Chemokine Coreceptor Usage by Diverse Primary Isolates of Human Immunodeficiency Virus Type 1

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defective CCR5 Δ32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defective CCR5 Δ32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.     

Neutralization Profiles of Primary Human Immunodeficiency Virus Type 1 Isolates in the Context of Coreceptor Usage

Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell.     

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.     

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.     

V3 Recombinants Indicate a Central Role for CCR5 as a Coreceptor in Tissue Infection by Human Immunodeficiency Virus Type 1

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.     

Antibody Neutralization-Resistant Primary Isolates of Human Immunodeficiency Virus Type 1

Although typical primary isolates of human immunodeficiency virus type 1 (HIV-1) are relatively neutralization resistant, three human monoclonal antibodies and a small number of HIV-1⁺ human sera that neutralize the majority of isolates have been described. The monoclonal antibodies (2G12, 2F5, and b12) represent specificities that a putative vaccine should aim to elicit, since in vitro neutralization has been correlated with protection against primary viruses in animal models. Furthermore, a neutralization escape mutant to one of the antibodies (b12) selected in vitro remains sensitive to neutralization by the other two (2G12 and 2F5) (H. Mo, L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. H. I. Parren, D. R. Burton, J. P. Moore, and D. D. Ho, J. Virol. 71:6869–6874, 1997), supporting the notion that eliciting a combination of such specificities would be particularly advantageous. Here, however, we describe a small subset of viruses, mostly pediatric, which show a high level of neutralization resistance to all three human monoclonal antibodies and to two broadly neutralizing sera. Such viruses threaten antibody-based antiviral strategies, and the basis for their resistance should be explored.     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.     

Chimeric Human Immunodeficiency Virus Type 1 Containing Murine Leukemia Virus Matrix Assembles in Murine Cells

Murine cells do not support efficient assembly and release of human immunodeficiency virus type 1 (HIV-1) virions. HIV-1-infected mouse cells that express transfected human cyclin T1 synthesize abundant Gag precursor polyprotein, but inefficiently assemble and release virions. This assembly defect may result from a failure of the Gag polyprotein precursor to target to the cell membrane. Plasma membrane targeting of the precursor is mediated by the amino-terminal region of polyprotein. To compensate for the assembly block, we substituted the murine leukemia virus matrix coding sequences into an infectious HIV-1 clone. Transfection of murine fibroblasts expressing cyclin T1 with the chimeric proviruses resulted in viruses that were efficiently assembled and released. Chimeric viruses, in which the cytoplasmic tail of the transmembrane subunit, gp41, was truncated to prevent potential interference between the envelope glycoprotein and the heterologous matrix, could infect human and murine cells. They failed to further replicate in the murine cells, but replicated with delayed kinetics in human MT-4 cells. These findings may be useful for establishing a murine model for HIV-1 replication.     

Continued Utilization of CCR5 Coreceptor by a Newly Derived T-Cell Line-Adapted Isolate of Human Immunodeficiency Virus Type 1

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates, Like HIV-1 Isolates, Frequently Use CCR5 but Show Promiscuity in Coreceptor Usage

Coreceptor usage of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to biological phenotype. The chemokine receptors CCR5 and CXCR4 are the major coreceptors that, together with CD4, govern HIV-1 entry into cells. Since CXCR4 usage determines the biological phenotype for HIV-1 isolates and is more frequent in patients with immunodeficiency, it may serve as a marker for viral virulence. This possibility prompted us to study coreceptor usage by HIV-2, known to be less pathogenic than HIV-1. We tested 11 primary HIV-2 isolates for coreceptor usage in human cell lines: U87 glioma cells, stably expressing CD4 and the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, and GHOST(3) osteosarcoma cells, coexpressing CD4 and CCR5, CXCR4, or the orphan receptor Bonzo or BOB. The indicator cells were infected by cocultivation with virus-producing peripheral blood mononuclear cells and by cell-free virus. Our results show that 10 of 11 HIV-2 isolates were able to efficiently use CCR5. In contrast, only two isolates, both from patients with advanced disease, used CXCR4 efficiently. These two isolates also promptly induced syncytia in MT-2 cells, a pattern described for HIV-1 isolates that use CXCR4. Unlike HIV-1, many of the HIV-2 isolates were promiscuous in their coreceptor usage in that they were able to use, apart from CCR5, one or more of the CCR1, CCR2b, CCR3, and BOB coreceptors. Another difference between HIV-1 and HIV-2 was that the ability to replicate in MT-2 cells appeared to be a general property of HIV-2 isolates. Based on BOB mRNA expression in MT-2 cells and the ability of our panel of HIV-2 isolates to use BOB, we suggest that HIV-2 can use BOB when entering MT-2 cells. The results indicate no obvious link between viral virulence and the ability to use a multitude of coreceptors.     

