Macrophages are thought to represent one of the first cell types in the body to be infected during the early stage of human immunodeficiency virus type 1 (HIV-1) transmission and represent a potential viral reservoir in vivo. Thus, an understanding of HIV-1 attachment to these cells is fundamental to the development of novel anti-HIV-1 therapies. Although one of the major targets of HIV-1 in vivo-CD4 ؉ T lymphocytes-express high CD4 levels, other major targets such as macrophages do not. We asked in this study whether this low CD4 level on macrophages is sufficient to support HIV-1 attachment to these cells or whether cell surface proteins other than CD4 are required for this process. We show that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to macrophages. Importantly, we find that heparan sulfate proteoglycans (HSPGs) serve as the main class of attachment receptors for HIV-1 on macrophages. Most importantly, we demonstrate that a single family of HSPGs, the syndecans, efficiently mediates HIV-1 attachment and represents an abundant class of attachment receptors on macrophages.

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies. Hepatitis C virus (HCV)2 is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFN␣ and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFN␣ is more effective than IFN␣ alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFN␣-independent manner (6 -10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and...

This study demonstrates that syndecan functions as an in trans HIV receptor. We show that syndecan, when expressed in nonpermissive cells, becomes the major mediator for HIV adsorption. This adsorption is mediated by the binding of gp120 to the heparan sulfate chains of syndecan. Although syndecan does not substitute for HIV entry receptors, it enhances the in trans infectivity of a broad range of primate lentiviruses including primary viruses produced from PBMCs. Furthermore, syndecan preserves virus infectivity for a week, whereas unbound virus loses its infectivity in less than a day. Moreover, we obtain evidence suggesting that the vast syndecan-rich endothelial lining of the vasculature can provide a microenvironment which boosts HIV replication in T cells.

Although the transport of human immunodeficiency virus type 1 (HIV-1) through the epithelium is critical for HIV-1 colonization, the mechanisms controlling this process remain obscure. In the present study, we investigated the transcellular migration of HIV-1 as a cell-free virus through primary genital epithelial cells (PGECs). The absence of CD4 on PGECs implicates an unusual entry pathway for HIV-1. We found that syndecans are abundantly expressed on PGECs and promote the initial attachment and subsequent entry of HIV-1 through PGECs. Although CXCR4 and CCR5 do not contribute to HIV-1 attachment, they enhance viral entry and transcytosis through PGECs. Importantly, HIV-1 exploits both syndecans and chemokine receptors to ensure successful cell-free transport through the genital epithelium. HIV-1-syndecan interactions rely on specific residues in the V3 of gp120 and specific sulfations within syndecans. We found no obvious correlation between coreceptor usage and the capacity of the virus to transcytose. Since viruses isolated after sexual transmission are mainly R5 viruses, this suggests that the properties conferring virus replication after transmission are distinct from those conferring cell-free virus transcytosis through the genital epithelium. Although we found that cell-free HIV-1 crosses PGECs as infectious particles, the efficiency of transcytosis is extremely poor (less than 0.02% of the initial inoculum). This demonstrates that the genital epithelium serves as a major barrier against HIV-1. Although one cannot exclude the possibility that limited passage of cell-free HIV-1 transcytosis through an intact genital epithelium occurs in vivo, it is likely that the establishment of infection via cell-free HIV-1 transmigration is a rare event.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log 10 after a 14-day oral treatment with 1200 mg twice daily (P < 0.0001) with an effect against the 3 genotypes (1, 3, and 4) represented in the study. In addition, the absence of viral rebound during treatment indicates that Debio-025 has a high barrier for the selection of resistance. In Debio-025-treated patients, cyclophilin B (CypB) levels in peripheral blood mononuclear cells decreased from 67 ؎ 6 (standard error) ng/mg protein (baseline) to 5 ؎ 1 ng/mg protein at day 15 (P < 0.01). Conclusion: Debio-025 induced a strong drop in CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs. (HEPATOLOGY 2008; 47:817-826.)

Background & Aims-The cyclophilin (Cyp) inhihibitors -cyclosporine A (CsA), NIM811, Debio 025 and SCY 635 -block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A.

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