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

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.

We have expressed a series of truncated spike (S) glycoproteins of SARS-CoV and found that the N-terminus 14-502 residuals were sufficient to bind to SARS-CoV susceptible Vero E6 cells. With this soluble S protein fragment as an affinity ligand, we screened HeLa cells transduced with retroviral cDNA library from Vero E6 cells and obtained a HeLa cell clone which could bind with the S protein. This cell clone was susceptible to HIV/SARS pseudovirus infection and the presence of a functional receptor for S protein in this cell clone was confirmed by the cell-cell fusion assay. Further studies showed the susceptibility of this cell was due to the expression of endogenous angiotensin-converting enzyme 2 (ACE2) which was activated by inserted LTR from retroviral vector used for expression cloning. When human ACE2 cDNA was transduced into NIH3T3 cells, the ACE2 expressing NIH3T3 cells could be infected with HIV/SARS pseudovirus. These data clearly demonstrated that ACE2 was the functional receptor for SARS-CoV.

Studies of SARS coronavirus (SARS-CoV)-the causative agent of severe acute respiratory syndrome (SARS)-have been hampered by its high transmission rate and the pathogenicity of this virus. To permit analysis of the host range and entry mechanism of SARS-CoV, we incorporated the humanized SARS-CoV spike (S) glycoprotein into HIV particles to generate a highly infectious SARS-CoV pseudotyped virus. The infection on Vero E6-a permissive cell line to SARS-CoV-could be neutralized by sera from convalescent SARS patients, and the entry was a pH-dependent process. With these highly infectious SARS-CoV pseudotypes, several cell lines derived from various tissues were revealed as susceptible to SARS-CoV, which were highly corresponding to the expression pattern of virus's receptor angiotensin-converting enzyme 2 (ACE2). In addition, we also demonstrated angiotensin 1 converting enzyme (ACE)-the homologue of ACE2 could not function as a receptor for SARS-CoV.

Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.Dengue virus (DV) is a flavivirus that is spread by mosquitoes and causes millions of cases of disease each year worldwide (2, 9, 17). DV infection can result in dengue hemorrhagic fever, a more lethal disease that leads to ϳ500,000 hospitalizations and ϳ12,500 deaths per year (10, 39). DV is currently endemic in more than 100 countries, including the United States (17), and the World Health Organization estimates that about 40% of the world's population lives in areas where dengue fever is endemic (39). As yet, there is no licensed DV vaccine or antiviral therapy. Studies of the molecular mechanisms of the virus life cycle are important to the development of new antiviral strategies.Flaviviruses such as DV are small, highly organized enveloped viruses with plus-sense single-stranded RNA genomes (reviewed in references 21 and 25). The flavivirus particle contains 3 structural proteins: a capsid protein, which associates with the genomic RNA to form the viral core, and two membrane proteins, the M protein and the membrane fusion protein E. Like many enveloped viruses, flaviviruses infect cells via endocytic uptake and a membrane fusion reaction triggered by the low pH within endosomes (38). Low-pH-triggered membrane fusion is mediated by conformational changes in the viral E protein, which converts from a prefusion E homodimer to a target membrane-inserted homotrimer. The structure of the DV E ectodomain in the prefusion form shows an elongated finger-like molecule with three domains (DI, DII, and DIII) composed primarily of ␤-sheets (22, 24, 42) ( Fig. 1A; see also Fig. 7). The central DI is connected to DII. The distal tip of DII contains the hydrophobic fusion loop, the region of E tha...

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Transmission of flaviviruses by hematophagous insects such as mosquitoes requires acquisition of the virus during blood feeding on the host, with midgut as the primary infection site. Here, we report that N-glycosylation of the E protein, which is conserved among most flaviviruses, is critical for the Zika virus (ZIKV) to invade the vector midgut by inhibiting the reactive oxygen species (ROS) pathway of the mosquito immune system. Our data further show that removal of the ZIKV E glycosylation site prevents mosquito infection by flaviviruses via the oral route, whereas there is no effect on infection by intrathoracic microinjection, which bypasses the midgut. Interestingly, the defect in infection of the mosquito midgut by the mutant virus through blood feeding is rescued by reduction of the ROS level by application of vitamin C, a well-known antioxidant. Therefore, our data demonstrate that ZIKV utilizes the glycosylation on the envelope to antagonize the vector immune defense during infection.