Lost small envelope protein expression from naturally occurring preS1 deletion mutants of hepatitis B virus is often accompanied by increased HBx and core protein expression as well as genome replication.

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.IMPORTANCEExpression 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.