Primary infection by a human immunodeficiency virus with atypical coreceptor tropism

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.     

Primary Human Immunodeficiency Virus Type 2 (HIV-2) Isolates Infect CD4-Negative Cells via CCR5 and CXCR4: Comparison with HIV-1 and Simian Immunodeficiency Virus and Relevance to Cell Tropism In Vivo

Cell surface receptors exploited by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) for infection are major determinants of tropism. HIV-1 usually requires two receptors to infect cells. Gp120 on HIV-1 virions binds CD4 on the cell surface, triggering conformational rearrangements that create or expose a binding site for a seven-transmembrane (7TM) coreceptor. Although HIV-2 and SIV strains also use CD4, several laboratory-adapted HIV-2 strains infect cells without CD4, via an interaction with the coreceptor CXCR4. Moreover, the envelope glycoproteins of SIV of macaques (SIV(MAC)) can bind to and initiate infection of CD4(-) cells via CCR5. Here, we show that most primary HIV-2 isolates can infect either CCR5(+) or CXCR4(+) cells without CD4. The efficiency of CD4-independent infection by HIV-2 was comparable to that of SIV, but markedly higher than that of HIV-1. CD4-independent HIV-2 strains that could use both CCR5 and CXCR4 to infect CD4(+) cells were only able to use one of these receptors in the absence of CD4. Our observations therefore indicate (i) that HIV-2 and SIV envelope glycoproteins form a distinct conformation that enables contact with a 7TM receptor without CD4, and (ii) the use of CD4 enables a wider range of 7TM receptors to be exploited for infection and may assist adaptation or switching to new coreceptors in vivo. Primary CD4(-) fetal astrocyte cultures expressed CXCR4 and supported replication by the T-cell-line-adapted ROD/B strain. Productive infection by primary X4 strains was only triggered upon treatment of virus with soluble CD4. Thus, many primary HIV-2 strains infect CCR5(+) or CXCR4(+) cell lines without CD4 in vitro. CD4(-) cells that express these coreceptors in vivo, however, may still resist HIV-2 entry due to insufficient coreceptor concentration on the cell surface to trigger fusion or their expression in a conformation nonfunctional as a coreceptor. Our study, however, emphasizes that primary HIV-2 strains carry the potential to infect CD4(-) cells expressing CCR5 or CXCR4 in vivo.     

Identification of a subset of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains able to exploit an alternative coreceptor on untransformed human brain and lymphoid cells

The chemokine receptors CCR5 and CXCR4 are the major coreceptors for human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). At least 12 other chemokine receptors or close relatives support infection by particular HIV and SIV strains on CD4(+) transformed indicator cell lines in vitro. However, the role of these alternative coreceptors in vivo is presently thought to be insignificant. Infection of cell lines expressing high levels of recombinant CD4 and coreceptors thus does not provide a true indication of coreceptor use in vivo. We therefore tested primary untransformed cell cultures that lack CCR5 and CXCR4, including astrocytes and brain microvascular endothelial cells (BMVECs), for naturally expressed alternative coreceptors functional for HIV and SIV infection. An adenovirus vector (Ad-CD4) was used to express CD4 in CD4(-) astrocytes and thus confer efficient infection if a functional coreceptor is present. Using a large panel of viruses with well-defined coreceptor usage, we identified a subset of HIV and SIV strains able to infect two astrocyte cultures derived from adult brain tissue. Astrocyte infection was partially inhibited by several chemokines, indicating a role for the chemokine receptor family in the observed infection. BMVECs were weakly positive for CD4 but negative for CCR5 and CXCR4 and were susceptible to infection by the same subset of isolates that infected astrocytes. BMVEC infection was efficiently inhibited by the chemokine vMIP-I, implicating one of its receptors as an alternative coreceptor for HIV and SIV infection. Furthermore, we tested whether the HIV type 1 and type 2 strains identified were able to infect peripheral blood mononuclear cells (PBMCs) via an alternative coreceptor. Several strains replicated in Delta32/Delta32 CCR5 PBMCs with CXCR4 blocked by AMD3100. This AMD3100-resistant replication was also sensitive to vMIP-I inhibition. The nature and potential role of this alternative coreceptor(s) in HIV infection in vivo is discussed.     

