Hepatitis B virus (HBV) is the prototype of hepadnaviruses, which can be subgrouped into orthohepadnaviruses infecting mammals, avihehepadnaviruses of birds, metahepadnaviruses of fish, and herpetohepadnaviruses of amphibians and reptiles. The middle (M) envelope protein and e antigen are new additions in the evolution of hepadnaviruses. They are alternative translation products of the transcripts for small (S) envelope and core proteins, respectively. For HBV, e antigen is converted from precore/core protein by removal of N-terminal signal peptide followed by furin-mediated cleavage of the basic C-terminus. This study compared old and newly discovered hepadnaviruses for their envelope protein and e antigen expression or processing. The S protein of bat hepatitis B virus (BHBV) and two metahepadnaviruses is probably myristoylated, in addition to two avihepadnaviruses. While most orthohepadnaviruses express a functional M protein with N-linked glycosylation near the amino-terminus, most metahepadnaviruses and herpetohepadnaviruses probably do not. These viruses and one orthohepadnavirus, the shrew hepatitis B virus, lack an open precore region required for e antigen expression. Potential furin cleavage sites (RXXR sequence) can be found in e antigen precursors of orthohepadnaviruses and avihepadnaviruses. Despite much larger precore/core proteins of avihepadnaviruses and their limited sequence homology with those of orthohepadnaviruses, their proximal RXXR motif can be aligned with a distal RXXR motif for orthohepadnaviruses. Thus, furin or another basic endopeptidase is probably the shared enzyme for hepadnaviral e antigen maturation. A precorederived cysteine residue is involved in forming intramolecular disulfide bond of HBV e antigen to prevent particle formation, and such a cysteine residue is conserved for both orthohepadnaviruses and avihepadnaviruses. All orthohepadnaviruses have an X gene, while all avihepadnaviruses can

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion.

Hepatitis B virus (HBV) transcribes co-terminal mRNAs of 0.7-3.5kb from the 3.2-kb covalently closed circular (ccc) DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pgRNA drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with majority of the S protein secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3’ preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. Human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric construct, or one mimicking cccDNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, which was accompanied by increased other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, replicative DNA, but impaired virion and L protein secretion. Highest intracellular L protein was rather achieved by mutants with residual S protein expression or retaining matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to high replication phenotype. Our findings could help explain why such deletions are selected at late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of hepatitis B surface antigen (HBsAg) by wild-type hepatitis B virus (HBV) are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed ability of the 3.5-kb pgRNA to diminish transcription of co-terminal RNAs of 2.4kb, 2.1kb, and 0.7kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here we found that many 3’ preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increased intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, replicative DNA, but lost virion secretion. These findings established biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion.