Two determinants of infectivity have been identified in the hepatitis B virus (HBV) envelope proteins: a pre-S1 receptor-binding site and an uncharacterized determinant in the antigenic loop (AGL), which is structurally related to the antigenic a-determinant. Infection would proceed through virus attachment to cell surface heparan sulfate (HS) proteoglycans (HSPGs) before pre-S1 engages a specific receptor for uptake. Using heparin binding and in vitro infection assays with hepatitis D virus as a surrogate for HBV, we established that HS binding is mediated by the AGL. Electrostatic interaction was shown to depend upon AGL residues R122 and K141, because their substitution with alanine modified the virus net-charge and prevented binding to heparin, attachment to hepatocytes, and infection. In addition to R122 and K141, the HS binding determinant was mapped to cysteines and prolines, which also define the conformational a-determinant. The importance of AGL conformation was further demonstrated by the concomitant loss of a-determinant and heparin binding upon treatment of viral particles with membraneimpermeable reducing agent. Furthermore, envelope proteins extracted from the viral membrane with a nonionic detergent were shown to conserve the a-determinant but to lose heparin affinity/avidity. Conclusion: Our findings support a model in which attachment of HBV to HSPGs is mediated by the AGL HS binding site, including only two positively charged residues (R122 and K141) positioned precisely in a three-dimensional AGL structure that is stabilized by disulfide bonds. HBV envelope proteins would individually bind to HS with low affinity, but upon their clustering in the viral membrane, they would reach sufficient avidity for a stable interaction between virus and cell surface HSPGs. Our data provide new insight into the HBV entry pathway, including the opportunity to design antivirals directed to the AGL-HS interaction. (HEPATOLOGY 2012;00:000-000) W orldwide, hepatitis B virus (HBV) affects more than 350 million chronically infected individuals whose infection may progress to severe liver disease, including cirrhosis and hepatocellular carcinoma.1 HBV has a very narrow host range and a prominent hepatotropism that, for the most part, is determined at viral entry. Although much information has been gathered in recent years in many aspects of the HBV replication cycle, the HBV entry pathway has not been fully explored due to cellular receptors that await identification. 2Three types of membrane-associated glycoproteins are present in the envelope of HBV virions, each bearing the HBV surface antigen (HBsAg): (1) the small envelope protein (S-HBsAg); (2) the middle protein (M-HBsAg), which differs from S-HBsAg by an N-terminal pre-S2 ectodomain; and (3) the large protein (L-HBsAg), which includes a further N-terminal pre-S1 extension. Surprisingly, the envelope proteins are produced in amounts far exceeding the need for virion assembly, leading to the production of empty subviral particles (SVPs) that outnumber virions...
The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant "a" determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the "a" determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the "a" determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the "a" determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan "a" determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.
Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery, the WHV carrier woodchucks were super-infected with WHV-enveloped HDV (wHDV). Six weeks later, the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time PCR assay and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues Conclusion Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Since others previously hypothesized that hepadnavirus-induced HCCs are resistant to re-infection with a hepadnavirus in vivo, our data suggest that if such a resistance exists, it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.
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