Antigenically distinct conformations of CXCR4

The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1 alpha and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.     

Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2.

The chemokine receptors CCR-5 and CXCR-4, and possibly CCR-3, are the principal human immunodeficiency virus type 1 (HIV-1) coreceptors, apparently interacting with HIV-1 envelope, in association with CD4. Cell lines coexpressing CD4 and these chemokine receptors were infected with a panel of seven primary HIV-2 isolates passaged in peripheral blood mononuclear cells (PBMC) and three laboratory HIV-2 strains passaged in T-cell lines. The CCR-5, CCR-3, and CXCR-4 coreceptors could all be used by HIV-2. The ability to use CXCR-4 represents a major difference between HIV-2 and the closely related simian immunodeficiency viruses. Most HIV-2 strains using CCR-5 could also use CCR-3, sometimes with similar efficiencies. As observed for HIV-1, the usage of CCR-5 or CCR-3 was observed principally for HIV-2 strains derived from asymptomatic individuals, while HIV-2 strains derived from AIDS patients used CXCR-4. However, there were several exceptions, and the patterns of coreceptor usage seemed more complex for HIV-2 than for HIV-1. The two T-tropic HIV-2 strains tested used CXCR-4 and not CCR-5, while T-tropic HIV-1 can generally use both. Moreover, among five primary HIV-2 strains all unable to use CXCR-4, three could replicate in CCR-5-negative PBMC, which has not been reported for HIV-1. These observations suggest that the CCR-5 coreceptor is less important for HIV-2 than for HIV-1 and indicate that HIV-2 can use other cell entry pathways and probably other coreceptors. One HIV-2 isolate replicating in normal or CCR-5-negative PBMC failed to infect CXCR-4+ cells or the U87MG-CD4 and sMAGI cell lines, which are permissive to infection by HIV-2 but not by HIV-1. This suggests the existence of several HIV-2-specific coreceptors, which are differentially expressed in cell lines and PBMC.     

Direct visualization of HIV-1 entry: mechanisms and role of cell surface receptors.

Highly fluorescent virions of T- and M-tropic HIV-1 strains were obtained by incorporation of the viral accessory protein Vpr, fused to the green fluorescent protein, in trans. The fluorescent virions displayed normal morphology, were infectious, and could be used for direct visualization of HIV-1 attachment and trafficking in various cell lines. More than 90% of the viral particles were found to enter the cells by direct membrane fusion in T-cells, CD4+ HeLa cells, and macrophages. Visualizing HIV-1 attachment and entry in the absence or presence of CD4 and/or the appropriate coreceptors indicated that CD4 is the major receptor for virus attachment in the case of JR-CSF and NL-4-3 HIV-1 isolates; however, the coreceptors are required for membrane fusion. Internalization of the coreceptor CXCR4 inhibited entry, but did not prevent virus binding suggesting that transient downregulation of the coreceptor(s) may not be the most efficient way of blocking HIV infection in vivo.     

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.     

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.

The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.     

Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism

Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4).     

Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity.

The CXCR-4 chemokine receptor and CD4 behave as coreceptors for cell line-adapted human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for dual-tropic HIV strains, which also use the CCR-5 coreceptor. The cell line-adapted HIV-1 strains LAI and NDK and the dual-tropic HIV-2 strain ROD were able to infect CD4+ cells expressing human CXCR-4, while only LAI was able to infect cells expressing the rat homolog of CXCR-4. This strain selectivity was addressed by using human-rat CXCR-4 chimeras. All chimeras tested mediated LAI infection, but only those containing the third extracellular domain (e3) of human CXCR-4 mediated NDK and ROD infection. The e3 domain might be required for the functional interaction of NDK and ROD, but not LAI, with CXCR-4. Alternatively, LAI might also interact with e3 but in a different way. Monoclonal antibody 12G5, raised against human CXCR-4, did not stain cells expressing rat CXCR-4. Chimeric human-rat CXCR-4 allowed us to map the 12G5 epitope in the e3 domain. The ability of 12G5 to neutralize infection by certain HIV-1 and HIV-2 strains is also consistent with the role of e3 in the coreceptor activity of CXCR-4. The deletion of most of the amino-terminal extracellular domain (e1) abolished the coreceptor activity of human CXCR-4 for ROD and NDK but not for LAI. These results indicate that HIV strains have different requirements for their interaction with CXCR-4. They also suggest differences in the interaction of dual-tropic HIV with CCR-5 and CXCR-4.     

