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Occludin a New HCV Entry Factor: Is the List of Essential Hepatitis C Virus Receptors Complete?
GASTROENTEROLOGY 2009;137:727–736
SELECTED SUMMARIES Gary R. Lichtenstein, Section Editor
STAFF OF CONTRIBUTORS Faten Aberra, Philadelphia, PA Nuzhat A. Ahmad, Philadelphia, PA Hans-Dieter Allescher, GermischPartenkirchen, Germany Jordi Bruix, Barcelona, Spain Antoni Castells, Catalonia, Spain William Chey, Ann Arbor, MI Massimo Colombo, Milan, Italy Jason Dominitz, Seattle, WA Anders Ekbom, Stockholm, Sweden Gary W. Falk, Cleveland, OH
James Farrell, Los Angeles, CA Lauren B. Gerson, Stanford, CA Michael Goggins, Baltimore, MD W. Ray Kim, Rochester, MN George Lau, Hong Kong, China Uma Mahadevan-Velayos, San Francisco, CA Peter Mannon, Birmingham, AL Oliver G. Opitz, Freiburg, Germany Julian Panes, Barcelona, Spain Henry Parkman, Philadelphia, PA
OCCLUDIN A NEW HCV ENTRY FACTOR: IS THE LIST OF ESSENTIAL HEPATITIS C VIRUS RECEPTORS COMPLETE? Ploss A, Evans MJ, Gaysinskaya VA, et al. (Center for the Study of Hepatitis C, The Rockefeller University, New York, New York). Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 2009;457:882– 886. Several human and nonhuman cell lines exist that are not susceptible to hepatitis C virus (HCV) infection despite expression of the currently known essential HCV receptors SR-BI, CD81, and claudin-1. The aim of the work by Ploss et al (Nature 2009;457:882– 886) was to identify additional essential entry factors rendering nonhuman cells susceptible to HCV infection. For that purpose, the authors used a cyclic, retrovirus-based repackaging screen of a cDNA library derived from the human hepatocarcinoma Huh-7.5 cell line that is highly HCV permissive. In brief, the cDNA library was packaged into vesicular stomatitis virus-G pseudoparticles (VSVGpp), which were used to transduce N3xF36 cells (mouse NIH3T3 cells expressing the known HCV receptors CD81, SR-BI, and claudin-1). The susceptibility of transduced N3xF36 cells to HCV was tested with HCV pseudoparticles (HCVpp). Infected N3xF36 clones were selected, expanded, and used to produce new VSVGpp encoding Huh-7.5 cDNA library inserts (cycling packaging rescue). The repackaged pseudoparticles were used to transduce naïve N3xF26 cells for subsequent rounds of screening. During multiple rounds of library transduction and selection, the authors found an increase in infectibility of N3xF36 cells to HCVpp. In 8 of 24 clones with increased susceptibility to HCVpp, a cDNA encoding for the tight junction protein occludin was found. To investigate if occludin expression is essential for HCV infection, the authors silenced occludin expression in the HCV-permissive cell lines Huh-7.5 and Hep3B cells
Scott Plevy, Chapel Hill, NC Raoul Poupon, Paris, France Eamonn Quigley, Cork, Ireland Shanthi Sitaraman, Atlanta, GA Nathan Subramaniam, Brisbane, Australia George Triadafilopoulos, Stanford, CA Thomas A. Ullman, New York, NY Kenneth K. Wang, Rochester, MN Stefan Zeuzem, Frankfurt, Germany
and observed a loss of permissiveness in both cell lines to HCVpp infection and HCVpp/HCVcc infection, respectively. Next, the authors showed that the renal carcinoma cell line 786-O which is naturally not permissive for HCV infection despite expression of CD81, SR-BI, and claudin-1 becomes susceptible to HCV after transduction of occludin. Furthermore, the authors showed that expression of all 4 factors conferred HCVpp susceptibility to mouse embryonic fibroblast cell lines STO5 and L929, the mouse hepatocyte cell lines AML12, Hepa1.6, and H2.35. By expressing every combination of human and murine receptors in Chinese hamster ovary cells the authors were able to show that human-specific HCV entry factor functions are associated with the presence of human CD81 and human occludin. Using chimeric mouse and human occludin molecules the authors mapped human-specific determinants to the 2nd extracellular loop of occludin. Overall, Ploss et al identified human occludin as new HCV entry factor essential for HCV infection together with the known HCV receptors human-CD81, SR-BI, and claudin-1. Mouse cells become susceptible for HCV infection when all 4 receptors are expressed. Comment. Attachment and cell entry of a virus into its
