Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3/SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17/gp160-CVT. Phenotypic analysis of the NL4.3/SPL2923 p17- and p17/gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3/SPL2923 towards SPL2923.     

The CXCR4 antagonist AMD3100 efficiently inhibits cell-surface-expressed human immunodeficiency virus type 1 envelope-induced apoptosis

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death by apoptosis in infected and uninfected cells. The envelope glycoprotein complex ([gp120/gp41](n)) of X4 HIV-1 isolates is involved in both infected and uninfected cell death via its interaction with cellular receptors CD4 and CXCR4. We studied the effect of the blockade of CXCR4 receptors by the agonist stromal derived factor (SDF-1alpha) and the antagonist bicyclam AMD3100 on apoptotic cell death of CD4(+) cells in different models of HIV infection. In HIV-infected CEM or SUP-T1 cultures, AMD3100 showed antiapoptotic activity even when added 24 h after infection. In contrast, other antiviral agents, such as zidovudine, failed to block apoptosis under these conditions. The antiapoptotic activity of AMD3100 was also studied in coculture of peripheral blood mononuclear cells or CD4(+) cell lines with chronically infected H9/IIIB cells. AMD3100 was found to inhibit both syncytium formation and apoptosis induction with 50% inhibitory concentrations ranging from 0.009 to 0.24 microg/ml, depending on the cell type. When compared to SDF-1alpha, AMD3100 showed higher inhibitory potency in all cell lines tested. Our data indicate that the bicyclam AMD3100 not only inhibits HIV replication but also efficiently blocks cell-surface-expressed HIV-1 envelope-induced apoptosis in uninfected cells.     

Role of human immunodeficiency virus (HIV) type 1 envelope in the anti-HIV activity of the betulinic acid derivative IC9564

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.     

Reduced fitness of HIV-1 resistant to CXCR4 antagonists

HIV-1 strains with a syncytium-inducing phenotype that use CXCR4 (X4 strains) have been associated with faster disease progression and AIDS. Antiviral agents designed to block CXCR4 may prevent the emergence of X4 HIV strains but resistant strains that maintain the X4 phenotype can be raised by sequential passage in cell cultures. We have demonstrated that a laboratory adapted strain (NL4-3) and a cloned clinical isolate (CI-1) of HIV-1 cultured in the presence of the CXCR4 antagonist, AMD3100, became resistant to the compound without a change in co-receptor use. These strains became resistant through divergence with respect to the wild-type virus. Conversely, a clinical isolate made resistant to AMD3100 switched co-receptor use from X4 to R5 through a change in diversity from the original virus population. When dual infection competition/heteroduplex tracking assays were performed, all AMD3100-resistant strains, regardless of co-receptor use showed a significantly diminished fitness compared with the wild-type virus. Single virus infections, at a similar multiplicity of infection, also indicated that the wild-type strains possess better replicative ability than their corresponding resistant strains. Thus, viral resistance development to a CXCR4 antagonist such as AMD3100 is associated with reduced viral fitness.     

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2- terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428- 440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)- synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.     

False-positive human immunodeficiency virus type 1 western blot tests in noninfected blood donors

Background: The manufacturers' criteria for a positive human immunodeficiency virus type 1 (HIV-1) Western blot (WB) test were recently revised to require reactivity to only two of the following bands: p24, gp41, and gp120/160. In a recent report, low-risk blood donors were identified in whom nonspecific reactivity to multiple env antigens in WB testing resulted in apparently false-positive WBs by these criteria. The present study was conducted to verify the existence of false-positive WBs among noninfected donors and to assess the extent of this problem. Study Design and Methods: Four donors classified as WB- positive on the basis of env-only (3 cases) or p24/env-only (1 case) patterns were investigated. Index and/or follow-up specimens were tested by polymerase chain reaction (PCR), by overlapping recombinant env antigens and synthetic peptides in enzyme immunoassays, and by deglycosylated and denatured antigen WBs. WB records from American Red Cross blood centers were reviewed to determine the frequency of env- only and p24/env-only patterns, relative to all positive WBs, from 1988 through 1993. Results: The four index-case donors denied risk and had stable WB reactivity during follow-up. HIV PCR was negative in all. Env reactivity was restricted to nonglycosylated gp41 epitopes; no gp120- specific reactivity was detected. For three of the four donors, env reactivity was mapped to a 20-amino acid N-terminal epitope of gp41. The rate of detecting WBs with these false-positive patterns increased from 0.6 percent of all positive WBs from 1988 to 1990 (4/776) to 8 percent in 1991 and 1992 (52/683), and then it declined to 6 percent in 1992 and 1993 (47/783). Env-only patterns predominated in 1991 and 1992, whereas p24/env-only patterns were more frequent following implementation of combined anti-HIV-1/HIV type 2 enzyme immunoassays in 1992. Conclusion: Low-risk blood donors can have false-positive results on WB tests. Increased detection of env-only and p24/env-only WBs appears related to the enhanced sensitivity of newer enzyme immunoassays to gp41 and p24 antibodies. Donors with these patterns should undergo follow-up testing to document the presence or absence of HIV infection.     