CXCR4 and CCR5 regulation and expression patterns on T- and monocyte-macrophage cell lineages: implications for susceptibility to infection by HIV-1

Chemokine receptor expression may vary dramatically among cell subsets. Therefore, the stage of differentiation and the lineage of CD4 cells may profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). However, the mechanisms of coreceptor competition for association with HIV-1 glycoproteins remain unknown. Here, we propose mathematical models that address the interdependence of the concentrations of CD4 and CCR5 for efficient infection by M-tropic HIV-1 as well as additional complications originated by coreceptor competition caused by posttranslational modifications that positively or negatively affect the coreceptor ability to form complexes with CD4 and/or HIV-1 envelope. Furthermore, since CCR5 and CXCR4 expression on human leukocytes designate these cells as HIV-1 potential targets, the expression of the major HIV-1 coreceptors are also dynamically modeled/quantified as function of the stage of cell differentiation. Results show that although coreceptor competition degree has limited influence on R5 strain infectivity, the infectivity of CXCR4-using isolates strongly depends on the CD4 expression, according to the coreceptor competition model proposed in Lee et al. [J. Virol. 74(11) (2000) 5016]. Understanding the role of in vivo alterations in CD4, CCR5 and CXCR4 densities on HIV-1 cell entry may help the development of optimal control strategies for AIDS pathogenesis.     

CXCR4 expression during lymphopoiesis: implications for human immunodeficiency virus type 1 infection of the thymus.

Human immunodeficiency virus type 1 (HIV-1) infection of the human thymus results in depletion of CD4-bearing thymocytes. This depletion is initially manifested in the immature CD4+/CD8+ thymocyte subset. To determine cellular factors involved in HIV infection in the thymus, we examined the expression of the recently identified viral coreceptor, CXCR4, on fresh human thymocytes and on human cells from SCID-hu (Thy/Liv) mice. CXCR4 is a member of the chemokine receptor family which is required along with CD4 for entry into the cell of syncytium-inducing (SI) HIV-1 strains. Our analyses show that CXCR4 expression is modulated during T-lymphoid differentiation such that immature thymocytes display an increased frequency and higher surface density of the coreceptor than do more mature cells. In addition, using an SI strain of HIV-1 which directs expression of a reporter protein on the surface of infected cells, we have found that the immature CD4+/CD8+ thymocytes that express the highest levels of both CD4 and CXCR4 are the cells that are preferentially infected and depleted by the virus in vitro. Thus, high levels of both primary receptor and coreceptor may allow efficient infection of the thymus by certain HIV-1 strains. This in part may explain the rapid disease progression seen in some HIV-infected children, where the thymus is actively involved in the production of new T lymphocytes.     

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.     

Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein.

The CC chemokine receptors CCR5, CCR2, and CCR3 and the CXC chemokine receptor CXCR4 have been implicated as CD4-associated cofactors in the entry of primary and cell line-adapted human immunodeficiency virus type 1 (HIV-1) strains. CXCR4 is also a receptor for T-cell-line-adapted, CD4-independent strains of HIV-2. With the exception of this latter example, little has been reported on the entry cofactors used by HIV-2 strains. Here we show that a CD4-dependent, T-cell-line-adapted HIV-2 strain uses CXCR4 and, to a lesser extent, CCR3 for fusion with and infectious entry into cells. In a cell-to-cell fusion assay, the envelope protein of this virus can utilize a wider repertoire of chemokine receptors to induce fusion. These include CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2, and CXCR4. Kinetic analysis indicated that cell lines expressing the receptors that support infection, CXCR4 and CCR3, form syncytia more rapidly than do cell lines expressing the other receptors. Nevertheless, although less efficient, fusion with CXCR2 expressing cells was specific, since it was inhibited by antibodies against CXCR2. The extensive use of chemokine receptors in cell-to-cell fusion has implications for understanding the molecular basis of CD4-chemokine receptor-induced lentivirus fusion and may have relevance for syncytium formation and the direct cell-to-cell transfer of virus in vivo.     

HIV coreceptors, cell tropism and inhibition by chemokine receptor ligands.

