Development of a novel dual CCR5-dependent and CXCR4-dependent cell-cell fusion assay system with inducible gp160 expression

In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC50 data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSO on this assay system were assessed, and a 2- to 3-fold increase in luciferase activity was observed in the presence of 0.05% to 2% DMSO. Although cell-cell fusion efficiency was enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Highly potent chimeric inhibitors targeting two steps of HIV cell entry

Blocking HIV-1 cell entry has long been a major goal of anti-HIV drug development. Here, we report a successful design of two highly potent chimeric HIV entry inhibitors composed of one CCR5-targeting RANTES (regulated on activation normal T cell expressed and secreted) variant (5P12-RANTES or 5P14-RANTES (Gaertner, H., Cerini, F., Escola, J. M., Kuenzi, G., Melotti, A., Offord, R., Rossitto-Borlat, I., Nedellec, R., Salkowitz, J., Gorochov, G., Mosier, D., and Hartley, O. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 17706-17711)) linked to a gp41 fusion inhibitor, C37. Chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed extremely high antiviral potency in single cycle and replication-competent viral assays against R5-tropic viruses, with IC(50) values as low as 0.004 nm. This inhibition was somewhat strain-dependent and was up to 100-fold better than the RANTES variant alone or in combination with unlinked C37. The chimeric inhibitors also fully retained the antiviral activity of C37 against X4-tropic viruses, and this inhibition can be further enhanced significantly if the target cell co-expresses CCR5 receptor. On human peripheral blood mononuclear cells, the inhibitors showed very strong inhibition against R5-tropic Ba-L strain and X4-tropic IIIB strain, with IC(50) values as low as 0.015 and 0.44 nm, which are 45- and 16-fold better than the parent inhibitors, respectively. A clear delivery mechanism requiring a covalent linkage between the two segments of the chimera was observed and characterized. Furthermore, the two chimeric inhibitors are fully recombinant and are easily produced at low cost. These attributes make them excellent candidates for anti-HIV microbicides. The results of this study also suggest a potent approach for optimizing existing HIV entry inhibitors or designing new inhibitors.     

HIV chemokine receptor inhibitors as novel anti-HIV drugs

The chemokine receptors CXCR4 and CCR5 are the main coreceptors used by the T-cell-tropic (CXCR4-using, X4) and macrophage-tropic (CCR5-using, R5) HIV-1 strains, respectively, for entering their CD4⁺ target cells. In this review, we focus on the function of these chemokine receptors in HIV infection and their role as novel targets for viral inhibition. Besides some modified chemokines with antiviral activity, several low-molecular weight CCR5 and CXCR4 antagonistic compounds have been described with potent antiviral activity. The best CXCR4 antagonists described are the bicyclam derivatives, which consistently block X4 but also R5/X4 viral replication in PBMCs. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both CXCR4 and CCR5 chemokine receptor inhibitors will be needed in combination and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle to obtain optimum antiviral therapeutic effects.     

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.     

The CCR5 and CXCR4 coreceptors are both used by human immunodeficiency virus type 1 primary isolates from subtype C

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses with different coreceptor usage profiles were isolated from 29 South African patients with advanced AIDS. All 24 R5 isolates were inhibited by the CCR5-specific agents, PRO 140 and RANTES, while the two X4 viruses and the three R5X4 viruses were sensitive to the CXCR4-specific inhibitor, AMD3100. The five X4 or R5X4 viruses were all able to replicate in peripheral blood mononuclear cells that did not express CCR5. When tested using coreceptor-transfected cell lines, one R5 virus was also able to use CXCR6, and another R5X4 virus could use CCR3, BOB/GPR15, and CXCR6. The R5X4 and X4 viruses contained more-diverse V3 loop sequences, with a higher overall positive charge, than the R5 viruses. Hence, some HIV-1 subtype C viruses are able to use CCR5, CXCR4, or both CXCR4 and CCR5 for entry, and they are sensitive to specific inhibitors of entry via these coreceptors. These observations are relevant to understanding the rapid spread of HIV-1 subtype C in the developing world and to the design of intervention and treatment strategies.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

Microglia Express CCR5, CXCR4, and CCR3, but of These, CCR5 Is the Principal Coreceptor for Human Immunodeficiency Virus Type 1 Dementia Isolates

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1β and SDF-1α, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1_DS-br, HIV-1_RC-br, and HIV-1_YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.     

Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide

BACKGROUND: The predominant mode of HIV-1 transmission is by heterosexual contact. The cervical/vaginal mucosa is the main port of HIV entry in women. A safe and effective topical microbicide against HIV is urgently needed to prevent sexual transmission. Hence, we evaluated griffithsin (GRFT), a 12.7 kDa carbohydrate-binding protein, both native and recombinant GRFT, potently inhibited both CXCR4-and CCR5-tropic HIV infection and transmission in vitro. METHODS: The antiviral efficacy of native and recombinant GRFT against CXCR4-and CCR5-tropic HIV and SHIV strains and SIVmac251 was evaluated by in vitro assays. We also evaluated the time course of antiviral activity and stability of GRFT in cervical/vaginal lavage as a function of pH 4-8. RESULTS: Griffithsin blocked CXCR4-and CCR5-tropic viruses at less than 1 nm concentrations and exhibited a high potency. GRFT was stable in cervical/vaginal lavage fluid and maintained a similar potency of anti-HIV activity. GRFT is not only a highly potent HIV entry inhibitor, but also prevents cell fusion and cell-to-cell transmission of HIV. CONCLUSIONS: The in vitro efficacy of GRFT revealed low cytotoxicity, high potency, rapid onset of antiviral activity and long-term stability in cervical/vaginal lavage. GRFT is an excellent candidate for anti-HIV microbicide development.     

