Mutations associated with occult hepatitis B virus infection result in decreased surface antigen expression in vitro

Abstract

Occult hepatitis B virus (HBV) infection is characterized by the absence of detectable hepatitis B surface antigen (HBsAg) in the serum, despite detectable HBV DNA. Investigations of the mechanisms underlying the development of occult HBV infection are lacking in the current literature, although viral mutations in the surface region, resulting in decreased HBsAg expression or secretion, represent one potential mechanism. Wild-type HBsAg expression vectors were constructed from genotype-matched chronic HBV sequences. Site-directed mutagenesis was then utilized to introduce three genotype A mutations - M103I, K122R and G145A - associated with occult HBV infection in vivo, alone and in combination, into the wild-type HBsAg vectors. Transfection of Huh7 and HepG2 cell lines was performed, and cell culture supernatants and cell lysates were collected over 7 days to assess the effects of these mutations on extracellular and intracellular HBsAg levels. The G145A mutation resulted in significantly decreased extracellular and intracellular HBsAg expression in vitro. The most pronounced reduction in HBsAg expression was observed when all three mutations were present. The mutations evaluated in vitro in the current study resulted in decreased HBsAg expression and potentially increased hepatic retention and/or decreased hepatic secretion of synthesized HBsAg, which could explain the lack of HBsAg detection that is characteristic of occult HBV infection in vivo.

Figure 1

Total ng of HBsAg produced by transfection of pCMV-HA alone, wild-type vector 1 alone or with mutations M103I, K122R, or G145A, were calculated for supernatants (A,C) and cell lysates (B,D) from Huh7 (A,B) and HepG2 (C,D) cells in duplicate and compared at days 3 (grey bars), 5 (white bars), and 7 (black bars) post-transfection. * p<0.05; ** p<0.01; ⁺ p<0.001; ⁺⁺ p<0.0001.

Figure 2

Total ng of HBsAg produced by transfection of pCMV-HA alone, wild-type vector 1 alone or with mutation combinations M103I+K122R, M103I+G145A, K122R+G145A, or M103I+K122R+G145A, were calculated for supernatants (A,C) and cell lysates (B,D) from Huh7 (A,B) and HepG2 (C,D) cells in duplicate and compared for days 3 (grey bars), 5 (white bars), and 7 (black bars) post-transfection. * p<0.05; ** p<0.01; ⁺ p<0.001; ⁺⁺ p<0.0001.