R5 Strains of Human Immunodeficiency Virus Type 1 from Rapid Progressors Lacking X4 Strains Do Not Possess X4-Type Pathogenicity in Human Thymus

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.     

Rabbit Cells Expressing Human CD4 and Human CCR5 Are Highly Permissive for Human Immunodeficiency Virus Type 1 Infection

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.     

Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains

The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.     

Different Tempo and Anatomic Location of Dual-Tropic and X4 Virus Emergence in a Model of R5 Simian-Human Immunodeficiency Virus Infection

We previously reported coreceptor switch in rhesus macaques inoculated intravenously with R5 simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N). Whether R5-to-X4 virus evolution occurs in mucosally infected animals and in which anatomic site the switch occurs, however, were not addressed. We herein report a change in coreceptor preference in macaques infected intrarectally with SHIV_SF162P3N. The switch occurred in infected animals with high levels of virus replication and undetectable antiviral antibody response and required sequence changes in the V3 loop of the gp120 envelope protein. X4 virus emergence was associated with an accelerated drop in peripheral CD4⁺ T-cell count but followed rather than preceded the onset of CD4⁺ T-cell loss. The conditions, genotypic requirements, and patterns of coreceptor switch in intrarectally infected animals were thus remarkably consistent with those found in macaques infected intravenously. They also overlapped with those reported for humans, suggestive of a common mechanism for coreceptor switch in the two hosts. Furthermore, two independent R5-to-X4 evolutionary pathways were identified in one infected animal, giving rise to dual-tropic and X4 viruses which differed in switch kinetics and tissue localization. The dual-tropic switch event predominated early, and the virus established infection in multiple tissues sites. In contrast, the switch to X4 virus occurred later, initiating and expanding mainly in peripheral lymph nodes. These findings help define R5 SHIV_SF162P3N infection of rhesus macaques as a model to study the mechanistic basis, dynamics, and sites of HIV-1 coreceptor switch.The human immunodeficiency virus (HIV) enters target cells via binding of the viral envelope glycoprotein to the CD4 receptor, triggering envelope conformational changes that allow for interaction with either the CCR5 or CXCR4 chemokine receptor (1, 3, 8, 15, 16, 18). Most HIV type 1 (HIV-1) transmissions are initiated with CCR5-using (R5) viruses (58, 68). With time, CXCR4-tropic (X4) viruses emerge and coexist with R5 viruses in close to 50% of subtype B-infected individuals, and this is accompanied by a rise in viremia, rapid CD4⁺ T-cell loss, and progression to disease (4, 7, 11, 34, 57, 65). The mechanistic basis and reasons for HIV-1 coreceptor switch, however, are still not well understood. Several factors including high viral load, low CD4⁺ T-cell numbers, reduced availability of CCR5⁺ cells, and progressive immune dysfunction have been proposed as playing important roles (48, 54). Since X4 virus emergence is associated with a faster rate of disease progression, insights into the determinants of HIV-1 coreceptor switch are of interest in understanding viral pathogenesis. Furthermore, with the introduction of CCR5 entry inhibitors as anti-HIV therapeutics (19, 23, 24, 38), there is a need not only to identify the presence of X4 variants in patients when treatment options are considered but also to understand the factors that influence X4 virus evolution. Although the majority of individuals failing on short-term CCR5 antagonist monotherapy harbor preexisting minor X4 variants (71), it is conceivable that given the right conditions and selective forces, inhibiting HIV-1 entry via CCR5 may drive the virus to evolve to CXCR4 usage and exacerbate disease. An animal model that faithfully recapitulates the process of coreceptor switch will be highly useful to study and identify the determinants and conditions that facilitate the change in coreceptor preference. In addition, an animal model provides the opportunity to track the kinetics of coreceptor switching at different anatomical sites, which may inform on the mechanisms of X4 virus emergence.In this regard, we recently reported coreceptor switch in two of nine rhesus macaques (RM) inoculated intravenously with simian-human immunodeficiency virus SF162P3N (SHIV_SF162P3N) that bears an HIV-1 CCR5-tropic Env (28, 29). In order to establish a reproducible model for coreceptor switch, however, it was crucial to document additional switching events. Furthermore, since the majority of HIV transmission occurs via mucosal surfaces, it was important to demonstrate coreceptor switch in macaques infected with R5 SHIV_SF162P3N by the mucosal route to validate this animal model in studying the in vivo evolution of HIV-1 coreceptor usage. Additionally, the tissue compartment(s) where CXCR4-using viruses evolve and expand is not well characterized. A recent study indicates that the thymus may play an important role in the evolution and/or amplification of coreceptor variants in pediatric HIV infection (56). Since the thymus is the primary source of T lymphopoiesis during early life (45) and since CXCR4 is the predominant coreceptor expressed on thymocytes (33, 64), this organ would seem to provide the ideal milieu for X4 amplification in infants and children. Indeed, we previously showed that whereas X4 SHIV infection of newborn RM resulted in severe thymic involution, R5 SHIV infection induced only a minor disruption in thymic morphology (55), lending support to the idea that the thymus is a preferred site for X4 replication in pediatric HIV infections. Nevertheless, thymopoietic function declines with age (17, 42, 60), and naïve T cells that express high levels of CXCR4 are also enriched in peripheral lymph nodes (5, 27, 36, 66). Thus, the role of the thymus and other lymphoid tissues in HIV-1 coreceptor switch in older individuals remains to be determined. To address these issues, we inoculated adult RM intrarectally (i.r.) with R5 SHIV_SF162P3N and performed frequent longitudinal blood and tissue samplings. Our goal was to document changes in coreceptor preference in mucosally infected macaques, as well as to obtain a more detailed picture of the kinetics and site of X4 virus evolution and amplification in vivo.     