Coreceptors of HIV infection and the development of HIV entry inhibitors: overview and targets]

In 1996 CXCR4 was identified as a coreceptor for HIV-1. This finding has lead to further identification of more than ten G-protein-coupled receptors (GPCRs) as coreceptors for HIV/SIV. Cell tropisms and coreceptor uses of HIV during the course of HIV infection are summarized. Promiscuous properties of correlations between chemokines and their chemokine receptor uses and also between variable amino acid sequences in the V3 region of HIV gp120 Env and HIV coreceptor uses are discussed. This promiscuous property of HIV-1 is claimed to be a possible cause of a difficulty in developing highly effective entry inhibitors and in addition to allow rapid appearance of immune escape HIV mutants. Representative agents that inhibit HIV entry with a special reference to inhibitors of coreceptor use and gp41 function are summarized. gp41 is discussed as a promising target for the development of effective entry inhibitors.     

Neutralizing antibodies to human immunodeficiency virus type-1 gp120 induce envelope glycoprotein subunit dissociation

The spectrum of the anti-human immunodeficiency virus (HIV) neutralizing immune response has been analyzed by the production and characterization of monoclonal antibodies (mAbs) to the viral envelope glycoproteins, gp41 and gp120. Little is known, however, about the neutralization mechanism of these antibodies. Here we show that the binding of a group of neutralizing mAbs that react with regions of the gp120 molecule associated with and including the V2 and V3 loops, the C4 domain and supporting structures, induce the dissociation of gp120 from gp41 on cells infected with the T cell line-adapted HIV-1 molecular clone Hx10. Similar to soluble receptor-induced dissociation of gp120 from gp41, the antibody-induced dissociation is dose- and time- dependent. By contrast, mAbs binding to discontinuous epitopes overlapping the CD4 binding site do not induce gp120 dissociation, implying that mAb induced conformational changes in gp120 are epitope specific, and that HIV neutralization probably involves several mechanisms.     

Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding

The human immunodeficiency virus (HIV) binds to the surface of T lymphocytes and other cells of the immune system via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. By analogy with certain other enveloped viruses, receptor binding by HIV may be followed by exposure of the hydrophobic NH2 terminus of its transmembrane glycoprotein, gp41, and fusion of the virus and cell membranes. A similar sequence of events is thought to take place between HIV-infected and uninfected CD4+ cells, resulting in their fusion to form syncytia. In this study, we have used a soluble, recombinant form of CD4 (sCD4) to model events taking place after receptor binding by the HIV envelope glycoproteins. We demonstrate that the complexing of sCD4 with gp120 induces conformational changes within envelope glycoprotein oligomers. This was measured on HIV-1-infected cells by the increased binding of antibodies to the gp120/V3 loops, and on the surface of virions by increased cleavage of this loop by an exogenous proteinase. At 37 degrees C, these conformational changes are coordinate with the dissociation of gp120/sCD4 complexes from gp41, and the increased exposure of gp41 epitopes. At 4 degrees C, gp120 dissociation from the cell surface does not occur, but increased exposure of both gp120/V3 and gp41 epitopes is detected. We propose that these events occurring after CD4 binding are integral components of the membrane fusion reaction between HIV or HIV-infected cells and CD4+ cells.     

