Yanjie Yi

A Dual-Tropic Primary HIV-1 Isolate That Uses Fusin and the β-Chemokine Receptors CKR-5, CKR-3, and CKR-2b as Fusion Cofactors

Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.

Regions in β-Chemokine Receptors CCR5 and CCR2b That Determine HIV-1 Cofactor Specificity

Macrophage-tropic (M-tropic) HIV-1 strains use the beta-chemokine receptor CCR5, but not CCR2b, as a cofactor for membrane fusion and infection, while the dual-tropic strain 89.6 uses both. CCR5/2b chimeras and mutants were used to map regions of CCR5 important for cofactor function and specificity. M-tropic strains required either the amino-terminal domain or the first extracellular loop of CCR5. A CCR2b chimera containing the first 20 N-terminal residues of CCR5 supported M-tropic envelope protein fusion. Amino-terminal truncations of CCR5/CCR2b chimeras indicated that residues 2-5 are important for M-tropic viruses, while 89.6 is dependent on residues 6-9. The identification of multiple functionally important regions in CCR5, coupled with differences in how CCR5 is used by M- and dual-tropic viruses, suggests that interactions between HIV-1 and entry cofactors are conformationally complex.

Macrophage activation through CCR5‐ and CXCR4‐mediated gp120‐elicited signaling pathways

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV‐1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV‐1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell‐surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV‐1 entry, CCR5 and CXCR4 are G protein‐coupled receptors that can activate multiple intracellular signaling pathways. HIV‐1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl–, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion‐related tyrosine kinase Pyk2, mitogen‐activated protein kinases (MAPK), and phosphoinositol‐3 kinase. Activation of the MAPK leads to gp120‐induced expression of chemokines such as monocyte chemoattractant protein‐1 and macrophage‐inflammatory protein‐1β and the proinflammatory cytokine tumor necrosis factor α. These responses establish a complex cytokine network, which may enhance or suppress HIV‐1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

Heterosexual Transmission of Human Immunodeficiency Virus Type 1 Subtype C: Macrophage Tropism, Alternative Coreceptor Use, and the Molecular Anatomy of CCR5 Utilization

ABSTRACT Human immunodeficiency virus type 1 transmission selects for virus variants with genetic characteristics distinct from those of donor quasispecies, but the biological factors favoring their transmission or establishment in new hosts are poorly understood. We compared primary target cell tropisms and entry coreceptor utilizations of donor and recipient subtype C Envs obtained near the time of acute infection from Zambian heterosexual transmission pairs. Both donor and recipient Envs demonstrated only modest macrophage tropism, and there was no overall difference between groups in macrophage or CD4 T-cell infection efficiency. Several individual pairs showed donor/recipient differences in primary cell infection, but these were not consistent between pairs. Envs had surprisingly broad uses of GPR15, CXCR6, and APJ, but little or no use of CCR2b, CCR3, CCR8, GPR1, and CXCR4. Donors overall used GPR15 better than did recipients. However, while several individual pairs showed donor/recipient differences for GPR15 and/or other coreceptors, the direction of the differences was inconsistent, and several pairs had unique alternative coreceptor patterns that were conserved across the transmission barrier. CCR5/CCR2b chimeras revealed that recipients as a group were more sensitive than were donors to replacement of the CCR5 extracellular loops with corresponding regions of CCR2b, but significant differences in this direction were not consistent within pairs. These data show that sexual transmission does not select for enhanced macrophage tropism, nor for preferential use of any alternative coreceptor. Recipient Envs are somewhat more constrained than are donors in flexibility of CCR5 use, but this pattern is not universal for all pairs, indicating that it is not an absolute requirement.

Preferential coreceptor utilization and cytopathicity by dual-tropic HIV-1 in human lymphoid tissue ex vivo

Many HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4(+) T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5(+)/CD4(+) T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4(+) T cell population in lymphoid tissue is associated with the use of CXCR4.

Depletion of CD4+ T cells abrogates post-peak decline of viremia in SIV-infected rhesus macaques

CD4+ T cells play a central role in the immunopathogenesis of HIV/AIDS, and their depletion during chronic HIV infection is a hallmark of disease progression. However, the relative contribution of CD4+ T cells as mediators of antiviral immune responses and targets for virus replication is still unclear. Here, we have generated data in SIV-infected rhesus macaques (RMs) that suggest that CD4+ T cells are essential in establishing control of virus replication during acute infection. To directly assess the role of CD4+ T cells during primary SIV infection, we in vivo depleted these cells from RMs prior to infecting the primates with a pathogenic strain of SIV. Compared with undepleted animals, CD4+ lymphocyte-depleted RMs showed a similar peak of viremia, but did not manifest any post-peak decline of virus replication despite CD8+ T cell- and B cell-mediated SIV-specific immune responses comparable to those observed in control animals. Interestingly, depleted animals displayed rapid disease progression, which was associated with increased virus replication in non-T cells as well as the emergence of CD4-independent SIV-envelopes. Our results suggest that the antiviral CD4+ T cell response may play an important role in limiting SIV replication, which has implications for the design of HIV vaccines.