CXCR4 as a Functional Coreceptor for Human Immunodeficiency Virus Type 1 Infection of Primary Macrophages

The coreceptors used by primary syncytium-inducing (SI) human immunodeficiency virus type 1 isolates for infection of primary macrophages were investigated. SI strains using only CXCR4 replicated equally well in macrophages with or without CCR5 and were inhibited by several different ligands for CXCR4 including SDF-1 and bicyclam derivative AMD3100. SI strains that used a broad range of coreceptors including CCR3, CCR5, CCR8, CXCR4, and BONZO infected CCR5-deficient macrophages about 10-fold less efficiently than CCR5⁺ macrophages. Moreover, AMD3100 blocked infection of CCR5-negative macrophages by these strains. Our results therefore demonstrate that CXCR4, as well as CCR5, is used for infection of primary macrophages but provide no evidence for the use of alternative coreceptors.     

Antibody from Patients with Acute Human Immunodeficiency Virus (HIV) Infection Inhibits Primary Strains of HIV Type 1 in the Presence of Natural-Killer Effector Cells

The partial control of viremia during acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by an HIV-1-specific cytotoxic T-lymphocyte (CTL) response and an absent or infrequent neutralizing antibody response. The control of HIV-1 viremia has thus been attributed primarily, if not exclusively, to CTL activity. In this study, the role of antibody in controlling viremia was investigated by measuring the ability of plasma or immunoglobulin G from acutely infected patients to inhibit primary strains of HIV-1 in the presence of natural-killer (NK) effector cells. Antibody that inhibits virus when combined with effector cells was present in the majority of patients within days or weeks after onset of symptoms of acute infection. Furthermore, the magnitude of this effector cell-mediated antiviral antibody response was inversely associated with plasma viremia level, and both autologous and heterologous HIV-1 strains were inhibited. Finally, antibody from acutely infected patients likely reduced HIV-1 yield in vitro both by mediating effector cell lysis of target cells expressing HIV-1 glycoproteins and by augmenting the release of beta-chemokines from NK cells. HIV-1-specific antibody may be an important contributor to the early control of HIV viremia.     

CD4-Immunoglobulin G2 Protects Hu-PBL-SCID Mice against Challenge by Primary Human Immunodeficiency Virus Type 1 Isolates

CD4-immunoglobulin G2 (IgG2) is a fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. Previous studies found that CD4-IgG2 potently neutralizes a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates in vitro and ex vivo. The current report demonstrates that CD4-IgG2 protects against infection by primary isolates of HIV-1 in vivo, using the hu-PBL-SCID mouse model. Passive administration of 10 mg of CD4-IgG2 per kg of body weight protected all animals against subsequent challenge with 10 mouse infectious doses of the laboratory-adapted T-cell-tropic isolate HIV-1_LAI, while 50 mg of CD4-IgG2 per kg protected four of five mice against the primary isolates HIV-1_JR-CSF and HIV-1_AD6. In contrast, a polyclonal HIV-1 Ig fraction exhibited partial protection against HIV-1_LAI at 150 mg/kg but no significant protection against the primary HIV-1 isolates. The results demonstrate that CD4-IgG2 effectively neutralizes primary HIV-1 isolates in vivo and can prevent the initiation of infection by these viruses.     

Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 × 10⁹ to 2 × 10⁹ liters/mol) against either a “cluster II”-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56⁺ cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (≥3 × 10⁸ liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.The in vivo role(s) of antibodies (Abs) that can direct antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus type 1 (HIV-1) Env-expressing cells in vitro remains unclear. In ADCC, anti-Env Abs direct effector cells to kill target cells bearing HIV-1 envelope on their surfaces; this is accomplished via specific binding of the Abs’ antigen-binding sites to Envs and their Fc regions to Fc receptors on the effector cells. Broadly strain reactive, ADCC-directing Abs arise early in the immune response to HIV-1 infection in vivo (14) and may be partially responsible for the initial clearance of viremia.Earlier in the HIV-1 epidemic, concerns were raised that shed soluble gp120 in HIV-1-infected individuals might bind to CD4⁺ cells, including uninfected ones, and could target these cells for “innocent bystander” killing by ADCC (6). However, effector cells armed with serum Abs able to direct ADCC in vitro against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three distinct gp120 epitopes to direct ADCC against uninfected CD4⁺ cells to which rgp120_SF2 had been adsorbed (i.e., innocent bystanders) was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells.The existing data from in vivo studies (reviewed in reference 1) supports the efficacy, rather than the pathogenicity, of ADCC-directing Abs against HIV-1. Consistent with this data is our recent characterization of two MAbs, 42F and 43F, isolated from a long-term survivor of HIV-1 infection (1); these MAbs directed significant levels of ADCC and defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120. Preliminary evidence indicated that concentrations of 42F- and 43F-like Abs in the serum of the donor were in the range required to direct high levels of ADCC, and these MAbs were shown to bind both oligomeric primary-isolate and laboratory-adapted Env efficiently (1).Because of the potential importance of ADCC-directing Abs against HIV-1, in this study we have evaluated ADCC directed against cells expressing HIV-1 Envs of primary or laboratory-adapted strains by a panel of human and chimpanzee anti-Env MAbs of different epitope specificities. Significant ADCC activities of selected MAbs against primary-isolate Env-expressing cells were demonstrated, and a new ADCC epitope in the V2 domain of gp120 was defined. Finally, a MAb’s ability to direct ADCC against a specific target cell type was shown to be dependent on additional factors beyond its ability to efficiently bind antigen on the target cell and its possession of an Fc region of the appropriate isotype to engage FcγR on effector cells.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Engagement of the CD4 Receptor Affects the Redistribution of Lck to the Immunological Synapse in Primary T Cells: Implications for T-Cell Activation during Human Immunodeficiency Virus Type 1 Infection

Understanding the molecular mechanisms underlying dysregulated immune responses in human immunodeficiency virus type 1 (HIV-1) infection is crucial for the control of HIV/AIDS. Despite the postulate that HIV envelope glycoprotein gp120-CD4 interactions lead to impaired T-cell responses, the precise mechanisms underlying such association are not clear. To address this, we analyzed Lck and F-actin redistribution into the immunological synapse in stimulated human primary CD4⁺ T cells from HIV-1-infected donors. Similar experiments were performed with CD4⁺ T cells from HIV-uninfected donors, which were exposed to anti-CD4 domain 1 antibodies, as an in vitro model of gp120-CD4 interactions, or aldithriol-inactivated HIV-1 virions before stimulation. CD4⁺ T cells from HIV-infected patients exhibited a two- to threefold inhibition of both Lck and F-actin recruitment into the synapse, compared to cells from uninfected donors. Interestingly, defective recruitment of Lck was ameliorated following suppressive highly active antiretroviral therapy. Engagement of the CD4 receptor on T cells from HIV-uninfected donors before anti-CD3/CD28 stimulation led to similar defects. Furthermore, the redistribution of Lck into lipid rafts was abrogated by CD4 preengagement. Our results suggest that the engagement of CD4 by HIV gp120 prior to T-cell receptor stimulation leads to dysregulation of early signaling events and could consequently play an important role in impaired CD4⁺ T-cell function.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

Mononuclear Phagocyte Differentiation, Activation, and Viral Infection Regulate Matrix Metalloproteinase Expression: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is mediated mainly by mononuclear phagocyte (MP) secretory products and their interactions with neural cells. Viral infection and MP immune activation may affect leukocyte entry into the brain. One factor that influences central nervous system (CNS) monocyte migration is matrix metalloproteinases (MMPs). In the CNS, MMPs are synthesized by resident glial cells and affect the integrity of the neuropil extracellular matrix (ECM). To ascertain how MMPs influence HAD pathogenesis, we studied their secretion following MP differentiation, viral infection, and cellular activation. HIV-1-infected and/or immune-activated monocyte-derived macrophages (MDM) and human fetal microglia were examined for production of MMP-1, -2, -3, and -9. MMP expression increased significantly with MP differentiation. Microglia secreted high levels of MMPs de novo that were further elevated following CD40 ligand-mediated cell activation. Surprisingly, HIV-1 infection of MDM led to the down-regulation of MMP-9. In encephalitic brain tissue, MMPs were expressed within perivascular and parenchymal MP, multinucleated giant cells, and microglial nodules. These data suggest that MMP production in MP is dependent on cell type, differentiation, activation, and/or viral infection. Regulation of MMP expression by these factors may contribute to neuropil ECM degradation and leukocyte migration during HAD.     