Efficient entry inhibition of human and nonhuman primate immunodeficiency virus by cell surface-expressed gp41-derived peptides

Membrane-anchored C-peptides (for example, maC46) derived from human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 effectively inhibit HIV-1 entry in cell lines and primary human CD4+ cells in vitro. Here we evaluated this gene therapy approach in animal models of AIDS. We adapted the HIV gp41-derived maC46 vector construct for use in rhesus monkeys. Simian immunodeficiency virus (SIV and SHIV) sequence-adapted maC46 peptides, and the original HIV-1-derived maC46 expressed on the surface of established cell lines blocked entry of HIV-1, SIVmac251 and SHIV89.6P. Furthermore, primary rhesus monkey CD4+ T cells expressing HIV sequence-based maC46 peptides were also protected from SIV entry. Depletion of CD8+ T cells from PBMCs enhanced the yield of maC46-transduced CD4+ T cells. Supplementation with interleukin-2 (IL-2) increased transduction efficiency, whereas IL-7 and/or IL-15 provided no additional benefit. Phenotypic analysis showed that maC46-transduced and expanded cells were predominantly central memory CD4+ T cells that expressed low levels of CCR5 and slightly elevated levels of CD62L, beta7-integrin and CXCR4. These findings show that maC46-based cell surface-expressed peptides can efficiently inhibit primate immunodeficiency virus infection, and therefore serve as the basis for evaluation of this gene therapy approach in an animal model for AIDS.     

Highly potent and selective inhibition of human immunodeficiency virus by the bicyclam derivative JM3100.

Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.     

Sequential immunizations with rgp120s from independent isolates of human immunodeficiency virus type 1 induce the preferential expansion of broadly crossreactive B cells

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is a dominant target against which the host's humoral immune response is directed. Unfortunately, gp120 proteins from different isolates of HIV are antigenically distinct, complicating the use of the envelope glycoprotein in vaccines designed to prevent acquired immunodeficiency syndrome. Using an enzyme-linked immunosorbent spot assay (ELISA), BALB/c mice immunized and boosted with recombinant purified gp120 were studied at the single cell level for their humoral immune response to HIV-1 envelope proteins. Approximately 90% of responding B cells produced antibodies reactive with the immunizing form of gp120 but not with gp120s from other strains of HIV. A novel sandwich ELISA was then used to analyze the frequency with which individual in vivo activated B cells produced antibodies that crossreacted with heterologous gp120s. Repeated immunizations with a single gp120 or with a mixture of different gp120s resulted in the activation of primarily mono-specific (noncrossreactive) B cells. In contrast, the sequential immunization of mice with recombinant purified envelope proteins from different strains of HIV (IIIB, SF2, and Zr6) induced the selective expansion of B cells producing highly crossreactive antibodies.     

Conferral of an antiviral state to CD4+ cells by a zipper motif envelope mutant of the human immunodeficiency virus type 1 transmembrane protein gp41.

We showed in a transient coexpression study that a single proline substitution for any of the five conserved leucine or isoleucine residues located in the envelope (Env) transmembrane protein gp41 zipper motif of the human immunodeficiency virus type 1 dominantly interferes with wild-type Env-mediated viral infectivity. In the present study, we intended to explore the feasibility of developing a genetic anti-HIV strategy targeting the zipper motif. Stable HeLa-CD4-LTR-beta-gal clones that harbored silent copies of Tat-regulated expression cassettes encoding the zipper motif Env mutants were first generated. Expression of any of the five Env mutants in transfectants interfered with exogenously expressed homologous HXB2 Env-mediated cytopathic effects. Mutant transfectants 566, 573, and 580 were further examined. Viral transmission mediated by the laboratory-adapted T cell-tropic HXB2 and NL4-3 viruses was greatly reduced in these transfectants compared with that observed in the env-defective control deltaKS and wt env transfectants. Moreover, viral replication mediated by the NL4-3 virus and a macrophage-tropic ADA-GG virus was delayed or reduced in human T cells harboring the mutant 566 or 580 env construct as opposed to those observed in cells harboring the control deltaKS or mutant 573 env construct. The wt and mutant Env proteins formed a hetero-oligomer when they were coexpressed. These results demonstrate that zipper motif Env mutants 566 and 580 confer an anti-HIV state to the host CD4+ cells, which indicates that dominant inhibitory mutants targeting the gp41 zipper motif might function as genetic anti-HIV agents to combat HIV-1 infection.     

