Hepatitis C virus (HCV) is a global challenge to public health. Several factors have been proven to be critical for HCV entry, including the newly identified claudin-1 (CLDN1). However, the mechanism of HCV entry is still obscure. Presently, among the 20 members of the claudin family identified in humans so far, CLDN1 has been the only member shown to be necessary for HCV entry. Recently, we discovered that Bel7402, an HCV-permissive cell line, does not express CLDN1 but expresses other members of claudin family. Among these claudins, CLDN9 was able to mediate HCV entry just as efficiently as CLDN1. We then examined if other members of the claudin family could mediate entry. We show that CLDN6 and CLDN9, but not CLDN2, CLDN3, CLDN4, CLDN7, CLDN11, CLDN12, CLDN15, CLDN17, and CLDN23, were able to mediate the entry of HCV into target cells. We found that CLDN6 and CLDN9 are expressed in the liver, the primary site of HCV replication. We also showed that CLDN6 and CLDN9, but not CLDN1, are expressed in peripheral blood mononuclear cells, an additional site of HCV replication. Through sequence comparison and mutagenesis studies, we show that residues N38 and V45 in the first extracellular loop (EL1) of CLDN9 are necessary for HCV entry.Hepatitis C virus (HCV) is the major cause of liver cirrhosis and hepatocellular carcinoma worldwide. Approximately 3% of the global population is infected with HCV, and at least 70% develop chronic hepatitis (13,17,32). In patients with chronic HCV infection, about 20% develop liver cirrhosis, about 5% of which go on to develop hepatocellular carcinoma (17). While HCV is generally confined to the liver, there is growing evidence suggesting that HCV can replicate in extrahepatic tissues including peripheral blood mononuclear cells (PBMCs) (4,15,23,24

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

The programmed death-1 (PD-1) molecule is mainly expressed on functionally "exhausted" CD8(+) T cells, dampening the host antitumor immune response. We evaluated the ratio between effective and regulatory T cells (Tregs) and PD-1 expression as a prognostic factor for operable breast cancer patients. A series of 218 newly diagnosed invasive breast cancer patients who had undergone primary surgery at Ruijin Hospital were identified. The influence of CD8(+) cytotoxic T lymphocytes, FOXP3(+) (Treg cell marker), and PD-1(+) immune cell counts on prognosis was analyzed utilizing immunohistochemistry. Both PD-1(+) immune cells and FOXP3(+) Tregs counts were significantly associated with unfavorable prognostic factors. In bivariate, but not multivariate analysis, high tumor infiltrating PD-1(+) cell counts correlated with significantly shorter patient survival. Our results suggest a prognostic value of the PD-1(+) immune cell population in such breast cancer patients. Targeting the PD-1 pathway may be a feasible approach to treating patients with breast cancer.

Highlights d MXRA8 has two Ig-like domains with an unexpected structural topology d MXRA8 binds in the ''canyon'' between two protomers of the CHIKV E spike d Both the two domains and hinge region of MXRA8 are involved in interaction d The stalk region of MXRA8 is critical for CHIKV virus entry