Chemokine Receptor Utilization by Human Immunodeficiency Virus Type 1 Isolates That Replicate in Microglia

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.     

Determinants of Entry Cofactor Utilization and Tropism in a Dualtropic Human Immunodeficiency Virus Type 1 Primary Isolate

Human immunodeficiency virus type 1 strain 89.6 is a dualtropic isolate that replicates in macrophages and transformed T cells, and its envelope mediates CD4-dependent fusion and entry with CCR5, CXCR-4, and CCR3. To map determinants of cofactor utilization by 89.6 and determine the relationship between cofactor use and tropism, we analyzed recombinants generated between 89.6 and T-cell-tropic (HXB) or macrophage-tropic (JRFL) strains. These chimeras showed that regions of 89.6 env outside V3 through V5 determine CXCR-4 utilization and T-cell line tropism as well as CCR5 utilization and macrophage tropism. However, the 89.6 env V3 domain also conferred on HXB the ability to use CCR5 for fusion and entry but not the ability to establish productive macrophage infection. CCR3 use was conferred on HXB by 89.6 env V3 or V3 through V5 sequences. While replacement of the 89.6 V3 through V5 region with HXB sequences abrogated CCR3 utilization, replacement of V3 or V4 through V5 separately did not. Thus, CCR3 use is determined by sequences within V3 through V5 and most likely can be conferred by either the V3 or the V4 through V5 domains. These results indicate that cofactor utilization and tropism in this dualtropic isolate are determined by complex interactions among multiple env segments, that distinct regions of the Env glycoprotein may be important for utilization of different chemokine receptors, and that determinants in addition to cofactor usage participate in postentry stages in the virus replication cycle that contribute to target cell tropism.     

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.     

Human Immunodeficiency Virus Type 1 Populations in Blood and Semen

Transmission of human immunodeficiency virus type 1 (HIV-1) usually results in outgrowth of viruses with macrophage-tropic phenotype and consensus non-syncytium-inducing (NSI) V3 loop sequences, despite the presence of virus with broader host range and the syncytium-inducing (SI) phenotype in the blood of many donors. We examined proviruses in contemporaneous peripheral blood mononuclear cells (PBMC) and nonspermatozoal semen mononuclear cells (NSMC) of five HIV-1-infected individuals to determine if this preferential outgrowth could be due to compartmentalization and thus preferential transmission of viruses of the NSI phenotype from the male genital tract. Phylogenetic reconstructions of ~700-bp sequences covering the second constant region through the fifth variable region (C2 to V5) of the viral envelope gene revealed distinct variant populations in the blood versus the semen in two patients with AIDS and in one asymptomatic individual (patient 613), whereas similar variant populations were found in both compartments in two other asymptomatic individuals. Variants with amino acids in the V3 loop that predict the SI phenotype were found in both AIDS patients and in patient 613; however, the distribution of these variants between the two compartments was not consistent. SI variants were found only in the PBMC of one AIDS patient but only in the NSMC of the other, while they were found in both compartments in patient 613. It is therefore unlikely that restriction of SI variants from the male genital tract accounts for the observed NSI transmission bias. Furthermore, no evidence for a semen-specific signature amino acid sequence was detected.     

Virion-Targeted Viral Inactivation of Human Immunodeficiency Virus Type 1 by Using Vpr Fusion Proteins

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4⁺ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4⁺ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6^gag domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.     

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.     

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.     

Zeta Chain of the T-Cell Receptor Interacts with nef of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2

A truncated version of the nef gene of simian immunodeficiency virus SIVmac239 capable of encoding amino acids 98 to 263 was used as bait to screen a cDNA library from activated lymphocytes in a yeast two-hybrid system. The zeta chain of the T-cell receptor (TCR_ζ) was found to interact specifically not only with truncated SIV nef in yeast cells but also with full-length glutathione S-transferase (GST)-SIVnef fusion protein in vitro. Coimmunoprecipitation of TCR_ζ with full-length SIV nef was demonstrated in transfected Jurkat cells and in Cos 18 cells which express the cytoplasmic domain of TCR_ζ fused to the external domain of CD8 via the CD8 transmembrane domain. Using a series of nef deletion mutants, we have mapped the binding site within the central core domain of nef (amino acids 98 to 235). Binding of TCR_ζ was specific for nef isolated from SIVmac239, SIVsmH4, and human immunodeficiency virus (HIV)-2ST and was not detected with nef from five different HIV-1 isolates. An active tyrosine kinase was coprecipitated with nef-TCR_ζ complexes from Jurkat cells but not from J.CAM1.6 cells which lack a functional Lck tyrosine kinase. These results demonstrate a specific association of SIV and HIV-2 nef, but not HIV-1 nef, with TCR_ζ.     