HIV-1 CRF01-AE、CRF07/08-BC和B′构造病毒株体外适应性比较

目的构建NL4-3env-EGFP/DsRed2前病毒,观察中国主要流行的HIV-1 CRF01-AE?CRF07/08-BC和B′亚型构造病毒株体外适应性。方法 5份HIV-1B′,CRF07/08-BC,CRF01-AE亚型感染的干血斑样本和1份Zambia C亚型PBMC DNA样本,经单基因组扩增技术获得env全长基因,克隆、亚克隆、转染等步骤构建14个NL4-3env-EGFP/DsRed2前病毒,按顺序进行配对竞争实验,流式细胞仪定量检测被感染细胞的数量。结果 CRF01-AE亚型前病毒适应性最强,适应性值W1,其次为B′,Zambia C,CRF07-BC/CRF08-BC,P0.05。结论可利用NL4-3env-EGFP/DsRed2构造前病毒,通过竞争实验比较流行病毒株的体外适应性,推测env基因对适应性的影响。     

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.     

HIV-1 proprotein processing as a target for gene therapy

The central role of endoconvertases and HIV-1 protease (HIV-1 PR) in the processing of HIV proproteins makes the design of specific inhibitors important in anti-HIV gene therapy. Accordingly, we tested native alpha(1) antitrypsin (alpha(1)AT) delivered by a recombinant simian virus-40-based vector, SV(AT), as an inhibitor of HIV-1 proprotein maturation. Cell lines and primary human lymphocytes were transduced with SV(AT) without selection and detectable toxicity. Expression of alpha(1)AT was confirmed by Northern blotting, immunoprecipitation and immunostaining. SV(AT)-transduced cells showed no evidence of HIV-1-related cytopathic effects when challenged with high doses of HIV-1(NL4-3). As measured by HIV-1 p24 assay, SV(AT)-transduced cells were protected from HIV-1(NL4-3) at challenge dose of 40 000 TCID(50) (MOI = 0.04). In addition, peripheral blood lymphocytes treated with SV(AT) were protected from HIV doses challenge up to 40 000 TCID(50) (MOI = 0.04). By Western blot analyses, the delivered alpha(1)AT inhibited cellular processing of gp160 to gp120 and decreased HIV-1 virion gp120. SV(AT) inhibited processing of p55(Gag) as well. Furthermore, high levels of uncleaved p55(Gag) protein were detected in HIV virus particles recovered from SV(AT)-transduced cells lines and primary lymphocytes. Thus, delivering alpha(1)AT using SV(AT) to human lymphocytes strongly inhibits replication of HIV-1, most likely by inhibiting the activities both of the cellular serine proteases involved in processing gp160 and of the aspartyl protease, HIV-1 PR, which cleaves p55(Gag). alpha(1)AT delivered by SV(AT) may represent a novel and effective strategy for gene therapy to interfere with HIV replication, by blocking a stage in the virus replicative cycle that has until now been inaccessible to gene therapeutic intervention.     

Inhibition of coreceptor-independent cell-to-cell human immunodeficiency virus type 1 transmission by a CD4-immunoglobulin G2 fusion protein

We have previously shown (J. Blanco et al., J. Biol. Chem. 279:51305-51314, 2004) that the contact between HIV producing cells and primary CD4(+) T cells may induce the uptake of human immunodeficiency virus (HIV) particles by target cells in the absence of HIV envelope-mediated membrane fusion or productive HIV replication. HIV uptake by CD4(+) T cells was dependent on cellular contacts mediated by the binding of gp120 to CD4 but was independent of the expression of the appropriate HIV coreceptor, CCR5 or CXCR4. Here, we have characterized the effect of agents blocking gp120 binding to CD4 on cell-to-cell HIV transmission. A recombinant CD4-based protein (CD4-immunoglobulin G2 [IgG2]), that is currently being evaluated in clinical trials, completely inhibited the uptake of HIV particles by CD4(+) T cells from persistently infected cells expressing R5, X4, or X4/T-20-resistant HIV-1 envelope glycoproteins. Consequently, both the release of viral particles from endocytic vesicles and the infection of reporter U87-CD4 cells were also prevented. The polyanionic anti-HIV agent dextran sulfate failed to prevent the intracellular uptake of virions by CD4(+) T cells. Indeed, it increased HIV uptake in a dose-dependent manner, suggesting functional differences between the specific gp120-targeting CD4-IgG2 agent and nonspecific HIV binding inhibitors. Thus, the inhibition of the specific interaction between gp120 and CD4 protein could be an effective strategy to inhibit HIV binding to CD4(+) T cells, and the mechanism by which CD4(+) T cells lacking the appropriate coreceptor may be converted in HIV carriers.