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

Abstract

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.

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

“…Indeed, Sourisseau et al 35) reported that various FLAG-tag-inserted OCLN constructs were expressed in human renal carcinoma 786-O cells, which are normally not able to support HCV entry owing to insufficient OCLN expression, 8) and that HCVpp entry into cells, mediated by some of the constructs, was inhibited by anti-FLAG antibodies. Previously, we have successfully developed, using a differential screening strategy for parental Huh7.5.1 cells and Huh7.5.1-derived CLDN1-knockout S7-A cells, anti-CLDN1 monoclonal antibodies that markedly inhibited HCV infection.…”

5-HT 2 Receptors Facilitate JC Polyomavirus Entry

“…Interestingly, there are many other examples of viruses that use different mechanisms for attachment and entry, including mammalian reovirus and human papillomavirus type 16 (HPV16) (54,58,59). Mammalian reovirus directly interacts with cell surface carbohydrate receptors and junctional adhesion molecule A (JAM-A) to direct attachment, and ␤1-integrins direct viral entry but do not function in viral attachment (54,60).…”

TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection

“…Our previous studies demonstrated that the cellular protein apolipoprotein E (apoE) is incorporated on the HCV envelope and mediates virion attachment to target cells by binding to the cell surface HSPGs, mainly the syndecan-1 (SDC1) proteoglycan (18)(19)(20). HCV E2 interacts with other cell surface receptors/ coreceptors, such as CD81, claudin, occludin, SR-BI, and LDLR, which all play crucial roles in HCV cell entry at postattachment steps (21)(22)(23)(24)(25)(26)(27)(28). Additionally, several other cellular proteins and pathways were found to be important for efficient HCV infection in cell culture, including epidermal growth factor receptor (EGFR), EphA2, Niemann-Pick C1 (NPC1L1), phosphatidylinositol 3-kinase (PI3K)-Akt, and CIDEB (29)(30)(31)(32).…”