Effect of cell polarization on hepatitis C virus entry

The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.     

Temporal Analysis of Hepatitis C Virus Cell Entry with Occludin Directed Blocking Antibodies

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.     

Hepatoma polarization limits CD81 and hepatitis C virus dynamics

Many viruses target the polarized epithelial apex during host invasion. In contrast, hepatitis C virus (HCV) engages receptors at the basal surface of hepatocytes in the polarized liver parenchyma. Hepatocyte polarization limits HCV entry by undefined mechanism(s). Given the recent reports highlighting a role for receptor mobility in pathogen entry, we studied the effect(s) of hepatocyte polarization on viral receptor and HCV pseudoparticle (HCVpp) dynamics using real‐time fluorescence recovery after photobleaching and single particle tracking. Hepatoma polarization reduced CD81 and HCVpp dynamics at the basal membrane. Since cell polarization is accompanied by changes in the actin cytoskeleton and CD81 links to actin via its C‐terminus, we studied the dynamics of a mutant CD81 lacking a C‐terminal tail (CD81_ΔC) and its effect(s) on HCVpp mobility and infection. CD81_ΔC showed an increased frequency of confined trajectories and a reduction of Brownian diffusing molecules compared to wild‐type protein in non‐polarized cells. However, these changes were notobserved in polarized cells. HCVpp showed a significant reduction in Brownian diffusion and infection of CD81_ΔC expressing non‐polarized cells. In summary, these data highlight the dynamic nature of CD81 and demonstrate a role for CD81 lateral diffusion to regulate HCV infection in a polarization‐dependent manner.     

The small GTPase Rab13 regulates assembly of functional tight junctions in epithelial cells

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell-cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.     

Claudin-6 and Occludin Natural Variants Found in a Patient Highly Exposed but Not Infected with Hepatitis C Virus (HCV) Do Not Confer HCV Resistance In Vitro

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.     

Impact of intra- and interspecies variation of occludin on its function as coreceptor for authentic hepatitis C virus particles

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.     

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer,Productive Infection,and Replication and Their Requirement for HCV Receptors

Hepatitis C virus (HCV) infection is believed to begin with interactions between cell-free HCV and cell receptors that include CD81, scavenger receptor B1 (SR-B1), claudin-1 (CLDN1), and occludin (OCLN). In this study, we have demonstrated that HCV spreading from infected hepatocytes to uninfected hepatocytes leads to the transfer of HCV and the formation of infection foci and is cell density dependent. This cell-cell contact-mediated (CCCM) HCV transfer occurs readily and requires all these known HCV receptors and an intact actin cytoskeleton. With a fluorescently labeled replication-competent HCV system, the CCCM transfer process was further dissected by live-cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and posttransfer. Importantly, the CCCM HCV transfer leads to productive infection of target cells. Taken together, these results show that CCCM HCV transfer constitutes an important and effective route for HCV infection and dissemination. These findings will aid in the development of new and novel strategies for preventing and treating HCV infection.     

Differential Expression of Candidate Virus Receptors in Human T Lymphocytes Prone or Resistant to Infection with Patient-Derived Hepatitis C Virus

Accumulated evidence implies that hepatitis C virus (HCV) infects not only the liver but also the immune system. A lymphocyte-specific CD5 molecule was recently identified as essential for infection of T cells with native, patient-derived HCV. To assess whether the proposed hepatocyte receptors may also contribute to HCV lymphotropism, expression of scavenger receptor-class B type 1 (SR-B1), claudin-1 (CLDN-1), claudin-6 (CLDN-6), occludin (OCLN), CD5 and CD81 was examined by real-time RT-PCR and the respective proteins quantified by immunoblotting in HCV-prone and resistant T cell lines, peripheral blood mononuclear cells (PBMC), primary T cells and their subsets, and compared to hepatoma Huh7.5 and HepG2 cells. SR-B1 protein was found in T and hepatoma cell lines but not in PBMC or primary T lymphocytes, CLDN-1 in HCV-resistant PM1 T cell line and hepatoma cells only, while CLDN-6 equally in the cells investigated. OCLN protein occurred in HCV-susceptible Molt4 and Jurkat T cells and its traces in primary T cells, but not in PBMC. CD5 was displayed by HCV-prone T cell lines, primary T cells and PBMC, but not by non-susceptible T and hepatoma cell lines, while CD81 in all cell types except HepG2. Knocking-down OCLN in virus-prone T cell line inhibited HCV infection, while de novo infection downregulated OCLN and CD81, and upregulated CD5 without modifying SR-B1 expression. Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells. This study narrowed the range of factors potentially utilized by HCV to infect T lymphocytes amongst those uncovered using laboratory HCV and Huh7.5 cells. Together with the demonstrated role for CD5 in HCV lymphotropism, the findings indicate that virus utilizes different molecules to enter hepatocytes and lymphocytes.     

