title: Attachment and Postattachment Receptors Important for Hepatitis C Virus Infection and Cell-to-Cell Transmission.
abstract: Hepatitis C virus (HCV) requires multiple receptors for its attachment to and entry into cells. Our previous studies found that human syndecan-1 (SDC-1), SDC-2, and T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) are HCV attachment receptors. Other cell surface molecules, such as CD81, Claudin-1 (CLDN1), Occludin (OCLN), SR-BI, and low-density lipoprotein receptor (LDLR), function mainly at postattachment steps and are considered postattachment receptors. The underlying molecular mechanisms of different receptors in HCV cell-free and cell-to-cell transmission remain elusive. In the present study, we used a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology, gene-specific small interfering RNAs, and a newly developed luciferase-based reporter system to quantitatively determine the importance of individual receptors in HCV cell-free and cell-to-cell transmission. Knockouts of SDC-1 and SDC-2 resulted in remarkable reductions of HCV infection and cell attachment, whereas SDC-3 and SDC-4 knockouts did not affect HCV infection. Defective HCV attachment to SDC-1 and/or SDC-2 knockout cells was completely restored by SDC-1 and SDC-2 but not SDC-4 expression. Knockout of the attachment receptors SDC-1, SDC-2, and TIM-1 also modestly decreased HCV cell-to-cell transmission. In contrast, silencing and knockout of the postattachment receptors CD81, CLDN1, OCLN, SR-BI, and LDLR greatly impaired both HCV cell-free and cell-to-cell transmission. Additionally, apolipoprotein E was found to be important for HCV cell-to-cell spread, but very-low-density lipoprotein (VLDL)-containing mouse serum did not affect HCV cell-to-cell transmission, although it inhibited cell-free infection. These findings demonstrate that attachment receptors are essential for initial HCV binding and that postattachment receptors are important for both HCV cell-free and cell-to-cell transmission.IMPORTANCEThe importance and underlying molecular mechanisms of cell surface receptors in HCV cell-free and cell-to-cell transmission are poorly understood. The role of some of the HCV attachment and postattachment receptors in HCV infection and cell-to-cell spread remains controversial. Using CRISPR-Cas9-mediated knockouts of specific cellular genes, we demonstrate that both SDC-1 and SDC-2, but not SDC-3 or SDC-4, are bona fide HCV attachment receptors. We also used a newly developed luciferase-based reporter system to quantitatively determine the importance of attachment and postattachment receptors in HCV cell-to-cell transmission. SDC-1, SDC-2, TIM-1, and SR-BI were found to modestly promote HCV cell-to-cell spread. CD81, CLDN1, OCLN, and LDLR play more important roles in HCV cell-to-cell transmission. Likewise, apolipoprotein E (apoE) is critically important for HCV cell-to-cell spread, unlike VLDL-containing mouse serum, which did not affect HCV cell-to-cell spread. These findings suggest that the mechanism(s) of HCV cell-to-cell spread differs from that of cell-free infection.
authors.affiliation: Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing, China.|Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
authors.forename: Huahao
authors.initials: H
authors.lastname: Fan
authors.affiliation: Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing, China.|Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
authors.forename: Luhua
authors.initials: L
authors.lastname: Qiao
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
authors.forename: Kyung-Don
authors.initials: KD
authors.lastname: Kang
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
authors.forename: Junfen
authors.initials: J
authors.lastname: Fan
authors.affiliation: Peking University School of Life Sciences, Beijing, China.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Wei
authors.affiliation: Department of Microbiology, Peking University School of Basic Medical Sciences, Beijing, China gluo@uab.edu.|Department of Microbiology, University of Alabama at Birmingham School of Medicine, Birmingham, Alabama, USA.
authors.forename: Guangxiang
authors.initials: G
authors.lastname: Luo
mesh_terms: D002460:Cell Line; D055785:Gene Knockdown Techniques; D055786:Gene Knockout Techniques; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*; D053585:Virus Attachment; D053586:Virus Internalization
chemical_list: D011991:Receptors, Virus
keywords: attachment receptor; cell-free infection; cell-to-cell transmission; hepatitis C virus; infection; postattachment receptor