Transmembrane Protein Aptamers That Inhibit CCR5 Expression and HIV Coreceptor Function

We have exploited the ability of transmembrane domains to engage in highly specific protein-protein interactions to construct a new class of small proteins that inhibit HIV infection. By screening a library encoding hundreds of thousands of artificial transmembrane proteins with randomized transmembrane domains (termed "traptamers," for transmembrane aptamers), we isolated six 44- or 45-amino-acid proteins with completely different transmembrane sequences that inhibited cell surface and total expression of the HIV coreceptor CCR5. The traptamers inhibited transduction of human T cells by HIV reporter viruses pseudotyped with R5-tropic gp120 envelope proteins but had minimal effects on reporter viruses with X4-tropic gp120. Optimization of two traptamers significantly increased their activity and resulted in greater than 95% inhibition of R5-tropic reporter virus transduction without inhibiting expression of CD4, the primary HIV receptor, or CXCR4, another HIV coreceptor. In addition, traptamers inhibited transduction mediated by a mutant R5-tropic gp120 protein resistant to maraviroc, a small-molecule CCR5 inhibitor, and they dramatically inhibited replication of an R5-tropic laboratory strain of HIV in a multicycle infection assay. Genetic experiments suggested that the active traptamers specifically interacted with the transmembrane domains of CCR5 and that some of the traptamers interacted with different portions of CCR5. Thus, we have constructed multiple proteins not found in nature that interfere with CCR5 expression and inhibit HIV infection. These proteins may be valuable tools to probe the organization of the transmembrane domains of CCR5 and their relationship to its biological activities, and they may serve as starting points to develop new strategies to inhibit HIV infection.     

R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.     

HIV and the chemokine system: 10 years later

The unexpected encounter, 10 years ago, between human immunodeficiency virus (HIV) and the chemokine system has dramatically advanced our understanding of the pathogenesis of AIDS, opening new perspectives for the development of effective prophylactic and therapeutic measures. To initiate infection, the HIV-1 external envelope glycoprotein, gp120, sequentially interacts with two cellular receptors, CD4 and a chemokine receptor (or coreceptor) like CCR5 or CXCR4. This peculiar two-stage receptor-interaction strategy allows gp120 to maintain the highly conserved coreceptor-binding site in a cryptic conformation, protected from neutralizing antibodies. The differential use of CCR5 and CXCR4 defines three HIV-1 biological variants (R5, R5X4, X4), which vary in their prevalence during the disease course. The evolutionary choice of HIV-1 to exploit chemokine receptors as cellular entry gateways has turned their chemokine ligands into endogenous antiviral factors that variably modulate viral transmission, disease progression and vaccine responses. Likewise, the natural history of HIV-1 infection is influenced by specific polymorphisms of chemokine and chemokine-receptor genes. The imminent clinical availability of coreceptor-targeted viral entry inhibitors raises new hope for bridging the gap towards a definitive cure of HIV infection.     

Multiple antiviral activities of cyanovirin-N: blocking of human immunodeficiency virus type 1 gp120 interaction with CD4 and coreceptor and inhibition of diverse enveloped viruses

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.     

In vivo evolution of X4 human immunodeficiency virus type 1 variants in the natural course of infection coincides with decreasing sensitivity to CXCR4 antagonists

CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) variants evolve from CCR5-restricted (R5) HIV-1 variants. Early after their first appearance in vivo, X4 HIV-1 variants additionally use CCR5. The ability to use CCR5 in addition to CXCR4 is generally lost late in infection. Here we studied whether this evolution of the coreceptor repertoire is also reflected in a changing sensitivity of X4 variants to CXCR4 antagonists such as peptide T22 and the synthetic compound AMD3100. We observed differences in the concentrations of CXCR4 antagonists needed to suppress replication of X4 HIV variants from different patients. In general, late X4 HIV variants were less sensitive to AMD3100 than were early R5X4 HIV variants. The differences between early R5X4 HIV variants and late X4 variants were less pronounced for T22-mediated inhibition. These results suggest an ongoing evolution of X4 virus variants toward more efficient usage of the cellular entry complex.     

V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.     

Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC(50), 1.8 to 7.3 microM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.     

The possible involvement of CXCR4 in the inhibition of HIV-1 infection mediated by DP178/gp41

The N- (N36/DP107) and C-terminal peptides (C34/DP178) from two alpha-helical domains of human immunodeficiency virus type 1 (HIV-1) gp41 inhibited HIV infection. A single-round infection using pseudotyped virus clarified that a greater amount of gp41-derived peptides was necessary for the inhibition of R5 virus (ADA) infection than for that of X4 virus (LAI) infection. Furthermore, R5X4 virus (89.6) infection via CCR5 needs more peptides for inhibition than its infection via CXCR4 does. A high sensitivity of X4 virus was partially ascribed to the inhibition of the 12G5 binding to CXCR4 by DP178LAI.