target cell are the first steps of viral infection and a prerequisite for replication. Because of the high turnover of infected cells in patients with chronic hepatitis C, de novo infection is not only the first essential step for development of acute infection, but also highly essential for persistence of HCV in the chronic phase of infection. It can be anticipated that inhibition of de novo infection could be an efficient approach to prevent and treat acute and chronic HCV-infection. For efficient inhibition of de novo infection, the exact knowledge of the first steps of HCV infection is necessary. The currently known essential HCV receptors such as CD81, SR-BI, and claudin-1 are not sufficient to explain HCV infection. The same
728
SELECTED SUMMARIES
group that previously discovered claudin-1 as an HCV receptor now identified occludin as a further HCV receptor. Their experiments conclusively show that non–HCVpermissive human and nonhuman cell lines become susceptible to HCV when all 4 molecules—CD81, SR-BI, claudin-1, and occludin—are expressed. Together, the results provide strong evidence that occludin is the final essential entry factor for HCV (Nature 2009;457:797– 798). The work by Ploss et al is an important step in the development of a small animal model for HCV. However, Ploss et al were not able to induce replication of HCVcc in mouse cells after infection, indicating that other hostspecific factors than CD81 and occludin are required for complete life cycle of HCV in mouse cells. Occludin is an approximately 60-kD integral membrane protein with 4 transmembrane domains and 2 extracellular loops that was first isolated in 1993 by Furuse et al (J Cell Biol 1993;123:1777–1788). Occludin is, like claudin, a major component of bicellular tight junctions that are located in the apical part of lateral membranes and comprise an elaborate network of paired strands, which form kissing points that eliminate extracellular space (for review, Curr Med Chem 2007;14:2482– 2488). Overall, the identification of occludin and claudin as essential HCV receptors highlight the significance of the tight junction complex and cell polarity for the cell entry process of HCV. Recently, it has been shown that claudin-1 and occludin are down-regulated during infection of Huh-7 cells (J Virol 2009;83:2011–2014). This may have important consequences for cellular physiology because occludin has also been shown to be involved in the cell signaling pathways including v-Raf, transforming growth factor- type I receptor, RhoA, MAPK, Akt, c-Yes, aPKC, and PI3-kinase (for review, Biochim Biophys Acta 2008;1778:588 – 600). The existence of different HCV receptors with distinct cell surface distributions and 2 receptors at bicellular connection points raises the question how HCV enters the cell. Overall, the current results suggest that HCV follows a coordinated entry pathway. DC-SIGN, L-SIGN, glycosaminoglycans, and the LDL receptor might serve as primary collectors of HCV particles for further targeting to SR-BI and CD81. HCV-E2/CD81 engagement has been shown to activate GTPase-dependent signaling pathways, leading to relocalization of the HCV-E2/CD81 complex to cell– cell contact areas where CD81 comes into contact with the tight junction proteins occludin and claudin (J Virol 2008;82:8316 – 8329). The last step is presumably endocytosis followed by the transport of the viral particle to the endosome. Penetration of the virus in the cytoplasm occurs putatively by membrane fusion between the viral envelope and the endosomal membrane triggering by low pH (for review, Hepatology 2006;44:527–535; Nat Rev Microbiol 2007;5:453– 463). The toxins produced by Vibrio cholerae and Clostridium perfringens exert their toxic effect by altering the tight
GASTROENTEROLOGY Vol. 137, No. 2
