Abstract
Hepatitis B virus (HBV) entry into hepatocytes is mediated via a high-affinity interaction between the preS1 glycoprotein and sodium/bile acid cotransporting polypeptide (NTCP). To date, in vitro model systems rely on high multiplicities of infection to achieve infection of cell lines overexpressing human NTCP. This study investigates a novel regulatory pathway for NTCP trafficking to the cell surface, induced by DMSO-mediated cellular differentiation. DMSO rapidly induces high cell surface expression of NTCP and results in increased susceptibility of cells to HBV infection. Additionally, DMSO treatment induces actin, as well as Tubulin reshaping within the cells. We show that direct disruption of the actin and Tubulin network directly enhances NTCP expression and the subsequent susceptibility of cells to HBV infection. DMSO induces these changes via alterations in the levels of cyclic (c)AMP, which participates in the observed actin rearrangements. Blocking of phosphodiesterases (PDEs), which degrade accumulated cAMP, had the same effect as DMSO differentiation and demonstrates that DMSO prevents phosphodiesterase-mediated cAMP degradation. This identifies adenylate cyclase as a novel target for blocking the entry of HBV via targeting the cell surface accumulation of NTCP.This article is part of the theme issue ‘Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses’.
Highlights
Hepatitis B virus (HBV) results in over 257 million chronic infections worldwide and even though there is a protective vaccine available, nearly 4 million new infections arise every year
Despite this apparent transcriptional overexpression, protein analysis of the former samples by western blot demonstrated that significantly less NTCP overexpression could be detected in HepG2 cells when cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) in the absence of DMSO
A similar kinetic was determined by immunofluorescence microscopy, where NTCP is detectable on the ER of cells at 4 h post-DMSO exposure, and it passes through the Golgi network onto the cell surface between 8 and 24 h post-DMSO exposure
Summary
Hepatitis B virus (HBV) results in over 257 million chronic infections worldwide and even though there is a protective vaccine available, nearly 4 million new infections arise every year. HBV entry into hepatocytes is separated into two distinct steps, where an initial low-affinity interaction with heparan sulfate proteoglycans (HSPGs), including the recently identified glypican 5 (GPC5) [9], on the hepatocyte surface, mediated by the preS1 region of the L (large) protein and the antigenic loop of the viral S (small) protein, is followed by the attachment of the virus to its high-affinity NTCP receptor [10]. This interaction results in endocytosis of the HBV particles and further viral infection [11]. This mechanism may explain why DMSO 2 addition is critical for achieving HBV susceptibility of cell lines overexpressing NTCP
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