Tight Junction Proteins Claudin-1 and Occludin Control Hepatitis C Virus Entry and Are Downregulated during Infection To Prevent Superinfection

A tight junction (TJ) protein, claudin-1 (CLDN1), was identified recently as a key factor for hepatitis C virus (HCV) entry. Here, we show that another TJ protein, occludin, is also required for HCV entry. Mutational study of CLDN1 revealed that its tight junctional distribution plays an important role in mediating viral entry. Together, these data support the model in which HCV enters liver cells from the TJ. Interestingly, HCV infection of Huh-7 hepatoma cells downregulated the expression of CLDN1 and occludin, preventing superinfection. The altered TJ protein expression may contribute to the morphological and functional changes observed in HCV-infected hepatocytes.     

Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLN-deficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.Hepatitis C virus (HCV), a member of the family Flaviviridae, is the causative agent of classically defined non-A, non-B hepatitis and is highly prevalent, with approximately 3% of the worldwide population infected (48). HCV infection often results in a chronic, life-long infection that can have severe health consequences, including hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. There is no HCV vaccine available, and the currently employed interferon-based treatment is inadequate as it has severe side effects and is effective only in half of the major genotype-infected individuals (22, 32). Specific anti-HCV inhibitors targeting the viral proteases and polymerase are currently being developed and will likely improve therapeutic options substantially. Undoubtedly, however, the emergence of viral resistance to such inhibitors will be a problem facing future HCV treatment options. As such, developing a spectrum of inhibitors targeting diverse steps in the virus life cycle, including HCV cell entry, is a priority for HCV research. Such inhibitors may be particularly useful following liver transplantation. Although HCV is the leading cause of liver transplants worldwide (10), the usefulness of such procedures is limited by subsequent universal graft reinfection and often accelerated disease progression (21). Even transiently inhibiting graft reinfection with HCV cell entry inhibitors could greatly improve the effectiveness of this procedure. Therefore, a greater understanding of HCV cell entry is required for the development of therapies targeting this stage of the viral life cycle.HCV host cell entry is a complex process that culminates in the clathrin-dependent endocytosis of the virion and low-pH-mediated fusion of viral and cellular lipid membranes in an early endosome (9, 12, 26, 27, 36, 51). The entry process requires the two viral envelope glycoproteins, E1 and E2, and many cellular factors, including glycosaminoglycans (GAGs) (3, 27), lipoproteins, the low-density lipoprotein receptor (LDL-R) (1, 38-40), tetraspanin CD81 (43), scavenger receptor class B type I (SR-BI) (47), and two tight junction proteins, claudin-1 (CLDN1) (17) and occludin (OCLN) (31, 44). The polarized nature of hepatocytes and the tight junction roles of OCLN and CLDN1 suggest an entry pathway similar to that of the group B coxsackieviruses, where the virion initially binds readily accessible factors that then provide a mechanism for migration of the virion into the tight junction region, just prior to internalization (14). Indeed, cellular factors are utilized by the incoming HCV virion in a temporal manner. At least GAGs and LDL-R appear to mediate virion binding (1, 3, 27, 38-40). Conflicting evidence has shown that SR-BI acts as either a binding (11) or postbinding entry factor (53), while CD81 (7, 13, 17, 27) and CLDN1 (17, 29) play postbinding roles in the HCV cell entry process. Although the kinetics of OCLN usage have not been clearly defined, this protein does not appear to play a role in virion binding (6). However, recent data showing that CD81 and CLDN1 may form complexes prior to infection (15, 24, 25, 28, 29, 35, 52) and imaging of the cell entry process (12) may contradict such a model.Human hepatocytes are the major target for HCV infection. While multiple blocks at a number of viral life cycle stages likely exist in other cell types, cell entry is one of the events limiting HCV tropism (45). Although species differences in SR-BI and CLDN1 may exert some influence on this selectivity (11, 23), CD81 and OCLN appear to be largely responsible for the restriction of HCV entry to cells from human and chimpanzee origin (7, 8, 20, 44). In fact, overexpression of the human versions of CD81 and OCLN, along with either mouse or human SR-BI and CLDN1, renders a mouse cell able to support HCV cell entry (44).We sought to provide greater insight into the species-specific restrictions of HCV cell entry and to elucidate the mechanism by which OCLN acts to mediate HCV cell entry. We examined the ability of OCLN proteins from a range of species to mediate HCV cell entry and how this function correlated with the degree of similarity to the human protein. A six-amino-acid portion of the second extracellular loop (EC2) of human OCLN was found to be responsible for the species-specific differences in entry factor function. OCLN proteins that were less functional than the human protein could be rendered fully functional by adding the human residues at these positions. Conversely, the ability of the human OCLN protein to mediate HCV cell entry was impaired by swapping this region with the corresponding sequence from species with less functional OCLN proteins. Comprehensive alanine scanning of the extracellular loops of human OCLN confirmed that the second half of EC2 was most important for the HCV cell entry process. Two cysteine residues that flank this region were found to be essential for HCV cell entry, suggesting that these residues may define a disulfide-linked subdomain of EC2. None of these amino acid changes influenced OCLN expression or localization, implying that they may serve to modulate an interaction with either another host protein or the incoming HCV virion.     

