The effect of the G1888A mutation of subgenotype A1 of hepatitis B virus on the translation of the core protein

Abstract

A distinctive characteristic of subgenotype A1 of hepatitis B virus is G1888A in the precore region. This transition introduces an out-of-frame AUG, creating an overlapping upstream open reading frame (uORF), terminating five nucleotides downstream from the core AUG. This uORF can potentially be translated into a seven amino acid peptide. In addition to stabilizing the encapsidation signal by forming a base pair with T1871, this mutation may affect translation of the core protein. The aim of this study was to use reporter constructs to determine whether G1888A had any modulating effect on core protein translation. The complete core gene with part of the precore of subgenotype A1 was cloned into the amino terminal of a green fluorescent protein (GFP) plasmid. Core/GFP fusion protein expression was measured using flow cytometry following transfection of Huh 7 cells. The introduction of uORF resulted in an 18.75% reduction of core gene expression. When the suboptimal Kozak sequence of the 1888 AUG was replaced with an optimal one, this reduction was enhanced (64.84%). By increasing the distance between the stop of the overlapping uORF and the core AUG, by a minimum of 15 nucleotides, core/GFP expression was almost doubled, indicating that stalling of ribosomes at the stop of the uORF may be interfering with initiation at the core AUG through steric hindrance. Our findings indicate that the G1888A mutation, may interfere with initiation at the downstream 1901 core AUG, decreasing core protein translation. This decrease may account for the relatively low viral loads seen in individuals infected with subgenotype A1.