Novel Gag-Pol Frameshift Site in Human Immunodeficiency Virus Type 1 Variants Resistant to Protease Inhibitors

Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease inhibitors have been shown to contain a mutation in the p1/p6 Gag precursor cleavage site. At the messenger RNA level, this mutation generates a U UUU UUU sequence that is reminiscent of the U UUU UUA sequence required for ribosomal frameshifting and Gag-Pol synthesis. To test whether the p1/p6 cleavage site mutation was generating a novel frameshift site, HIV sequences were inserted in translation vectors containing a chloramphenicol acetyltransferase (CAT) reporter gene requiring −1 frameshifting for expression. All sequences containing the original HIV frameshift site supported the synthesis of CAT but expression was increased 3- to 11-fold in the presence of the mutant p1/p6 sequence. When the original frameshift site was abolished by mutation, expression remained unchanged when using constructs containing the mutant p1/p6 sequence, whereas it was decreased 2- to 4.5-fold when using wild-type p1/p6 constructs. Similarly, when introduced into HIV molecular clones, the p1/p6 mutant sequence supported Gag-Pol synthesis and protease activity in the absence of the original frameshift site, indicating that this sequence could also promote ribosomal frameshifting in virus-expressing cells.     

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.     

Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.Human immunodeficiency virus type 1 (HIV-1) and HIV-2 are related human retroviruses that show various biological and structural differences. HIV-2 is found mainly in West Africa, whereas HIV-1 is spreading throughout the world. HIV-2 is less transmissible, and HIV-2-positive patients exhibit longer clinical latency periods than individuals infected with HIV-1 (23). A recent report has also shown that the mortality in HIV-2-infected individuals is only twice as high as in the uninfected population and, in the majority of adults, survival is not affected by HIV-2 status (31).Although the two viruses are similar in genomic organization, various genetic and enzymatic differences have been found at many stages of the retroviral life cycle. They differ significantly in terms of amino acid sequence, the more conserved being the Pol and Gag sequences, which exhibit less than 60% homology (17).Despite these differences, epidemiological data and animal studies have shown some evidence of cross-protection between the two viral infections. Travers et al. reported that HIV-2-infected women had a lower incidence of HIV-1 infection than did HIV-seronegative women in a cohort of commercial sexual workers in Dakar (37), and rhesus macaques immunized with a recombinant HIV-1 poxvirus vaccine are protected against HIV-2 challenge (2). These studies, though not conclusive (1, 6), suggest that differences in the virus may not necessarily preclude the development of defensive immunity to a subsequent pathogenic infection, an old-fashioned concept pioneered by Jenner, who used cowpox to vaccinate against human smallpox.The immunological basis of cross-protection is largely unknown, and a clear understanding of the role played by the humoral or cell-mediated immune response in HIV protection is still lacking. However, mounting evidence suggests that cytotoxic T-lymphocyte (CTL) response could be the key element. Indeed, the protection afforded in animal models against simian (13) and feline (12) immunodeficiency virus infections is closely correlated with the induction of specific CTL response, and HIV-1 and HIV-2 HLA-B35-restricted cross-reactive CTLs have been postulated to confer protection against repeated HIV exposure (33).CTLs recognize short viral peptides, 8 to 11 amino acids long, that are generated by the intracellular processing of endogenously synthesized viral antigens within the infected cells, which are expressed at the cell surface in the binding groove of HLA class I molecules. The specificity of the T-cell response is determined by the interaction of the antigen-specific T-cell receptor (TCR) with the peptide-HLA complex, and this interaction, together with non-antigen-specific signals, activates the CTLs (15).The presence of cross-reactive CTLs able to lyse HIV-1- or HIV-2-infected cells should be dependent on the extent of conservation between the two viruses within the epitopes selected by particular HLA class I molecules. It is well known that amino acid substitutions within the epitopes can abrogate the CTL response by inhibiting either HLA binding or TCR recognition (32). However, a number of recent studies have shown that T cells can recognize apparently unrelated peptides (10, 41), and crystallographic data have shown physical limits to the TCR epitope specificity due to the limited size of contact between the TCR and the peptide (14), suggesting a flexibility in T-cell recognition of antigen (19).Some individuals with a particular HLA profile which is responsible for presentation of the viral antigen and for selection of the T-cell repertoire may possess a CTL response not affected by mutations within the epitope, as has been demonstrated in subjects with HLA alleles B27 (28) and B35 (33). In these cases, amino acid substitutions within the HIV-1 and -2 epitopes were tolerated by the CTLs.In this study, we have investigated the extent of cross-reacting CTLs between HIV-2 and HIV-1 in a group of HIV-2-infected subjects with different HLA class I types. We have shown that despite differences in amino acid sequence between the two viruses, the majority of HIV-2-positive subjects possess CTLs which are able to recognize HIV-1 Gag protein.Furthermore, analysis of HLA profiles and the fine specificity of the cytotoxic response demonstrated that HLA-B5801-positive subjects show broad cross-recognition of HIV-1 isolates. These subjects mounted a CTL response that tolerated extensive amino acid substitutions within an HLA-B5801-restricted HIV-1 epitope.     

