Abstract
Cell entry of enveloped viruses relies on the fusion between the viral and plasma or endosomal membranes, through a mechanism that is triggered by a cellular signal. Here we used a combination of computational and experimental approaches to unravel the main determinants of hepatitis B virus (HBV) membrane fusion process. We discovered that ERp57 is a host factor critically involved in triggering HBV fusion and infection. Then, through modeling approaches, we uncovered a putative allosteric cross-strand disulfide (CSD) bond in the HBV S glycoprotein and we demonstrate that its stabilization could prevent membrane fusion. Finally, we identified and characterized a potential fusion peptide in the preS1 domain of the HBV L glycoprotein. These results underscore a membrane fusion mechanism that could be triggered by ERp57, allowing a thiol/disulfide exchange reaction to occur and regulate isomerization of a critical CSD, which ultimately leads to the exposition of the fusion peptide.
Highlights
47 Hepatitis B is a major public health problem; it affects over 250 million people worldwide and 850,000 deaths occur each year as a result of hepatitis B complications (WHO, March 2015)
We found that cell-cell fusion could be detected for either indicator cell type to the same extent as for Huh[7] cells (Figure 1C). Altogether, these results indicated that cell-cell fusion mediated by hepatitis B virus (HBV) GPs is independent of acid pH and requires neither heparan sulfates proteoglycans (HSPGs) nor NTCP receptor, which underscores an alternative fusion trigger. 134 The preS1 domain of HBsAg harbors a critical determinant of membrane fusion
153 detected a cell-cell fusion activity at the same level than for wt HBV GPs (Figure 1D). This indicated that M is not necessary for membrane fusion, in agreement with previous results (Ni et al, 2010; Sureau et al, 1994) showing that M is dispensable for infectivity of viral particles (Figure 1-figure supplement 3). Altogether, these results suggested that the determinants of membrane fusion are harbored within L and S GPs. 160 aiming to identify a fusion peptide in either protein, we used a computational approach to 161 pinpoint regions of the HBV GPs that may potentially interact with membrane bilayers
Summary
47 Hepatitis B is a major public health problem; it affects over 250 million people worldwide and 850,000 deaths occur each year as a result of hepatitis B complications (WHO, March 2015). When we tested the pT7HB2.7Mless plasmid, which induces co-expression of S and L only (“noM” in Figure 1D-1F), we detected a cell-cell fusion activity at the same level than for wt HBV GPs (Figure 1D) This indicated that M is not necessary for membrane fusion, in agreement with previous results (Ni et al, 2010; Sureau et al, 1994) showing that M is dispensable for infectivity of viral particles (Figure 1-figure supplement 3). Aiming to validate our prediction that an additional disulfide bond between the two β-strands could, by stabilizing the 298-313 β-hairpin motif, prevent membrane fusion from occurring, we produced HDV particles carrying the individual (T303C or G308C) and double (T303C/G308C) mutations in HBV GPs. By measuring HDV RNAs in cell supernatants, we found that all mutants could produce comparable levels of viral particles relative to wt virus (Figure 5A), suggesting absence of gross alterations of HBV GP conformation that would otherwise preclude virion assembly (Abou-Jaoudé and Sureau, 2007).
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