Specific amino acid substitutions in the S protein prevent its excretion in vitro and may contribute to occult hepatitis B virus infection

Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the pre-seroconversion window period. The mechanisms leading to OBI remain largely unknown. The potential role of specific amino acid substitutions in the S protein from OBI on HBsAg production and excretion was examined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfected with plasmids containing the S gene of eight HBsAg+ controls and 18 OBI clones. The intracellular (IC)/extracellular (EC) HBsAg production ratio was ∼1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strains clones: pattern 1 defined as IC/EC ratio 1.0 in 5/18 OBI clones; pattern 2 with detectable IC but low or undetectable EC HBsAg (IC/EC: 7.0-800) in 6/18 OBIs; and pattern 3 with both low or undetectable IC and EC HBsAg in 7/18 clones. Intracellular immunofluorescence staining showed that, in pattern 2, HBsAg was concentrated around the nucleus suggesting retention in the endoplasmic reticulum. Substitutions M75T, Y100S or P178R were present in 4/6 pattern 2 OBI clones. Site-directed mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introduced in a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies expressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of two OBI strains may contribute significantly to the OBI phenotype