Coreceptor Utilization by Human Immunodeficiency Virus Type 1 Is Not a Primary Determinant of Neutralization Sensitivity

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses.Although CD4 had long been recognized as the cellular receptor to which the human immunodeficiency virus type 1 (HIV) envelope protein binds (9, 21, 22), it had also been recognized that expression of CD4 alone is insufficient to render nonhuman cells susceptible to HIV infection (4, 5, 22). Similarly, different HIV isolates display different abilities to infect CD4-positive human macrophages, T lymphocytes, and established T-cell lines (31, 32, 35), suggesting that additional molecules may be responsible for cell tropism specificity. During the past year, cellular molecules that act in conjunction with CD4 have been identified as required cofactors for HIV envelope protein-mediated binding and entry (1, 6, 10–12, 14). These HIV coreceptors are members of the superfamily of seven-transmembrane segment G-protein-coupled receptors and act primarily as cellular receptors for chemokines.The discovery of cellular coreceptors for HIV has provided new perspectives for understanding these early events in HIV infection (see review in reference 2). Thus, phenotypically distinct isolates of HIV utilize as coreceptors different chemokine receptor molecules. Although all primary isolates of HIV infect primary T lymphocytes, some also infect cells of the macrophage lineage (31, 32). These monocyteropic isolates utilize the CCR5 chemokine receptor, whose natural ligands include the chemokines RANTES, MIP-1α, and MIP-1β (1, 6, 10–12). Monocytropic isolates do not induce syncytia in primary lymphocyte culture and do not infect established T-cell lines (31). During the late course of HIV infection, syncytium-inducing (SI) primary viruses often arise from the population of monocytropic viruses (31, 32). These SI primary isolates no longer infect macrophages, and they utilize both CCR5 and another chemokine receptor, CXCR4 (7, 33, 38). CXCR4, whose natural chemokine ligand is SDF-1 (3, 27), was originally identified by Feng et al. as the cofactor used by laboratory-adapted viruses (14). In fact, the common laboratory viruses (IIIb/LAI, LAV, and RF) are unable to utilize CCR5 coreceptor (1, 6, 10–12), presumably reflecting the lack of CCR5 expression in most established T-cell lines (1, 13). Although some primary isolates utilize additional chemokine receptor molecules, notably CCR3 and CCR2b (6, 11, 18), the relationship between these coreceptors and viral phenotypes is less clear. The ability to utilize CCR5 coreceptor, however, is unique to primary-isolate (PI) viruses.Paralleling these differences in coreceptor utilization and cell tropism are differences in sensitivity to virus neutralization. Although laboratory-adapted isolates of HIV can be potently neutralized by sera elicited by recombinant gp120 (rgp120) protein, primary isolates are largely refractory to neutralization by rgp120 vaccine sera (23, 37). Similarly, PI viruses are significantly more resistant than T-cell line-adapted (TCLA) viruses to neutralization by gp120-directed monoclonal antibodies (MAbs) (25, 37) and to inhibition by soluble forms of CD4 (8). We and others have demonstrated that neutralization sensitivity develops concomitantly with adaptation of primary isolates to persistent growth in established T-cell lines (24, 37). By studying pedigreed PI and TCLA viruses (168P and 168C, respectively), we have shown that adaptation renders the TCLA virus sensitive not only to rgp120 vaccine sera and CD4 immunoadhesin but also to MAbs directed to the V3 loop of gp120 (37). However, the basis for this increase in neutralization sensitivity remains unclear.In this report, we explore the relationship between neutralization sensitivity and coreceptor utilization, especially with regard to changes that accompany adaptation. We examined neutralization sensitivity of the well-characterized SI primary isolate 168P under experimental conditions where infection can be directed via either the CXCR4 or the CCR5 pathway. The pedigreed TCLA derivative 168C utilizes only CXCR4 and was sensitive to neutralization by the panel of V3-directed MAbs used in these assays. However, the primary isolate 168P remained refractory to neutralization regardless of coreceptor pathway taken. Our findings suggest that envelope protein structure, and not coreceptor utilization, is the primary determinant of differential neutralization sensitivity in PI and TCLA viruses.