Use of G-Protein-Coupled and -Uncoupled CCR5 Receptors by CCR5 Inhibitor-Resistant and -Sensitive Human Immunodeficiency Virus Type 1 Variants

Small-molecule CCR5 inhibitors such as vicriviroc (VVC) and maraviroc (MVC) are allosteric modulators that impair HIV-1 entry by stabilizing a CCR5 conformation that the virus recognizes inefficiently. Viruses resistant to these compounds are able to bind the inhibitor-CCR5 complex while also interacting with the free coreceptor. CCR5 also interacts intracellularly with G proteins, as part of its signal transduction functions, and this process alters its conformation. Here we investigated whether the action of VVC against inhibitor-sensitive and -resistant viruses is affected by whether or not CCR5 is coupled to G proteins such as Gα_i. Treating CD4⁺ T cells with pertussis toxin to uncouple the Gα_i subunit from CCR5 increased the potency of VVC against the sensitive viruses and revealed that VVC-resistant viruses use the inhibitor-bound form of Gα_i-coupled CCR5 more efficiently than they use uncoupled CCR5. Supportive evidence was obtained by expressing a signaling-deficient CCR5 mutant with an impaired ability to bind to G proteins, as well as two constitutively active mutants that activate G proteins in the absence of external stimuli. The implication of these various studies is that the association of intracellular domains of CCR5 with the signaling machinery affects the conformation of the external and transmembrane domains and how they interact with small-molecule inhibitors of HIV-1 entry.     

Modulation of Sp1 Phosphorylation by Human Immunodeficiency Virus Type 1 Tat

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67:6224–6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.     

Target Cell Populations of Human Immunodeficiency Virus Type 1 in Peripheral Blood Lymphocytes with Different Chemokine Receptors at Various Stages of Disease Progression

We studied the distribution of human immunodeficiency virus type 1 (HIV-1) DNA in CCR5-positive and -negative peripheral blood lymphocyte populations in HIV-1-infected individuals. While HIV-1 DNA in the CCR5-positive population showed no correlation with CD4 count, the increase of total HIV-1 DNA with lower CD4 count was mainly contributed by the increase of HIV-1 DNA in the CCR5-negative population. This might indicate the change in coreceptor usage from CCR5 to CXCR4 in later stages of disease progression. However, some of the samples with a high viral DNA load in the CCR5-negative population did not have any characteristic of the V3 loop sequence that is compatible with CXCR4 usage or the syncytium-inducing (SI) phenotype. We also did not find any known characteristic change predictive of the SI phenotype in V1 and V2 sequences. Our findings showed that there might be a shift in target cell populations during disease progression, and this shift was not necessarily associated with the genetic changes characteristic of CXCR4 usage.     

