Hepatitis B virus (HBV) possesses a partially double-stranded DNA genome that encodes four overlapping open reading frames (ORF), including C, X, P, and S . The S-ORF encodes three surface proteins, L, M, and S-protein (HBsAg), through alternative translation initiation (ATI) from three in-framed start codons [6, 15, 17, 18].
Naturally occurring mutations of surface proteins identified by clinical screening have been extensively documented in recent years [1, 3, 7, 14, 19]. However, certain amino acid residues bearing critical function for virus viability are highly conserved and mutations rarely occur among the various HBV genotypes, which makes clinical screening insufficient to address the importance of these highly conserved residues. In this report, site-directed mutagenesis of highly conserved residues in the S-domain revealed two methionines, mutations of which decreased HBV genome replication level without compromising the overlapping p-gene product.
Hepatoma cell line Huh7 cells maintenance and plasmids transfection were performed as previously described .
All HBV genome-derived sequences were cloned using polymerase chain reaction (PCR) from pAdr-1 , sequences of primers used in this study are listed in Table-1.
To introduce mutations into the S-ORF in pHBV1.3 , two-step PCR was performed. The first round of PCR used primer sets 1.3up/R75 and F75/1.3dn, respectively. The subsequent PCR products were mixed in equal molar as template and amplified using primer set 1.3up/1.3dn to generate M75T mutant HBsAg encoding gene, by converting ATG to ACG. The final PCR product was digested with EcoR I and Spe I and replaced the corresponding fragment of pHBV1.3 to generate pHBV1.3-75 (Fig. 1A). Similarly, pHBV1.3-103 was generated with primer sets 1.3up/ R103 and F103/1.3dn (Fig. 1A). pHBV1.3-75-103 bearing M75T and M103T was constructed as pHBV1.3-103, except pHBV1.3-75 was used as initial template (Fig. 1A).
Figure 1. Plasmid constructs. A: pHBV1.3 and derived plasmids encoding M75T or/and M103T within HBsAg. B: pSG and derived plasmids encoding M75T or/and M103T within HBsAg. C: Plasmids expressing truncated HBsAg, in which coding sequences of either 1-74 (pS75) or 1-102 (pS103) AA on N-terminal of HBsAg were removed.
To label HBsAg with EGFP, HBsAg encoding genes were amplified by PCR using primer set Gup/Gdn. The resultant fragment was cloned in frame into pEGFP-N1 (Clontech) between NheI and ApaI, to generate pSG harboring egfp-fused HBsAg (Fig. 1B). To introduce mutations into pSG, site-directed mutagenesis was performed. Plasmids pSG-75, pSG-103, and pSG-75-103, each bearing either M75T, M103T or dual mutations, were generated using similar methods as described above, with primer sets Gup/R75, F75/ Gdn, Gup/R103, and F103/Gdn (Fig. 1B).
Two pcDNA3.1-based (Invitrogen) vectors expressing truncated HBsAg with the HA tag were generated using primer sets 3.1-75/3.1dn and 3.1-103/3.1dn, which spanned HBsAg from M75 (pS75) or M103 (pS103) to the C-terminal, respectively (Fig. 1C).
All the plasmids were verified by sequencing.
HBV replicative intermediates were purified from intracellular core particles of cells transfected with indicated plasmids, and submitted to Southern-blot assay as previously described .
Table 1. Primers used in this study
HBsAg and HBV e antigen (HBeAg) in the supernatant or cell lysates from indicated HBV-bearing plasmids transfected Huh7 cells were analyzed by ELISA kit (Kehua) according to the manufacturer's instructions.
Huh7 cells cultured on coverslips were transfected with indicated plasmids. At 48 hour post transfection (hpt), cells were submitted to fluorescent microscopic assay by using an Olympus IX51 inverted microscope.
1163 HBsAg sequences (genotypes from A to H) were retrieved from the Hepatitis virus database (http://s2as02.genes.nig.ac.jp/), and aligned to pAdr-1. The conservation of ATG start codons in front of each putative ORF on the S-ORF was calculated using a Perl script (available upon request).
Huh7 cells transfected with indicated plasmids were harvested at 48 hpt. 20 μg total cell lysates of each transfected cells were submitted to Western blot with anti-GFP antibody (Sigma).