junction indicating that tight junction proteins are potential targets for therapy. Tight junction modulators are currently developed for drug delivery. Because of the essential roles for HCV infection, these compounds targeting the tight junction could also be effective for treatment of hepatitis C (for review, Curr Med Chem 2007;14: 2482–2488). However, 2 tight junction molecules involved in HCV entry also raise the question of potential cell-to-cell infection. If this was the case, it would have major implication whether entry inhibitors could act as antiviral drugs. HCV plus and minus strand RNA has been detected in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, suggesting that HCV is a potential lymphotropic virus. Whether HCV only attaches to PBMC or whether HCV replicates in PBMC is not yet completely understood. A recent work by Marukian et al used HCVcc to investigate susceptibility of peripheral blood lymphocytes to HCV infection. The study showed that no HCV replication was achievable in B or T lymphocytes, monocytes, macrophages, or dendritic cells from healthy donors (Hepatology 2008;48:1843–1850). The identification of the 4 essential receptors supports this finding. Claudin-1 and SR-BI were not detectable in any subset of blood cells. Occludin is typically not expressed in peripheral blood cells; however, it has been shown to be expressed in T cells under certain conditions (Inflammation 1998;22:573– 582). Marukian et al reported that B-cells transduced for CD81, SR-BI, and claudin-1 expression did not become infectable with HCVpp. Occludin could be the missing receptor for infection of blood cells; however, Marukian et al also showed that lymphocytes do not support replication of transfected HCV-RNA, indicating that multiple blocks in HCV infection of lymphocytes exist. Thus, it is unlikely that the 4th receptor occludin makes blood cells susceptible for HCV infection. Altogether, the recent identification of occludin as 4th essential HCV receptor is a hallmark in the understanding of HCV pathophysiology. The discovery opens new options for development of new treatment approaches and small animal models for HCV. BERND KRONENBERGER, MD STEFAN ZEUZEM, MD
GUT PERMEABILITY IN IRRITABLE BOWEL SYNDROME: MORE LEAKS ADD TO SLIGHTLY INFLAMED BOWEL SYNDROME CONSPIRACY THEORY Piche T, Barbara G, Aubert P, et al. (INSERM U 913, Hopital Hotel Dieu, Nantes, France). Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut 2009;58:196 –201.
SELECTED SUMMARIES Gary R. Lichtenstein, Section Editor
STAFF OF CONTRIBUTORS Faten Aberra, Philadelphia, PA Nuzhat A. Ahmad, Philadelphia, PA Hans-Dieter Allescher, GermischPartenkirchen, Germany Jordi Bruix, Barcelona, Spain Antoni Castells, Catalonia, Spain William Chey, Ann Arbor, MI Massimo Colombo, Milan, Italy Jason Dominitz, Seattle, WA Anders Ekbom, Stockholm, Sweden Gary W. Falk, Cleveland, OH
James Farrell, Los Angeles, CA Lauren B. Gerson, Stanford, CA Michael Goggins, Baltimore, MD W. Ray Kim, Rochester, MN George Lau, Hong Kong, China Uma Mahadevan-Velayos, San Francisco, CA Peter Mannon, Birmingham, AL Oliver G. Opitz, Freiburg, Germany Julian Panes, Barcelona, Spain Henry Parkman, Philadelphia, PA
OCCLUDIN A NEW HCV ENTRY FACTOR: IS THE LIST OF ESSENTIAL HEPATITIS C VIRUS RECEPTORS COMPLETE? Ploss A, Evans MJ, Gaysinskaya VA, et al. (Center for the Study of Hepatitis C, The Rockefeller University, New York, New York). Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 2009;457:882– 886. Several human and nonhuman cell lines exist that are not susceptible to hepatitis C virus (HCV) infection despite expression of the currently known essential HCV receptors SR-BI, CD81, and claudin-1. The aim of the work by Ploss et al (Nature 2009;457:882– 886) was to identify additional essential entry factors rendering nonhuman cells susceptible to HCV infection. For that purpose, the authors used a cyclic, retrovirus-based repackaging screen of a cDNA library derived from the human hepatocarcinoma Huh-7.5 cell line that is highly HCV permissive. In brief, the cDNA library was packaged into vesicular stomatitis virus-G pseudoparticles (VSVGpp), which were used to transduce N3xF36 cells (mouse NIH3T3 cells expressing the known HCV receptors CD81, SR-BI, and claudin-1). The susceptibility of transduced N3xF36 cells to HCV was tested with HCV pseudoparticles (HCVpp). Infected N3xF36 clones were selected, expanded, and used to produce new VSVGpp encoding Huh-7.5 cDNA library inserts (cycling packaging rescue). The repackaged pseudoparticles were used to transduce naïve N3xF26 cells for subsequent rounds of screening. During multiple rounds of library transduction and selection, the authors found an increase in infectibility of N3xF36 cells to HCVpp. In 8 of 24 clones with increased susceptibility to HCVpp, a cDNA encoding for the tight junction protein occludin was found. To investigate if occludin expression is essential for HCV infection, the authors silenced occludin expression in the HCV-permissive cell lines Huh-7.5 and Hep3B cells