Coreceptor utilization by pedigreed PI and TCLA viruses.

Cross-sectional surveys of coreceptor use have shown that primary SI isolates generally utilize CXCR4 and CCR5 coreceptors, whereas unrelated laboratory-adapted isolates utilize only CXCR4 (1, 6, 7, 10–12, 14, 33, 38). We wished to confirm this trend in a longitudinal study of adaptation. We previously described the adaptation of the SI primary isolate 168P to persistent growth in the FDA/H9 T-cell line and the concomitant development of neutralization sensitivity in the resulting TCLA virus 168C (37). In the present study, the ability of these pedigreed viruses to utilize specific coreceptors was tested by infection of U87 human glioma cell lines expressing CD4 (U87-CD4) and the specific coreceptor (19).For this assay, virus stocks were prepared from cell culture supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) (168P) or FDA/H9 cells (168C) and standardized to yield a submaximal number of foci of infection on U87-CD4-CXCR4 cells (approximately 100 to 200 foci/96-well microplate culture). To confirm coreceptor specificity, in some assays CCR5 chemokines (each at 500 ng/ml) were added to cells 1 h prior to infection. After 2 days of incubation, cell monolayers were fixed with methanol-acetone and immunochemically stained with HIV immunoglobulin (HIVIG) (29), anti-human ABC kit (Biomeda Corp.), and diaminobenzidine substrate.Figure ​Figure11 confirms the ability of the SI 168P virus to utilize both CXCR4 and CCR5 and the subsequent loss of this latter specificity in the 168C TCLA virus. Infection was dependent on coreceptor expression, and both PI and TCLA viruses could also utilize CCR3 (data not presented). Open in a separate windowFIG. 1Coreceptor utilization by pedigreed PI and TCLA 168 viruses. U87-CD4 cell lines expressing CXCR4 (▪) or CCR5 () were used to define the ability of 168P and 168C viruses to utilize the respective coreceptor. CCR5 utilization was further tested by the addition to U87-CD4-CCR5 cells of CCR5-specific chemokines (RANTES, MIP-1α, and MIP-1β; R&D Systems) (□). For details, see text. ∗, no foci were observed.In keeping with the determined coreceptor specificity, infection could be blocked by addition of coreceptor-specific ligands. Thus, 168P virus infection of CCR5-expressing cells was blocked by the CCR5-specific ligands RANTES, MIP-1α, and MIP-1β (1, 6, 10–12) (Fig. ​(Fig.1).1). Similarly, infection of CXCR4-expressing U87-CD4 cells by either virus could be blocked by the CXCR4-specific chemokine ligand SDF-1 (3, 27) (data not presented).

Coreceptor pathway and neutralization sensitivity.