HIV is a persistent virus that survives and replicates despite an onslaught by the host's immune system. A strategy for cell entry, requiring the use of two receptors, has evolved that may help evade neutralizing antibodies. HIV and SIV usually require both CD4 and a seven transmembrane (7TM) coreceptor for infection. At least eleven different 7TM coreceptors have been identified that confer HIV and/or SIV entry. For HIV-1, the major coreceptors are CCR5 and CXCR4, while the role of other coreceptors for replication and cell tropism in vivo is currently unclear. Polymorphisms in the CCR5 gene that reduce CCR5 expression levels, protect against disease progression, suggesting that drugs targeted to CCR5 could be effective. Such therapies however will not work if HIV simply adapts to use alternative coreceptors. In the light of these themes, this review will discuss the following topics: (i) the coreceptors used by primary HIV-1 and HIV-2 viruses, (ii) the properties and coreceptors of HIV-2 strains that infect cells without CD4, (iii) the role of coreceptors in HIV cell tropism and particularly macrophage infection and (iv) the properties of chemokine receptor ligands that block HIV infection.     

Spontaneous Mutations in the env Gene of the Human Immunodeficiency Virus Type 1 NDK Isolate Are Associated with a CD4-Independent Entry Phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts in trans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4-3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.     

HIV coreceptors,cell tropism and inhibition by chemokine receptor ligands

HIV is a persistent virus that survives and replicates despite an onslaught by the host's immune system. A strategy for cell entry, requiring the use of two receptors, has evolved that may help evade neutralizing antibodies. HIV and SIV usually require both CD4 and a seven transmembrane (7TM) coreceptor for infection. At least eleven different 7TM coreceptors have been identified that confer HIV and/ or SIV entry. For HIV-1, the major coreceptors are CCR5 and CXCR4, while the role of other coreceptors for replication and cell tropism in vivo is currently unclear. Polymorphisms in the CCR5 gene that reduce CCR5 expression levels, protect against disease progression, suggesting that drugs targeted to CCR5 could be effective. Such therapies however will not work if HIV simply adapts to use alternative coreceptors. In the light of these themes, this review will discuss the following topics: (i) the coreceptors used by primary HIV-1 and HIV-2 viruses, (ii) the properties and coreceptors of HIV-2 strains that infect cells without CD4, (iii) the role of coreceptors in HIV cell tropism and particularly macrophage infection and (iv) the properties of chemokine receptor ligands that block HIV infection.     

Genetically Divergent Strains of Human Immunodeficiency Virus Type 2 Use Multiple Coreceptors for Viral Entry

Several members of the seven-transmembrane chemokine receptor family have been shown to serve, with CD4, as coreceptors for entry by human immunodeficiency virus type 1 (HIV-1). While coreceptor usage by HIV-1 primary isolates has been studied by several groups, there is only limited information available concerning coreceptor usage by primary HIV-2 isolates. In this study, we have analyzed coreceptor usage of 15 primary HIV-2 isolates, using lymphocytes from a donor with nonfunctional CCR5 (CCR5 −/−; homozygous 32-bp deletion). Based on the infections of PBMCs, seven of these primary isolates had an absolute requirement for CCR5 expression, whereas the remaining eight exhibited a broader coreceptor usage. All CCR5-requiring isolates were non-syncytium inducing, whereas isolates utilizing multiple coreceptors were syncytium inducing. Blocking experiments using known ligands for chemokine receptors provided indirect evidence for additional coreceptor utilization by primary HIV-2 isolates. Analysis of GHOST4 cell lines expressing various chemokine receptors (CCR1, CCR2b, CCR3, CCR4, CCR5, CXCR4, BONZO, and BOB) further defined specific coreceptor usage of primary HIV-2 isolates. The receptors used included CXCR4, CCR1-5, and the recently described receptors BONZO and BOB. However, the efficiency at which the coreceptors were utilized varied greatly among the various isolates. Analysis of V3 envelope sequences revealed no specific motif that correlated with coreceptor usage. Our data demonstrate that primary HIV-2 isolates are capable of using a broad range of coreceptors for productive infection in vitro. Additionally, our data suggest that expanded coreceptor usage by HIV-2 may correlate with disease progression.     

Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.