Scott Plevy, Chapel Hill, NC Raoul Poupon, Paris, France Eamonn Quigley, Cork, Ireland Shanthi Sitaraman, Atlanta, GA Nathan Subramaniam, Brisbane, Australia George Triadafilopoulos, Stanford, CA Thomas A. Ullman, New York, NY Kenneth K. Wang, Rochester, MN Stefan Zeuzem, Frankfurt, Germany
and observed a loss of permissiveness in both cell lines to HCVpp infection and HCVpp/HCVcc infection, respectively. Next, the authors showed that the renal carcinoma cell line 786-O which is naturally not permissive for HCV infection despite expression of CD81, SR-BI, and claudin-1 becomes susceptible to HCV after transduction of occludin. Furthermore, the authors showed that expression of all 4 factors conferred HCVpp susceptibility to mouse embryonic fibroblast cell lines STO5 and L929, the mouse hepatocyte cell lines AML12, Hepa1.6, and H2.35. By expressing every combination of human and murine receptors in Chinese hamster ovary cells the authors were able to show that human-specific HCV entry factor functions are associated with the presence of human CD81 and human occludin. Using chimeric mouse and human occludin molecules the authors mapped human-specific determinants to the 2nd extracellular loop of occludin. Overall, Ploss et al identified human occludin as new HCV entry factor essential for HCV infection together with the known HCV receptors human-CD81, SR-BI, and claudin-1. Mouse cells become susceptible for HCV infection when all 4 receptors are expressed. Comment. Attachment and cell entry of a virus into its
target cell are the first steps of viral infection and a prerequisite for replication. Because of the high turnover of infected cells in patients with chronic hepatitis C, de novo infection is not only the first essential step for development of acute infection, but also highly essential for persistence of HCV in the chronic phase of infection. It can be anticipated that inhibition of de novo infection could be an efficient approach to prevent and treat acute and chronic HCV-infection. For efficient inhibition of de novo infection, the exact knowledge of the first steps of HCV infection is necessary. The currently known essential HCV receptors such as CD81, SR-BI, and claudin-1 are not sufficient to explain HCV infection. The same
728
SELECTED SUMMARIES
group that previously discovered claudin-1 as an HCV receptor now identified occludin as a further HCV receptor. Their experiments conclusively show that non–HCVpermissive human and nonhuman cell lines become susceptible to HCV when all 4 molecules—CD81, SR-BI, claudin-1, and occludin—are expressed. Together, the results provide strong evidence that occludin is the final essential entry factor for HCV (Nature 2009;457:797– 798). The work by Ploss et al is an important step in the development of a small animal model for HCV. However, Ploss et al were not able to induce replication of HCVcc in mouse cells after infection, indicating that other hostspecific factors than CD81 and occludin are required for complete life cycle of HCV in mouse cells. Occludin is an approximately 60-kD integral membrane protein with 4 transmembrane domains and 2 extracellular loops that was first isolated in 1993 by Furuse et al (J Cell Biol 1993;123:1777–1788). Occludin is, like claudin, a major component of bicellular tight junctions that are located in the apical part of lateral membranes and comprise an elaborate network of paired strands, which form kissing points that eliminate extracellular space (for review, Curr Med Chem 2007;14:2482– 2488). Overall, the identification of occludin and claudin as essential HCV receptors highlight the significance