Expression and Use of Human Immunodeficiency Virus Type 1 Coreceptors by Human Alveolar Macrophages

Human immunodeficiency virus type 1 (HIV-1) requires, in addition to CD4, coreceptors of the CC or CXC chemokine families for productive infection of T cells and cells of the monocyte-macrophage lineage. Based on the hypothesis that coreceptor expression on alveolar macrophages (AM) may influence HIV-1 infection of AM in the lung, this study analyzes the expression and utilization of HIV-1 coreceptors on AM of healthy individuals. AM were productively infected with five different primary isolates of HIV-1. Levels of surface expression of CCR5, CXCR4, and CD4 were low compared to those of blood monocytes, but CCR3 was not detectable. mRNA for CCR5, CXCR4, CCR2, and CCR3 were all detectable, but to varying degrees and with variability among donors. Expression of CCR5, CXCR4, and CCR2 mRNA was downregulated following stimulation with lipopolysaccharide (LPS). In contrast, secretion of the chemokines RANTES, MIP-1alpha, and MIP-1beta was upregulated with LPS stimulation. Interestingly, HIV-1 replication was diminished following LPS stimulation. Infection of AM with HIV-1 in the presence of the CC chemokines demonstrated blocking of infection. Together, these studies demonstrate that AM can be infected by a variety of primary HIV-1 isolates, AM express a variety of chemokine receptors, the dominant coreceptor used for HIV entry into AM is CCR5, the expression of these receptors is dependent on the state of activation of AM, and the ability of HIV-1 to infect AM may be modulated by expression of the chemokine receptors and by chemokines per se.     

Enhancement of Human Immunodeficiency Virus Type 1 Infection by the CC-Chemokine RANTES Is Independent of the Mechanism of Virus-Cell Fusion

We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus. Furthermore, RANTES enhances the infectivity of HIV-1 pseudotyped with the envelope glycoprotein of murine leukemia virus or vesicular stomatitis virus, showing that the mechanism of enhancement is independent of the route of virus-cell fusion. The enhancing effects of RANTES are not mediated via CCR5 or other known chemokine receptors and are not mimicked by MIP-1α or MIP-1β. The N-terminally modified derivative aminooxypentane RANTES (AOP-RANTES) efficiently inhibits HIV-1 infection via CCR5 but otherwise mimics RANTES by enhancing viral infectivity. There are two mechanisms of enhancement: one apparent when target cells are pretreated with RANTES (or AOP-RANTES) for several hours, and the other apparent when RANTES (or AOP-RANTES) is added during virus-cell absorption. We believe that the first mechanism is related to cellular activation by RANTES, whereas the second is an increase in virion attachment to target cells.     

Variants from the Diverse Virus Population Identified at Seroconversion of a Clade A Human Immunodeficiency Virus Type 1-Infected Woman Have Distinct Biological Properties

Development of effective therapeutics to prevent new infections with human immunodeficiency type 1 (HIV-1) is predicated on an understanding of the properties that provide a selective advantage to a transmitted viral population. In contrast to the homogeneous virus population that typifies early HIV-1 infection of men, the viral population in women recently infected with clade A HIV-1 is genetically diverse, based on evaluation of the envelope gene. A longitudinal study of viral envelope evolution in several women suggested that representative envelope variants detected at seroconversion had distinct biological properties that affected viral fitness. To test this hypothesis, a full-length, infectious molecular clone, Q23-17, was obtained from an infected woman 1 year following seroconversion, and chimeric viruses containing envelope genes representative of seroconversion and 27-month-postseroconversion populations were constructed. Dendritic cells (DC) could transfer infection of seroconversion variant Q23ScA, which dominated the viral population in the year following seroconversion, and the closely related 1-year isolate Q23-17 to resting peripheral blood mononuclear cells (PBMC). In contrast, resting PBMC exposed to DC pulsed with Q23ScB, which was detected infrequently in samples after seroconversion, or the 27-month chimeras were inconsistently infected. Additionally, quiescent PBMC infected with Q23ScA or Q23-17 proliferated more robustly than uninfected cells or cells infected with the other envelope chimeras in response to immobilized anti-CD3. Stimulation with tetanus toxoid led to an increased proportion of CD45RA+ cells and a decreased expression of CD28 on CD45RO+ cells in cultures of Q23-17-infected PBMC. These data demonstrate that variants from the heterogeneous seroconversion clade A HIV-1 population in a Kenyan woman have distinct biological features that may influence viral pathogenesis.