Role of RNA secondary structure in emergence of compartment specific hepatitis B virus immune escape variants

To investigate the role of subgenotype specific RNA secondary structure in the compartment specific selection of hepatitis B virus (HBV) immune escape mutations.

This study was based on the analysis of the specific observation of HBV subgenotype A1 in the serum/plasma, while subgenotype A2 with G145R mutation in the peripheral blood leukocytes (PBLs). Genetic variability found among the two subgenotypes was used for prediction and comparison of the full length pregenomic RNA (pgRNA) secondary structure and base pairings. RNA secondary structures were predicted for 37 °C using the Vienna RNA fold server, using default parameters. Visualization and detailed analysis was done using RNA shapes program.

In this analysis, using similar algorithm and conditions, entirely different pgRNA secondary structures for subgenotype A1 and subgenotype A2 were predicted, suggesting different base pairing patterns within the two subgenotypes of genotype A, specifically, in the HBV genetic region encoding the major hydrophilic loop. We observed that for subgenotype A1 specific pgRNA, nucleotide 358^U base paired with 1738^A and nucleotide 587^G base paired with 607^C. However in sharp contrast, in subgenotype A2 specific pgRNA, nucleotide 358^U was opposite to nucleotide 588^G, while 587^G was opposite to 359^U, hence precluding correct base pairing and thereby lesser stability of the stem structure. When the nucleotides at 358^U and 587^G were replaced with 358^C and 587^A respectively (as observed specifically in the PBL associated A2 sequences), these nucleotides base paired correctly with 588^G and 359^U, respectively.

The results of this study show that compartment specific mutations are associated with HBV subgenotype specific alterations in base pairing of the pgRNA, leading to compartment specific selection and preponderance of specific HBV subgenotype with unique mutational pattern.