of the tight junction complex and cell polarity for the cell entry process of HCV. Recently, it has been shown that claudin-1 and occludin are down-regulated during infection of Huh-7 cells (J Virol 2009;83:2011–2014). This may have important consequences for cellular physiology because occludin has also been shown to be involved in the cell signaling pathways including v-Raf, transforming growth factor- type I receptor, RhoA, MAPK, Akt, c-Yes, aPKC, and PI3-kinase (for review, Biochim Biophys Acta 2008;1778:588 – 600). The existence of different HCV receptors with distinct cell surface distributions and 2 receptors at bicellular connection points raises the question how HCV enters the cell. Overall, the current results suggest that HCV follows a coordinated entry pathway. DC-SIGN, L-SIGN, glycosaminoglycans, and the LDL receptor might serve as primary collectors of HCV particles for further targeting to SR-BI and CD81. HCV-E2/CD81 engagement has been shown to activate GTPase-dependent signaling pathways, leading to relocalization of the HCV-E2/CD81 complex to cell– cell contact areas where CD81 comes into contact with the tight junction proteins occludin and claudin (J Virol 2008;82:8316 – 8329). The last step is presumably endocytosis followed by the transport of the viral particle to the endosome. Penetration of the virus in the cytoplasm occurs putatively by membrane fusion between the viral envelope and the endosomal membrane triggering by low pH (for review, Hepatology 2006;44:527–535; Nat Rev Microbiol 2007;5:453– 463). The toxins produced by Vibrio cholerae and Clostridium perfringens exert their toxic effect by altering the tight
GASTROENTEROLOGY Vol. 137, No. 2
junction indicating that tight junction proteins are potential targets for therapy. Tight junction modulators are currently developed for drug delivery. Because of the essential roles for HCV infection, these compounds targeting the tight junction could also be effective for treatment of hepatitis C (for review, Curr Med Chem 2007;14: 2482–2488). However, 2 tight junction molecules involved in HCV entry also raise the question of potential cell-to-cell infection. If this was the case, it would have major implication whether entry inhibitors could act as antiviral drugs. HCV plus and minus strand RNA has been detected in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, suggesting that HCV is a potential lymphotropic virus. Whether HCV only attaches to PBMC or whether HCV replicates in PBMC is not yet completely understood. A recent work by Marukian et al used HCVcc to investigate susceptibility of peripheral blood lymphocytes to HCV infection. The study showed that no HCV replication was achievable in B or T lymphocytes, monocytes, macrophages, or dendritic cells from healthy donors (Hepatology 2008;48:1843–1850). The identification of the 4 essential receptors supports this finding. Claudin-1 and SR-BI were not detectable in any subset of blood cells. Occludin is typically not expressed in peripheral blood cells; however, it has been shown to be expressed in T cells under certain conditions (Inflammation 1998;22:573– 582). Marukian et al reported that B-cells transduced for CD81, SR-BI, and claudin-1 expression did not become infectable with HCVpp. Occludin could be the missing receptor for infection of blood cells; however, Marukian et al also showed that lymphocytes do not support replication of transfected HCV-RNA, indicating that multiple blocks in HCV infection of lymphocytes exist. Thus, it is unlikely that the 4th receptor occludin makes blood cells susceptible for HCV infection. Altogether, the recent identification of occludin as 4th essential HCV receptor is a hallmark in the understanding of HCV pathophysiology. The discovery opens new options for development of new treatment approaches and small animal models for HCV. BERND KRONENBERGER, MD STEFAN ZEUZEM, MD
GUT PERMEABILITY IN IRRITABLE BOWEL SYNDROME: MORE LEAKS ADD TO SLIGHTLY INFLAMED BOWEL SYNDROME CONSPIRACY THEORY Piche T, Barbara G, Aubert P, et al. (INSERM U 913, Hopital Hotel Dieu, Nantes, France). Impaired intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut 2009;58:196 –201.