Preferential Use of CXCR4 by R5X4 Human Immunodeficiency Virus Type 1 Isolates for Infection of Primary Lymphocytes

ABSTRACT Coreceptor specificity of human immunodeficiency virus type 1 (HIV-1) strains is generally defined in vitro in cell lines expressing CCR5 or CXCR4, but lymphocytes and macrophages are the principal targets in vivo. CCR5-using (R5) variants dominate early in infection, but strains that use CXCR4 emerge later in a substantial minority of subjects. Many or most CXCR4-using variants can use both CXCR4 and CCR5 (R5X4), but the pathways that are actually used to cause infection in primary cells and in vivo are unknown. We examined several R5X4 prototype and primary isolates and found that they all were largely or completely restricted to CXCR4-mediated entry in primary lymphocytes, even though lymphocytes are permissive for CCR5-mediated entry by R5 strains. In contrast, in primary macrophages R5X4 isolates used both CCR5 and CXCR4. The R5X4 strains were also more sensitive than R5 strains to CCR5 blocking, suggesting that interactions between the R5X4 strains and CCR5 are less efficient. These results indicate that coreceptor phenotyping in transformed cells does not necessarily predict utilization in primary cells, that variability exists among HIV-1 isolates in the ability to use CCR5 expressed on lymphocytes, and that many or most strains characterized as R5X4 are functionally X4 in primary lymphocytes. Less efficient interactions between R5X4 strains and CCR5 may be responsible for the inability to use CCR5 on lymphocytes, which express relatively low CCR5 levels. Since isolates that acquire CXCR4 utilization retain the capacity to use CCR5 on macrophages despite their inability to use it on lymphocytes, these results also raise the possibility that a CCR5-mediated macrophage reservoir is required for sustained infection in vivo.

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP–neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Chemokine receptor utilization and macrophage signaling by human immunodeficiency virus type 1 gp120: Implications for neuropathogenesis

Human immunodeficiency virus type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 for entry. Macrophages and microglia (M/M) are the principal productively infected brain cells in HIV encephalopathy (HIVE), and neuronal injury is believed to result both from direct effects of viral proteins and indirect effects mediated by macrophage activation and secretion of neurotoxic products. In vitro, direct injury by the viral envelope glycoprotein gp120 can be mediated by neuronal CXCR4, but most HIV-1 isolates from the central nervous system (CNS) studied to date use CCR5 (R5 strains) rather than CXCR4 (X4 or R5X4 strains). Additionally, it remains unknown how HIV induces M/M activation and neurotoxin secretion. To address these issues, the authors analyzed a CNS-derived primary isolate, TYBE, and showed that it uses CXCR4 only and replicates efficiently in macrophages through CXCR4-mediated entry. The authors also showed that both R5 and X4 gp120 activate intracellular signals in macrophages through CCR5 and CXCR4, including calcium elevations; K+, Cl−and nonselective cation channel activation; phosphorylation of the nonreceptor tyrosine kinase Pyk2; and activation of p38 and SAPK/JNK mitogen-activated protein kinases (MAPKs). Finally, the authors showed that macrophages stimulated with gp120 produce soluble factors through MAPK-dependent pathways, including β-chemokines implicated in HIVE pathogenesis. The findings emphasize that both X4 and R5 HIV-1 isolates may contribute to HIVE pathogenesis, and that gp120/chemokine receptor interactions in M/M trigger specific signal transduction pathways that may affect M/M function and provide a mechanism underlying CNS injury.

An unusual syncytia-inducing human immunodeficiency virus type 1 primary isolate from the central nervous system that is restricted to CXCR4, replicates efficiently in macrophages, and induces neuronal apoptosis.

Macrophage/microglia cells are the principal targets for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS). Prototype HIV-1 isolates from the CNS are macrophage (M)-tropic, non-syncytia-inducing (NSI), and use CCR5 for entry (R5 strains), but whether syncytia-inducing (SI) CXCR4-using X4 strains might play a role in macrophage/microglia infection and neuronal injury is unknown. To explore the range of features among HIV-1 primary isolates from the CNS, the authors analyzed an HIV-1 strain (TYBE) from cerebrospinal fluid of an individual with acquired immunodeficiency syndrome (AIDS) that was unusual because it was SI. Like other CNS isolates, HIV-1/TYBE replicated to high level in primary human macrophages, but, in contrast to CNS prototypes, TYBE used CXCR4 exclusively to infect macrophages. A functional TYBE env clone confirmed the X4 phenotype and displayed a highly charged V3 sequence typical of X4 strains. Supernatant from TYBE-infected primary human macrophages induced apoptosis of neurons. Thus, TYBE represents a novel type of CNS-derived HIV-1 isolate that is CXCR4-restricted yet replicates efficiently in macrophages and induce neuronal injury. These results demonstrate that HIV-1 variants in the CNS may possess a broader range of biological characteristics than generally appreciated, raise the possibility that X4 strains may participate in AIDS neuropathogenesis, and provide a prototype clade B HIV-1 strain that replicates efficiently in primary macrophages through the exclusive use of CXCR4 as a coreceptor.