The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

Abstract

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides-estimated at three to four per virion-is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S-and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.Hepatitis B virus (HBV) remains a major public health concern worldwide, affecting more than 350 millions of chronically infected individuals. Since the discovery of HBV, substantial information has been gathered on the viral replication cycle, but our understanding of the viral entry mechanism remains limited, and the identity of the receptor(s) for HBV is still unknown (15). HBV displays a very narrow host range, which is likely determined at viral entry by a highly specific interaction between the HBV envelope proteins and receptors at the surface of human hepatocytes. The envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (SHBsAg) are membrane-spanning glycoproteins that differ from each other by the size of their N-terminal ectodomain (21). L-HBsAg contains a N-terminal pre-S1, central pre-S2, and C-terminal S domains. M-HBsAg is shorter than L-HBsAg in lacking pre-S1, whereas S-HBsAg consists of the S domain only (Fig. 1). Envelope protein synthesis occurs at the endoplasmic reticulum (ER) membrane. Empty subviral particles (SVPs) assemble from aggregates at a pre-Golgi membrane and exit the cell through the secretory pathway (36). Assembly of mature HBV virions requires, in addition to S-HBsAg, the presence of L-HBsAg as a matrix protein for nucleocapsid envelopment (6). Recent findings indicate that HBV viri...

Impaired Virion Secretion by Hepatitis B Virus Immune Escape Mutants and Its Rescue by Wild-Type Envelope Proteins or a Second-Site Mutation

“…In contrast, the T118A, P120T, and N131S mutants displayed efficient virion secretion. Very similar results were obtained when virions were immunoprecipitated with R254, a rabbit polyclonal antibody targeting the preS1 domain of the L protein (data not shown) (33). Most mutants with low HBsAg values in culture supernatant (suggestive of impaired HBsAg secretion or detection or reduced HBsAg stability) were also impaired in virion secretion.…”

“…Interestingly, many immune escape mutations also severely impaired infectivity of hepatitis delta virus (41), which hijacks HBV envelope proteins for release from and entry into hepatocytes. The immune escape mutations were detrimental to infectivity when present on the S but not the L protein (33). Similarly, we found that introducing the I110M, G119E, and R169P mutations into the S protein impaired HBV virion secretion but that virion secretion was unaltered when the same mutations were introduced to L and M proteins only (29).…”

“…Similarly, we found that introducing the I110M, G119E, and R169P mutations into the S protein impaired HBV virion secretion but that virion secretion was unaltered when the same mutations were introduced to L and M proteins only (29). Moreover, I110, G119, C124, K141, P142, C147, and C149 are critical for both virion formation and viral infectivity, with the notable exception that the G145R mutation did not compromise infectivity despite severely impairing virion formation (29,33,41). This maintenance of infectivity might explain its higher prevalence compared to other immune escape mutations.…”

Sodium Taurocholate Cotransporting Polypeptide Mediates Woolly Monkey Hepatitis B Virus Infection of Tupaia Hepatocytes

“…Therefore, studies to demonstrate the same receptor engagement of WMHBV and HBV are of great importance to consolidate the use of WMHBV as a surrogate for HBV. On the other hand, investigation into whether WMHBV utilizes NTCP as a receptor will shed light on the likelihood of NTCP's orthologs serving as a common receptor for all known HBVs from nonhuman primates.The pre-S1 domain of the L protein is a key determinant of HBV entry (22)(23)(24). Synthetic myristoylated peptides corresponding to the pre-S1 N-terminal domain of HBV are sufficient to bind to the human or Tupaia receptor NTCP (6).…”

“…The pre-S1 domain of the L protein is a key determinant of HBV entry (22)(23)(24). Synthetic myristoylated peptides corresponding to the pre-S1 N-terminal domain of HBV are sufficient to bind to the human or Tupaia receptor NTCP (6).…”