Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.     

Cenicriviroc,a Novel CCR5 (R5) and CCR2 Antagonist,Shows In Vitro Activity against R5 Tropic HIV-2 Clinical Isolates

Background

Maraviroc activity against HIV-2, a virus naturally resistant to different HIV-1 antiretroviral drugs, has been recently demonstrated. The aim of this study was to assess HIV-2 susceptibility to cenicriviroc, a novel, once-daily, dual CCR5 and CCR2 antagonist that has completed Phase 2b development in HIV-1 infection.

Methods

Cenicriviroc phenotypic activity has been tested using a PBMC phenotypic susceptibility assay against four R5-, one X4- and one dual-tropic HIV-2 clinical primary isolates. All isolates were obtained by co-cultivation of PHA-activated PBMC from distinct HIV-2-infected CCR5-antagonist-naïve patients included in the French HIV-2 cohort and were previously tested for maraviroc susceptibility using the same protocol. HIV-2 tropism was determined by phenotypic assay using Ghost(3) cell lines.

Results

Regarding the 4 R5 HIV-2 clinical isolates tested, effective concentration 50% EC₅₀ for cenicriviroc were 0.03, 0.33, 0.45 and 0.98 nM, similar to those observed with maraviroc: 1.13, 0.58, 0.48 and 0.68 nM, respectively. Maximum percentages of inhibition (MPI) of cenicriviroc were 94, 94, 93 and 98%, similar to those observed with maraviroc (93, 90, 82, 100%, respectively). The dual- and X4-tropic HIV-2 strains were resistant to cenicriviroc with EC50 >1000 nM and MPI at 33% and 4%, respectively.

Conclusions

In this first study assessing HIV-2 susceptibility to cenicriviroc, we observed an in vitro activity against HIV-2 R5-tropic strains similar to that observed with maraviroc. Thus, cenicriviroc may offer a once-daily treatment opportunity in the limited therapeutic arsenal for HIV-2. Clinical studies are warranted.     

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

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

细胞内表达CCR5Delta32蛋白对HIV-1感染抑制作用的研究

人CCR5Delta32突变个体能有效抵制HIV-1感染,主要是由于该个体淋巴细胞内表达的CCR5Delta32突变蛋白能通过反式显性失活效应(TDN)抑制细胞表面HIV-1辅受体CCR5和CXCR4的产生．通过构建CCR5Delta32慢病毒载体,体外转染人外周血单个核细胞(PBMCs),研究细胞内表达CCR5Delta32蛋白对HIV-1感染的抑制作用．结果表明,表达CCR5Delta32蛋白的人PBMCs对HIV-1 R5、X4及R5X4毒株感染均具有显著的抑制作用．这些工作为后续的AIDS基因治疗研究奠定了基础．     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Patterns of CCR5, CXCR4, and CCR3 Usage by Envelope Glycoproteins from Human Immunodeficiency Virus Type 1 Primary Isolates

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.     

Primary intestinal epithelial cells selectively transfer R5 HIV-1 to CCR5+ cells

The upper gastrointestinal tract is a principal route of HIV-1 entry in vertical transmission and after oral-genital contact. The phenotype of the newly acquired virus is predominantly R5 (CCR5-tropic) and not X4 (CXCR4-tropic), although both R5 and X4 viruses are frequently inoculated onto the mucosa. Here we show that primary intestinal (jejunal) epithelial cells express galactosylceramide, an alternative primary receptor for HIV-1, and CCR5 but not CXCR4. Moreover, we show that intestinal epithelial cells transfer R5, but not X4, viruses to CCR5+ indicator cells, which can efficiently replicate and amplify virus expression. Transfer was remarkably efficient and was not inhibited by the fusion blocker T-20, but was substantially reduced by colchicine and low (4 degrees C) temperature, suggesting endocytotic uptake and microtubule-dependent transcytosis of HIV-1. Our finding that CCR5+ intestinal epithelial cells select and transfer exclusively R5 viruses indicates a mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Allosteric regulation of CCR5 by guanine nucleotides and HIV-1 envelope

L-Selectin-mediated rolling of leukocytes on endothelial cells is an important step for lymphocyte homing and an early event in the immune response to pathogens or inflammatory stimuli. We have previously elucidated intracellular signaling cascades upon L-selectin engagement resulting in activation of Ras, Rac and JNK as well as cytoskeletal changes, oxygen release, ceramide synthesis and receptor capping. Activation of the src-tyrosine kinase p56lck is followed by phosphorylation of the L-selectin molecule and MAP-K. Here we show a tyrosine kinase dependent phosphorylation of the Cbl adapter protein after L-selectin engagement in lymphocytes. Phosphorylation of Cbl was absent in Jurkat cells that are pharmacologically treated with tyrosine kinase inhibitors and in lck-deficient JCaM cells. There is an activation induced association of tyrosine phosphorylated Cbl with Grb2 and CrkL, respectively, but not CrkII. Therefore, the adapter protein Cbl plays a role in L-selectin signaling and might modulate immune function by the specific recruitment of signaling molecules to multiprotein complexes.     

Inhibition of early steps of HIV-1 replication by SNF5/Ini1

To replicate, human immunodeficiency virus, type 1 (HIV-1) needs to integrate a cDNA copy of its RNA genome into a chromosome of the host cell, a step controlled by the viral integrase (IN) protein. Viral integration involves the participation of several cellular proteins. SNF5/Ini1, a subunit of the SWI/SNF chromatin remodeling complex, was the first cofactor identified to interact with IN. We report here that SNF5/Ini1 interferes with early steps of HIV-1 replication. Inhibition of SNF5/Ini1 expression by RNA interference increases HIV-1 replication. Using quantitative PCR, we show that both the 2-long terminal repeat circle and integrated DNA forms accumulate upon SNF5/Ini1 knock down. By yeast two-hybrid assay, we screened a library of HIV-1 IN random mutants obtained by PCR random mutagenesis using SNF5/Ini1 as prey. Two different mutants of interaction, IN E69G and IN K71R, were impaired for SNF5/Ini1 interaction. The E69G substitution completely abolished integrase catalytic activity, leading to a replication-defective virus. On the contrary, IN K71R retained in vitro integrase activity. K71R substitution stimulates viral replication and results in higher infectious titers. Taken together, these results suggest that, by interacting with IN, SNF5/Ini1 interferes with early steps of HIV-1 infection.     

Heterozygous defect in HIV-1 coreceptor CCR5 and chemokine production

CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus 1 (HIV-1). An inactive CCR5 allele with a 32-nucleotide deletion (CCR5Delta32) has been described that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. We found the allele CCR5Delta32 to be not rare in 399 Swiss blood donors with a frequency of 0.080. To assess the influence of defective CCR5 on production of its ligands we determined the capacity to produce the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES in comparison with the production of the CXC chemokine IL-8 which does not bind to CCR5. Production of chemokines was determined during endotoxin stimulation of whole-blood samples ex vivo. Both, basal and LPS-induced chemokine production in 32 blood donors heterozygous for CCR5Delta32 were not significantly different when compared with 55 blood donors who were homozygous for the wild type CCR5 allele.     

Transmitted/Founder and Chronic HIV-1 Envelope Proteins Are Distinguished by Differential Utilization of CCR5

Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4⁺ T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.     

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

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