Proteomic,functional and motif‐based analysis of C‐terminal Src kinase‐interacting proteins

C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.     

MEAN inhibits hepatitis C virus replication by interfering with a polypyrimidine tract‐binding protein

MEAN (6‐methoxyethylamino‐numonafide) is a small molecule compound, and here, we report that it effectively inhibits hepatitis C virus (HCV) infection in an HCV cell culture system using a JC1‐Luc chimeric virus, with a 50% effective concentration (EC50) of 2.36 ± 0.29 μM. Drug combination usage analyses demonstrated that MEAN was synergistic with interferon α, ITX5061 and ribavirin. In addition, MEAN effectively inhibits N415D mutant virus and G451R mutant viral infections. Mechanistic studies show that the treatment of HCV‐infected hepatocytes with MEAN inhibits HCV replication but not translation. Furthermore, treatment with MEAN significantly reduces polypyrimidine tract‐binding protein (PTB) levels and blocks the cytoplasmic redistribution of PTB upon infection. In the host cytoplasm, PTB is directly associated with HCV replication, and the inhibition of HCV replication by MEAN can result in the sequestration of PTB in treated nuclei. Taken together, these results indicate that MEAN is a potential therapeutic candidate for HCV infection, and the targeting of the nucleo‐cytoplasmic translocation of the host PTB protein could be a novel strategy to interrupt HCV replication.     

Splicing Diversity of the Human OCLN Gene and Its Biological Significance for Hepatitis C Virus Entry

Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver, the fifth most common malignancy worldwide, and the third leading cause of cancer-related death, after cancers of lung and stomach (WHO Mortality Database [http://www.who.int/healthinfo/morttables/en/index.html]). The estimated incidence of new HCC cases is about 500,000 to 1,000,000 annually, with mortality of 600,000 cases per year on a global scale (12, 16, 17, 20, 24). Various risk factors for HCC include infection with hepatitis C virus (HCV) or hepatitis B virus (HBV), toxic exposures (alcohol and aflatoxins), metabolic disease (diabetes, nonalcoholic fatty liver disease, and hereditary hemochromatosis), and immune-related conditions such as primary biliary cirrhosis and autoimmune hepatitis (15).The only established in vivo model for the study of HCV infection in an immunocompetent host is the chimpanzee (23). The inability of HCV to infect animals other than humans and chimpanzees has severely hampered efforts in developing a useful small animal model for the disease, specific antiviral therapies, and an effective vaccine against HCV-mediated liver cancer (18, 23).In the United States, chronic HCV infection is the major etiological agent of liver cancer. Among individuals infected with HCV, approximately 80% develop chronic HCV infection, of which 20% will progress to cirrhosis, and 1 to 5% will progress to liver cancer (14). Genetic factors might affect the risk of liver cancer by modifying the susceptibility to HCV infection and viral clearance. Recent studies identified occludin (OCLN), an integral tight junction (TJ) protein, as one of the key factors for HCV entry into cells (8, 18). HCV infectivity was exclusively mediated by the second extracellular loop (EC2) of the OCLN MARVEL membrane-associating domain (18). This domain is found in proteins involved in lipid-rich membrane apposition events, such as cell fencing contacts and formation of vesicular particles (19). OCLN also has a large intracellular protein (ELL) domain, found in C-terminal parts of OCLN and in the ELL family of RNA polymerase II elongation factors (7), but its role in HCV infection is unclear.We hypothesized that splicing diversity, generating multiple functionally distinct OCLN protein isoforms, might modulate susceptibility to HCV infection. Six splicing forms of OCLN and two distinct promoters, P1 and P2, have been described in cell lines (4, 5, 9, 10, 13). In the present study, we explored the splicing diversity of OCLN in normal human liver and observed variable expression of known and novel isoforms. Additionally, in vitro infectivity assays proved some of these forms to be nonpermissive for HCV infection. Our study suggests that naturally occurring splicing forms of OCLN might modify the outcome of HCV infection in humans.     

