (HHV-6) on cultured human neural cells: oligodendrocytes and microglia.

Human herpesvirus-6 (HHV-6) is a betaherpesvirus that has been frequently associated with pediatric encephalitis. In 1995 Challoner et al reported that HHV-6 variant B (HHV-6B) was linked to multiple sclerosis (MS) due to the presence of viral DNA and antigen in the oligodendrocytes surrounding MS plaques. These findings led us to examine HHV-6B's in vitro tropism for primary neural cells. HIV-6B mediated cell-to-cell fusion in cultured adult oligodendroglia. Infection of oligodendrocytes was further confirmed by transmission electron microscopy (EM), which showed the presence of intracellular HHV-6 particles, and by PCR for HHV-6 DNA. However, the release of infectious virus was low or undetectable in multiple experiments. Microglia were also susceptible to infection by HHV-6B, as demonstrated by an antigen capture assay. We did not detect infection of a differentiated neuronal cell line (NT2D). Our findings suggest that HHV-6B infection of oligodendrocytes and/or microglia could potentially play a role in neuropathogenesis.

Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex.

The Vpr protein of human immunodeficiency virus type 1 (HIV-1) performs a number of functions that are associated with the nucleus. Vpr enhances the nuclear import of postentry viral nucleoprotein complexes, arrests proliferating cells in the G2 phase of the cell cycle, and acts as a modest transcriptional activator. For this paper, we have investigated the nuclear import of Vpr. Although Vpr does not encode a sequence that is recognizable as a nuclear localization signal (NLS), Vpr functions as a transferable NLS both in somatic cells and in Xenopus laevis oocytes. In certain contexts, Vpr also mediates substantial accumulation at the nuclear envelope and, in particular, at nuclear pore complexes (NPCs). Consistent with this, Vpr is shown to interact specifically with nucleoporin phenylalanine-glycine (FG)-repeat regions. These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr's functions may take place at the NPC.

Chemokine receptors in the human brain and their relationship to HIV infection.

Chemokine receptors have been recently identified as the important co-factors which in conjunction with CD4, mediate entry of HIV into its target cells. The brain is one of the most prominent targets of HIV infection, where it leads to HIV encephalitis (HIVE) and HIV-associated dementia. Knowledge of the distribution, physiology, and pathology of chemokines and chemokine receptors in the human brain is fundamental for understanding the pathogenesis of the interaction between HIV and the central nervous system (CNS). There is also increasing evidence that chemokine receptors expression in the CNS increases during pathological, especially inflammatory, conditions. The major co-factors for HIV infection, CCR5, CCR3, and CXCR4 have been detected in the human brain in a variety of cell types including microglia, astrocytes, neurons, and vascular endothelial cells. Furthermore, antibodies to chemokine receptors can also block HIV infectivity in cultured CNS cells. This indicates that chemokine receptors are likely to have a functional role in the pathogenesis of HIVE.