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

Abstract

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

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Compartmentalization; G145R; Hepatitis B; Peripheral blood leukocytes; RNA secondary structure; pgRNA.

Figure 1

Comparative diagram showing the differences between different aspects of the predicted secondary structures of the pgRNA, specific for HBV subgenotype A1 and subgenotype A2. A: Predicted minimum free energy (mfe) structures, coloured by base-pairing probabilities (according to the rainbow scale shown in the middle, denoting base pair probabilities from 0 to 1). Colour of the unpaired regions denotes the probability of being unpaired; B: Mountain plot representing the mfe structure (red line), the thermodynamic ensemble of RNA structures (green line), and the centroid structure (blue line). Positional entropy for each position is presented below the mountain plot; C: Dot-plot showing the base-pairing probabilities of the two predictions.

Figure 2

Diagram showing the genetic variability and the part of the predicted mfe structure, corresponding to the genetic region encoding the major hydrophilic loop of subgenotype A1. A: Consensus nucleotide sequence (corresponding to positions 341 to 660 of the HBV genome) and predicted amino acid sequence (corresponding to residues 63 to 168 of the HBsAg). Subgenotype A1 specific nucleotides (505^C, 514^C, 616^A and 619^T) and two variable nucleotides (358^T and 587^G) are indicated by arrowheads; B: Detailed base pairing pattern of the mfe pgRNA structure, specific for subgenotype A1. Aforementioned variable sites are encircled by pink circles and indicated by arrows and numbers correspond to their nucleotide position in the HBV genome. HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen.

Figure 3

Diagram showing the genetic variability and the part of the predicted mfe structure, corresponding to the genetic region encoding the major hydrophilic loop of subgenotype A2. A: Consensus nucleotide sequence (corresponding to positions 341 to 660 of the HBV genome) and predicted amino acid sequence (corresponding to residues 63 to 168 of the HBsAg). Subgenotype A2 specific nucleotides (505^T, 514^A, 616^G and 619^C) and two co-evolving nucleotides (358^C and 587^A) are indicated by arrowheads; B: Detailed base pairing pattern of the mfe pgRNA structure, specific for subgenotype A2. Aforementioned variable sites are encircled by pink circles and indicated by arrows and numbers correspond to their nucleotide position in the HBV genome. Part of the mfe structure is amplified in the inset for better visualization of the base pairing. HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen.