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The second extracellular loop dictates Occludin-mediated HCV entry.

ABSTRACT: Recent findings have implicated tight junction (TJ) protein Occludin (OCLN) as an essential factor for hepatitis C virus (HCV) to enter human hepatocytes. To gain insights into OCLN-mediated HCV entry, we created a panel of OCLN deletion mutants and found that without impairing OCLN's cell surface localization, removal of the extracellular loop 2 (EL2) from OCLN abolished both its ability to mediate HIV-HCV pseudotypes' (HCVpp) entry as well as its ability to coprecipitate HCV glycoprotein E2. Recombinant OCLN EL2, however, failed to robustly bind soluble E2 (sE2) in pull-down assays. Subsequent studies revealed that OCLN formed complex with Dynamin II, an important GTPase for endocytosis, in an EL2-dependent fashion. HCVpp, as well as cell culture grown HCV (HCVcc), was sensitive to Dynamin knockdown or inhibition. We conclude that OCLN EL2 dictates the Dynamin-dependent HCV entry. Furthermore, OCLN could function to bridge virions to Dynamin-dependent endocytic machineries.

SUBMITTER: Liu S

PROVIDER: S-EPMC2946412 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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The second extracellular loop dictates Occludin-mediated HCV entry.

Liu Shufeng S Kuo Wayne W Yang Wei W Liu Weiqun W Gibson Gregory A GA Dorko Kenneth K Watkins Simon C SC Strom Stephen C SC Wang Tianyi T

Virology 20100906 1

Recent findings have implicated tight junction (TJ) protein Occludin (OCLN) as an essential factor for hepatitis C virus (HCV) to enter human hepatocytes. To gain insights into OCLN-mediated HCV entry, we created a panel of OCLN deletion mutants and found that without impairing OCLN's cell surface localization, removal of the extracellular loop 2 (EL2) from OCLN abolished both its ability to mediate HIV-HCV pseudotypes' (HCVpp) entry as well as its ability to coprecipitate HCV glycoprotein E2. R ...[more]

PMID: 20822789

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Project description:Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although PEDV was long believed to replicate in the intestinal epithelium by using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells results in disruption of the tight junctional distribution of occludin to its intracellular location. Overexpression of occludin in target cells makes them more susceptible to PEDV infection, whereas ablation of occludin expression by use of small interfering RNA (siRNA) in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the postbinding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization are closely coupled. However, the macropinocytosis inhibitors could not impede virus replication once the virus had entered host cells. This suggests that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection during late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells to each other, thereby forming intercellular seals. Here we investigate, for the first time, the role of the tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.

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The second extracellular loop dictates Occludin-mediated HCV entry.

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The second extracellular loop dictates Occludin-mediated HCV entry.

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