Fig 5 Immunofluorescence staining selleck inhibitor of HBsAg in cells transfected with wild-type and mutated M88-cl4 sequences. The position and nature of the OBI-specific amino acid substitutions introduced into the genotype B control M88-cl4 are indicated. Several modeling programs predicted very similar arrangements of the S protein in the bilayer lipid membrane of the endoplasmic reticulum (ER). Persson and Argos’ model as well as SOSUI’s and DAS’s identified the same four transmembrane (TM) domains for wild-type S. However, SOSUI predicted a fifth TM domain (amino acids 149 to 171) that we considered doubtful. Nevertheless, the three amino acid substitutions identified as critical to HBsAg excretion had identical locations predicted by the three models.
M75T was located in the cytosolic loop separating TM1 and TM2, Y100S was at the end of TM2, and P178R was in TM3. HBsAg excretion deficit as potential contributor to OBI phenotype. Amino acid substitutions impairing HBsAg excretion reflected by HBsAg quantification in vitro have been identified in individual OBI clones. However, HBVs in HBsAg-positive as well as OBI strains circulate as quasispecies of related variants not necessarily represented by a single clone tested in vitro. The genetic diversity and the relevance of specific mutations as potential explanation for the OBI phenotype were therefore examined by sequencing multiple clones from several donor samples containing pattern 2 clones (Fig. 6). Fig 6 Phylogenetic analysis of the S amino acid sequences of multiple clones from OBI and non-OBI strains.
Phylogenetic analysis was performed as previously described (10). HBV reference sequences of genotypes/subgenotypes A1-3 and B-H are identified by their … The diversity of patterns was shown in one control (M92; two clones with pattern 2 and one clone with pattern 3) and three OBIs: TW0498 (one pattern 2 and one pattern 3), TW8964 (one pattern 1 and one pattern 3) and HK6794 (3 pattern 1, one pattern 2, and one pattern 3) (Fig. 6). In OBI HK6794, the average amino acid diversity between clones was 20 residues (range, 11 to 32 residues) without a significant relationship with the HBsAg phenotype (data not shown). The Y100S mutations responsible for pattern 2 of clone 2 were present in a subcluster of five clones associated with clone 2, but Y100F was present in all 12 other clones.
In contrast, in OBIs HK01556, HK3110, Brefeldin_A and HK3475, genetic diversity was very limited (Fig. 6), and each of the 8 to 12 clones contained Y100S (HK01556) or P178R (HK3110 and HK3475). For these three OBI strains, it is likely that these two mutations shown to prevent HBsAg excretion contribute to the OBI phenotype. DISCUSSION High mutation rates in the HBsAg in OBI strains from various geographical origins have been reported (4, 5, 8, 10).