Determinants Flanking the CD4 Binding Loop Modulate Macrophage Tropism of Human Immunodeficiency Virus Type 1 R5 Envelopes

Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env) in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic. Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33) and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N) conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to identify additional determinants. The determinants of macrophage tropism that were identified included residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3 loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4 contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need to expose critical CD4 contact residues to the immune system.Human immunodeficiency virus type 1 (HIV-1) requires interactions between viral envelope glycoproteins and cell surface CD4 and coreceptors to trigger fusion and entry into cells. HIV-1 R5 viruses that specifically use CCR5 as a coreceptor are those predominantly transmitted (3). Yet, our knowledge of R5 virus variation in different biological properties is still limited. In vivo, HIV-1 infection is limited mostly to cells that express CD4 and appropriate coreceptors. Thus, HIV-1 infects CD4⁺ T cells, monocyte/macrophage lineage cells, and dendritic cells. CCR5 is expressed on each of these cell lineages, although on T cells, CCR5 is restricted mainly to RO45⁺ memory cells (1, 16). Early in infection, R5 viruses target and decimate mucosal CD4⁺ memory T cells (2, 18, 26). R5 viruses are also predominant in tissues in which monocyte/macrophage lineage cells are prevalent, and several reports have described the presence of highly macrophage-tropic R5 viruses in brain tissue (11, 12, 20, 22). Previously, we used PCR to amplify HIV-1 envelope genes directly from patient tissues. We found that R5 virus envelopes amplified from brain tissue frequently conferred highly efficient infection of macrophages, while the majority of those from lymph nodes, blood, and semen infected macrophages inefficiently (20, 22). Although those studies examined relatively few infected individuals, they demonstrated over 1,000-fold variation in macrophage-tropic HIV-1 R5 viruses. Such variation is likely to have a significant impact on transmission and pathogenesis.The envelope (env) determinants of R5 macrophage-tropic strains are poorly understood. Several studies have shown that highly macrophage-tropic brain envelopes are able to exploit low levels of CD4 on macrophages for infection, consistent with an enhanced interaction between gp120 and CD4. Dunfee et al. reported that an asparagine residue at position 283 in the C2 part of the CD4 binding site was present in 41% of envelope sequences from brain tissue specimens of patients with HIV-associated dementia and in only 8% of envelopes from non-HIV-associated dementia brain tissue (8). The same study showed that the presence of N283 (rather than the more conserved T283) led to an increased affinity of gp120 for CD4, probably because the side chain of asparagine could more readily form a hydrogen bond with Q40 on CD4. However, our previous data showed that N283 is not the only determinant of macrophage infectivity, since several macrophage-tropic R5 envelopes from brain and semen specimens lacked N283, while non-macrophage-tropic envelopes from lymph node specimens carrying N283 were identified (22). Dunfee et al. also reported that a glycosylation site at residue 386, close to the CD4 binding loop, influenced exposure of the CD4 binding site and had an impact on macrophage tropism and sensitivity to the CD4 binding site antibody b12 (9). We have recently confirmed a role for N386 in resistance to the CD4 binding site monoclonal antibody (MAb) b12. However, resistance was dependent on the presence of a proximal residue, R373, which acted together with N386 to block b12 (7).Here, we have further investigated envelope determinants of macrophage tropism by preparing chimeric envelopes from highly macrophage-tropic and non-macrophage-tropic R5 envelopes from brain and lymph node specimens from the same subject. We show that R5 macrophage tropism is controlled by several determinants in gp120 that are focused on amino acids flanking the CD4 binding loop, with a contribution from residues in the V3 loop.     

Adaptation of a CCR5-Using, Primary Human Immunodeficiency Virus Type 1 Isolate for CD4-Independent Replication

The gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and chemokine receptors on the target cell. Primary, clinical HIV-1 isolates require interaction with CD4 to allow gp120 to bind the CCR5 chemokine receptor efficiently. We adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for the adaptation were limited to alteration of glycosylation addition sites in the V2 loop-V1-V2 stem. The gp120 glycoproteins of the adapted viruses bound CCR5 directly, without prior interaction with CD4. Thus, a major function of CD4 binding in the entry of primary HIV-1 isolates can be bypassed by changes in the gp120 V1-V2 elements, which allow the envelope glycoproteins to assume a conformation competent for CCR5 binding.