Hepatitis B virus polymerase suppresses translation of pregenomic RNA via a mechanism involving its interaction with 5′ stem–loop structure

Abstract

The pregenomic RNA (pgRNA) of hepadnaviruses serves a dual role: as mRNA for the core (C) and polymerase (P) synthesis and as an RNA template for viral genome replication. A question arises as to how these two roles are regulated. We hypothesized that the P protein could suppress translation of the pgRNA via its interaction with 5' stem-loop structure (epsilon or encapsidation signal). Consistent with the hypothesis, we observed up-regulation of the C protein level in the absence of the P protein expression in a physiological context. Importantly, translational suppression depended on the 5' epsilon sequence. Furthermore, the impact of the P protein on ongoing translation of the C ORF was directly demonstrated by polysome distribution analysis. We conclude that the P protein suppresses translation of the pgRNA via a mechanism involving its interaction with the 5' epsilon sequence, a finding that implicates the coordinated switch from translation to genome replication.

Incorporation of Eukaryotic Translation Initiation Factor eIF4E into Viral Nucleocapsids via Interaction with Hepatitis B Virus Polymerase

“…The HBV Pol expression construct, from which HBV Pol with three copies of the Flag epitope at N terminus is expressed, was described previously (19). The pgRNA expression plasmid was derived from the pCMV-HBV/30 plasmid, the wild-type HBV replicon construct, from which both the C (core) open reading frame and the (Pol) open reading frame were inactivated by an introduction of a stop codon and a frameshift mutation, respectively, as described previously (24).…”

“…It is not clear, however, whether and how the dual functions of the pgRNA are coordinated. Recently, we demonstrated that HBV Pol directs genome replication by suppressing the translation of the pgRNA (19). Therefore, we speculated that the translation suppression of the pgRNA could precede encapsidation.…”

“…Importantly, the recognition of the 5Ј stem-loop structure ( Fig. 1), called the encapsidation signal (ε or epsilon), by HBV Pol is critical for the translation suppression (19). In addition, the ability of Pol to specifically recognize the 5Ј ε also is critical for the initiation of viral reverse transcription as well as the assembly of replication-competent nucleocapsids (11,15,23).…”

“…The epsilon sequence, initially defined as the RNA packaging signal that directs the specific encapsidation of the pgRNA into nucleocapsids, subsequently was found to be the origin of reverse transcription as well (20). Taking these findings together, the binding of Pol to ε thus triggers three critical steps in hepadnaviral replication, (i) the initiation of reverse transcription (23), (ii) translation suppression (19), and (iii) nucleocapsid assembly (11,15), leading to the selective incorporation of both Pol and pgRNA into the viral nucleocapsids.…”

Genetic dissection of naturally occurring basal core promoter mutations of hepatitis B virus reveals a silent phenotype in the overlapping X gene

“…HepG2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (Gibco-BRL) and 10 mg gentamicin ml 21 at 37 uC in 5 % CO 2 and passaged every third day. Cells were transfected using polyethylenimine (PEI; 25 kDa; Sigma-Aldrich) as described previously (Ryu et al, 2008). Briefly, cells were plated at 40-50 % confluency.…”