In previous work, we demonstrated that the PI 168P virus is refractory to neutralization by HIV MN gp120 vaccine sera and by several well-characterized V3-directed murine MAbs which strongly neutralize infectivity of the TCLA 168C virus (37). In the present study, we extended the panel of MAbs to include two V3-directed human MAbs, 257-D and 268-D (17). These well-characterized human MAbs recognize core epitopes at the crown of the V3 loop of gp120 (KRIHI and HIGPGR, respectively), linear sequences known to be present in both 168P and 168C envelope proteins (37). These epitope predictions were confirmed by gp120 capture enzyme-linked immunosorbent assay (ELISA) (26) which demonstrated equal binding to envelope protein in detergent-solubilized 168P and 168C virions (data not presented). Sensitivity to neutralization by these human MAbs was determined in a standard assay using PHA-activated PBLs (37). MAbs 257-D and 268-D were found to potently neutralize 168C but fail to neutralize 168P (Fig. ​(Fig.2).2). This pattern of neutralization sensitivity is similar to that previously described for the V3-directed murine MAb 50.1 (30, 36, 37). Open in a separate windowFIG. 2Neutralization sensitivity of 168 viruses in PBL culture. Virus neutralization assays in PHA-stimulated PBL culture were performed as previously described (37). 168P (○, •) and 168C (□, ▪) virus stocks were standardized to yield submaximal extents of virus spread during the 5-day infection. CCR5-specific chemokines (•, ▪) were added as described for Fig. ​Fig.1.1. The V3-directed MAbs are indicated. p24 antigen was determined by p24 antigen capture ELISA (SAIC Frederick) and was normalized to infected cell control values (168P, 190 ng/ml [170 ng/ml with chemokines]; 168C, 36 ng/ml [33 ng/ml with chemokines]).To examine whether sensitivity to neutralization was affected by the coreceptor pathway utilized in infection of PBLs, we used inhibitory concentrations of CCR5-specific chemokine ligands RANTES, MIP-1α, and MIP-1β in order to restrict infection to the CXCR4 pathway. Addition of these chemokines to the PBL cultures did not affect virus growth, nor did it affect sensitivity to neutralization by the V3-directed human MAbs (Fig. ​(Fig.2).2). To the extent that CCR5 blockade was complete, these results suggest that the simple availability of the CCR5 pathway is not a factor in the resistance of PI viruses to neutralization.To strengthen this conclusion, we examined neutralization sensitivity in human U87-CD4 cell lines expressing only CXCR4 or CCR5. Using this method, we confirmed that the SI 168P virus remained refractory to neutralization by human MAbs 257-D and 268-D as well as by the murine MAb 50.1, regardless of whether infection occurred via CXCR4 or CCR5 (Fig. ​(Fig.3).3). These results suggest that availability of the CCR5 pathway is not a primary determinant for the resistance of PI viruses to neutralization. The TCLA 168C virus utilized CXCR4 only and was sensitive to neutralization. Open in a separate windowFIG. 3Neutralization sensitivity of 168 viruses in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. 168P (○, •) and 168C (▪) viruses were used to infect U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1.1. The V3-directed MAbs were incubated with virus for 1 h prior to infection.

Molecularly cloned PI and TCLA envelope genes.

To understand better the changes that accompany adaptation and those that determine coreceptor utilization and neutralization sensitivity, we molecularly cloned the envelope genes of the 168P and 168C viruses. High-fidelity XL PCR (rTth and Vent DNA polymerases; PE Applied Biosystems) and primers envA and envN (15) were used to amplify a 3.1-kb region of proviral DNA encoding the rev and envelope genes. PCR products were isolated by unidirectional T/A cloning in the eucaryotic expression vector pCR3.1-Uni (Invitrogen). Expression in pCR3.1-Uni is driven by the cytomegalovirus immediate-early promoter. Multiple clones were isolated from each virus, and transient transfection studies in COS-7 cells confirmed the surface expression and fusion competence of all clones tested (data not presented).DNA sequence analysis demonstrated that all 168C molecular clones analyzed encoded the three adaptation-associated amino acid changes previously identified by PCR sequencing of the 168C virus population (V2, I166R; C2, I282N; and V3, G318R) (37). Two molecular clones of each 168P and 168C envelope were subjected to complete DNA sequence analysis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF035532","term_id":"2827997","term_text":"AF035532"}}AF035532 to {"type":"entrez-nucleotide","attrs":{"text":"AF035534","term_id":"2828001","term_text":"AF035534"}}AF035534). Molecular clones 168C23 and 168C60 were identical throughout the envelope gene. Molecular clones 168P5 and 168P23 differed from each other and from the previously determined sequence at four to five positions distinct from those associated with adaptation. These scattered changes within the primary virus quasispecies are considered inconsequential at the present level of analysis; the significance of the three adaptation-associated changes is under separate investigation.Functional analysis of these molecularly cloned envelope genes was performed by incorporation of the molecularly cloned envelope protein into pseudotyped HIV virions. We used an envelope-defective provirus derived from the molecularly cloned NL4-3 provirus (kindly provided by I. S. Y. Chen, University of California, Los Angeles). The pNLthyΔBgl provirus (28) contains a BglII-BglII deletion within the envelope gene and a substitution of the viral nef gene with a cDNA encoding the murine Thy1.2 cell surface protein. The simian virus 40 ori was subsequently introduced into the plasmid to generate pSVNLthyΔBgl (27a). Cotransfection of COS-7 cells (16, 20) with pSVNLthyΔBgl provirus and the envelope expression plasmid resulted in the production of pseudotyped HIV virions. Culture supernatants were harvested 3 days posttransfection, filtered, and used to infect U87-CD4 cell lines expressing coreceptor. Cells infected by virions bearing the complementing envelope protein were identified by immunostaining for murine Thy1.2 or HIV proteins.As anticipated, the molecularly cloned envelope proteins recapitulated the coreceptor specificity of the parental virus population (see the legend to Fig. ​Fig.4).4). Pseudotyped virions containing 168C60 were able to infect only U87-CD4 cells expressing CXCR4, while virions containing 168P23 envelope were able to infect U87-CD4 cells expressing either CCR5 or CXCR4. Thus, the viral envelope protein appears to be the major, if not sole, determinant of viral coreceptor use. These findings also indicate that dual coreceptor use is a direct property of the envelope protein complex and not a result of a mixture of distinct envelope proteins in the SI virus population. This conclusion is corroborated by the failure of CCR5-specific chemokine ligands to diminish 168P virus infection in PBL culture (Fig. ​(Fig.22).Open in a separate windowFIG. 4Neutralization sensitivity of pseudotyped virions in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. Pseudotyped virions were derived by cotransfection of COS-7 cells with pSVNLthyΔBgl provirus and plasmid expressing 168P23 (○, •) or 168C60 (▪) envelope protein. Virion preparations were incubated with U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1;1; V3-directed MAbs were added as indicated. The number of foci was normalized to control values (60 to 100 foci/well for U87-CD4-CXCR4 cells; 10 foci/well for U87-CD4-CCR5 cells). ∗, no foci were observed.Finally, we wished to determine the neutralization sensitivity of pseudotyped virions containing the molecularly cloned 168P23 and 168C60 envelope proteins and to confirm that coreceptor pathway is not a primary determinant of neutralization sensitivity. We found that infection of U87-CD4-CXCR4 cells by pseudotyped virions containing 168C60 envelope protein was sensitive to neutralization by MAbs 257-D, 268-D, and 50.1 at concentrations comparable to those determined in assays using 168C virus (Fig. ​(Fig.4).4). Pseudotyped virions containing 168P23 envelope protein remained refractory to neutralization by all three V3-directed MAbs, regardless of the coreceptor expressed by the U87-CD4 cell line. In summary, we examined the relationship between coreceptor utilization and sensitivity to neutralization by V3-directed MAbs. The observed dichotomy in the sensitivity to neutralization of PI and TCLA viruses had suggested a discrete difference between these viruses, and we tested one hypothesis: that PI viruses are refractory to neutralization as a result of their unique ability to utilize the CCR5 coreceptor. We examined neutralization sensitivity of a well-characterized SI primary isolate under experimental conditions wherein the virus was forced to utilize either CCR5 or CXCR4 for infection. We showed that coreceptor pathway is not a direct determinant of neutralization sensitivity. The primary virus envelope protein remained refractory to neutralization by V3-directed MAbs regardless of the coreceptor pathway utilized. Similarly, coreceptor utilization did not affect neutralization sensitivity by soluble CD4 (34) or HIVIG (data not presented).In discarding the otherwise attractive hypothesis that PI viruses escape neutralization through their unique ability to utilize CCR5, we are left to consider the as yet undefined structural differences between the envelope protein complex of PI and TCLA viruses. Several studies have suggested that critical determinants in the envelope protein of PI viruses are less accessible than those of TCLA viruses and that it is this differential access that determines neutralization sensitivity (reviewed in reference 25). By contrast, our studies have indicated similar binding of V3-directed MAbs to PBLs infected with neutralization-resistant isolate 168P or neutralization-sensitive isolate 168C (37). Thus, the basis for the differential neutralization sensitivity of PI and TCLA viruses remains unresolved.Our present studies also do not address whether changes in coreceptor utilization and/or neutralization sensitivity are necessarily linked as a consequence of adaptation. The analysis of independently derived PI and TCLA viruses may allow further separation of these viral phenotypes. Subsequent dissection of the amino acid changes that distinguish pedigreed PI and TCLA envelope proteins will help to define the structural bases underlying the changes that accompany adaptation.     

Amino-Terminal Substitutions in the CCR5 Coreceptor Impair gp120 Binding and Human Immunodeficiency Virus Type 1 Entry

The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4⁺ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.     

Intrinsic Human Immunodeficiency Virus Type 1 Resistance of Hematopoietic Stem Cells Despite Coreceptor Expression

Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.     

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-1β and SDF-1α, 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-1_DS-br, HIV-1_RC-br, and HIV-1_YU-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.     

Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.     

The Retroviruses Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Adopt Radically Different Strategies To Regulate Promoter-Proximal Polyadenylation

Maximal gene expression in retroviruses requires that polyadenylation in the 5' long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5' LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5' LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5' LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.