Biological implications of hepatitis B virus mutations

MBAYED, V.; FLICHMAN, D.; TORRES, C.; PEZZANO, S.; TADEY, L; CAMPOS, R.

Buenos Aires, Argentina

Simposio; 2nd Symposium IMBS (International Master in Biomedical Sciences); 2009

Background Hepatitis B virus (HBV) is a globally distributed human pathogen. Over 75% of the world's population lives in areas where the levels of infections are high. It is estimated that more than 2 billion people have been infected with HBV, and about 350 million live with chronic infection. HBV has a partially double-stranded DNA genome of around 3200 nt, it replicates through a retrotranscription step and contains four partially overlapping open reading frames. This virus diverged in the eight genotypes (A-H) described so far that show a distinctive geographic distribution. Genotypes F and H are thought to be indigenous to America since they were found in the aboriginal population, mainly in Central and South America. Genotype F isolates from different geographical regions were classified into four subgenotypes (F1-F4). During the course of chronic infection a large proportion of HBV surface antigen (HBsAg) carriers seroconvert from HBeAg to anti-HBe which represents a late phase in the natural history of chronic infection. The study of viral mutations has become a fashionable issue to address many questions regarding the relations between the major HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection, specifically during HBeAg seroconversion. Aim The aim of our work was to analyze mutations along the viral genome that might display different biological implications a) mutations affecting HBeAg expression, b) genetic differences involved in transcription regulation of the pregenomic/precore RNA. Material and Methods Patients This retrospective study includes: 70 untreated HBeAg negative/anti-HBe positive chronic HBsAg carriers followed up at least three times yearly; 10 patients with acute symptomatic self-limited infection (AH) by HBV genotype F whose sera were obtained at 15-day intervals from the day of beginning of symptoms, before and after HBeAg seroconversion. Sequencing and genotyping PCR products covering the S and the Basic Core Promoter (BCP) and Precore (pC) regions were purified and direct sequences were obtained. Phylogenetic analysis of the S region was performed in order to determine the genotype of the samples. Mutations associated with the HBeAg seroconversion were characterized in the BCP/pC region. Analysis of transcription factors binding sites: The nucleotide sequences of subgenotypes F1 and F4 were studied by in silico analysis with MatInspector programme (TRANSFAC database) in order to characterize the transcription factors binding sites. Result 1. Mutations associated with HBeAg seroconversion: On the chronic HBeAg negative patients substitutions abrogating the protein expression were observed in most of the cases, being the most prevalent A1762T, G1764A and G1896A. Other includes: C1817T (n=1), T1847A (n=2), G1897A (n=1). Nucleotide insertion in 1846 (n=4), in 1939 (n=3) and 1839, 1845 and 1847 in one case each; deletion on nucleotide 1845 (n=1). On the other hand, in the acute infections none of these mutations were observed during the HBeAg seroconversion process. Result 2. Binding of transcription factors to regulatory regions (in silico analysis): Consensus sequence was obtained for each subgenotype (F1 and F4) and regions involved in the regulation of transcription (nt 1580-1900) of the ARN (pC and Pregenomic) were studied. Specific features of subgenotype are independent of HBeAg status. Different binding sites of transcription factors were found for each subgenotype. Discussion 1- Mutations on the BCP/pC region are the hallmark of chronic HBeAg negative/anti-HBe positive HBV infection. In this study, mutations affecting the HBeAg expression occurred in the great majority of the individuals (92.9%). Mutations affecting HBeAg expression were biased by the genotype. In particular, in the Pc region : G1896A was rare on genotype A, intermediate on genotype F1b and rather high on genotypes D and F4 while other substitutions, insertions and deletions affecting the HBeAg expression were more prevalent in genotypes A and F1b. It is well established that the G1896A mutation is favoured on genotypes showing Timidine at position 1858 (B, C2, D, E, F1 and F4) since stabilizes the encapsidation signal and potentially stimulates viral replication. Nevertheless, the mechanism followed by subgenotype F1b to regulate HBeAg expression is not fully explained by the structure of the encapsidation signal. 2- On the other hand, among the acute infections we did not find changes in the viral genomes that could be directly linked to a molecular base for the seroconversion event. This reinforces the role of the host's immune response in the control of the viral replication and therefore the protein expression in acute self-limited infections. 3- The genetic region involved in the regulation of pC and pregenomic RNA transcription showed subgenotype specificity, suggesting that different regulatory mechanisms might be acting according to the subgenotype, that eventually may have clinical impact.