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.     

Requirement of cytosolic phospholipase A2 gamma in lipid droplet formation

Lipid droplet (LD) accumulation in hepatocytes is a typical character of steatosis. Hepatitis C virus (HCV) infection, one of the risk factors related to steatosis, induced LD accumulation in cultured cells. However, the mechanisms of which HCV induce LD formation are not fully revealed. Previously we identified cytosolic phospholipase A2 gamma (PLA2G4C) as a host factor upregulated by HCV infection and involved in HCV replication. Here we further revealed that PLA2G4C plays an important role in LD biogenesis and refined the functional analysis of PLA2G4C in LD biogenesis and HCV assembly. LD formation upon fatty acid and HCV stimulation in PLA2G4C knockdown cells was impaired and could not be restored by complementation with PLA2G4A. PLA2G4C was tightly associated in the membrane with the domain around the amino acid residues 260–292, normally in ER but relocated into LDs upon oleate stimulation. Mutant PLA2G4C without enzymatic activity was not able to restore LD formation in PLA2G4C knockdown cells. Thus, both the membrane attachment and the enzymatic activity of PLA2G4C were required for its function in LD formation. The participation of PLA2G4C in LD formation is correlated with its involvement in HCV assembly. Finally, PLA2G4C overexpression itself led to LD formation in hepatic cells and enhanced LD accumulation in the liver of high-fat diet (HFD)-fed mice, suggesting its potential role in fatty liver disease.     

Scarface,a secreted serine protease‐like protein,regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo

Cell–matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. Here, in Drosophila, we analyse the role of the secreted serine protease‐like protein Scarface in germ‐band retraction and dorsal closure—morphogenetic processes that rely on the activity of integrins and laminins. We present evidence that scarface is regulated by c‐Jun amino‐terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. We propose that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context.     

PopW of Ralstonia solanacearum,a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine‐rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380‐amino‐acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non‐host), PopW induced a rapid tissue collapse via a heat‐stable but protease‐sensitive HR‐eliciting activity. PopW has an N‐terminal harpin domain (residues 1–159) and a C‐terminal pectate lyase (PL) domain (residues 160–366); its HR‐eliciting activity depends on its N‐terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB‐dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW‐deficient mutant retained the ability of wild‐type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall‐associated, hrpB‐dependent, two‐domain harpin that is conserved across the R. solanacearum species complex.     

ProP‐ProP and ProP‐phospholipid interactions determine the subcellular distribution of osmosensing transporter ProP in Escherichia coli

Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C‐terminal domain. Orthologues with and without a C‐terminal, α‐helical coiled‐coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)‐dependent manner. The roles of phospholipids and the C‐terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C‐terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled‐coil formation, the C‐terminal domain bound liposomes and ProP concentrated at the cell poles in a CL‐independent manner. The presence of the coiled‐coil replaced those phenomena with CL‐dependent binding and localization. The effects of amino acid replacements on lipid association of the C‐terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C‐terminal domain with the anionic lipid‐enriched membrane at the cell poles. The coiled‐coil domain present on only some orthologues renders that phenomenon CL‐dependent.