The HBx protein contains regulatory (or negative regulatory) and transactivation (orcoactivation) domains (Fig. 1B). The regulatory domain (1 to 50 aa) is dispensable for HBx activity and represses HBx transactivation activity (18). Consensus sequence analysis of regulatory domain shows that region 1M to 20P is highly conserved, while the region 31S to 40P is variable, except 32G and 35G residues which are highly conserved among all HBV genotypes.
The transactivation domain (51 to 154 aa) is essential for augmentation effects on HBV transcription and replication (19). The region 52H to 65S amino acids is critical for augmentation effect in HBV replication (19). Consensus analysis shows that this HLSLRGLPVCAFSSmotif is highly conserved among all HBV genotypes. Deletion of 141L to 154A (last 14) amino acids of transactivation domain did not affect transactivation property (20). It has been demonstrated that 132F to 140K and 137C residues are crucial for transactivation of HBx (20). The consensus sequence analysis shows that this FVLGGCRHK motif and 137C residue are completely conserved among all HBV genotypes. It could be inferred that designing anti-HBV siRNA or inhibitor of these region might target HBx from all HBV genotypes. However a natural mutant of HBx (HBxDelta127) without FVLGGCRHK motif and 137C, has been reported from chronichepatitis B, liver cirrhosis and HCC patients, which can induce growth and proliferation of hepatoma cells (21, 22). The HBx protein, due to presence of BH3-like motif (110A to 135G) in the C-terminal region, directly associates with anti-apoptotic Bcl-2 family proteins and induce elevated cytosolic calcium levels and promote viral DNA replication (23–25). Consensus sequence analysis showed 111Y, 113K to 115C, 117F,120W to 122E, and 132F to 135G residues were completely conserved in BH3-like motif of HBx in all HBV genotypes. Among 13 XaaP motifs in HBx protein, 10D11P, 19R20P, 28R29P, 45V46P in regulatory domain and 58L59P, 67G68P, 89L90Pmotifs in transactivation domain were completely conserved in all HBV genotypes.
The HBc protein is composed of N-terminal (1 to 140 aa), linker (141 to 149 aa), and C-terminal (150 to 183 or 185 aa depending on the strains) domains (26)(Fig. 3B). The N-terminal domain (NTD) is critical and sufficient for capsid assembly (27, 28). Consensus sequence analysis of CTD shows that region 1M to 11A of all HBV genotypesis completely conserved, except genotype G that contains additional 12 amino acids RTTLPYGLFGLD insertion. This insertion has pleiotropic effects on core protein expression, HBV replication, and virion secretion (29). Insertion of RTTLPYGLFGLD motif enhanced core protein levels independent of viral genotype, augments replication in genotype G, while impairs replication in genotype A and D (Gutelius et al., 2011).The region 13V to 39R (or 25V to 51R of genotype G) is highly conserved among all HBV genotypes. The NTD carries proteaselike sequence 30L to 35S (or 42L to 47Sof genotype G) which resembles to retroviral proteases (30). Consensuses sequence analysis shows that this LLDTAS motif is highly conserved among all HBV genotypes. The region between 74 to 101 amino acids is considered as hypervariable which might lead to development of liver injury (31, 32). Consensuses sequence analysis also shows that 74X and 179X residues are most variable among all HBV genotypes.
The linker domain STLPETTVV can interfere with NTD, pgRNA packaging in sequence-independent manner, viral DNA synthesis in sequence independent manner (during first step of reverse transcription to initiate single strand DNA) and in sequence dependent manner (during second step of reverse transcription that is extensive plus strand DNA synthesis to generate relaxed circular DNA), and virion secretion in sequence dependent manner (26). Presence of only five amino acids ETTVV in linker region were sufficient to generate single stranded DNA synthesis (26). The consensus sequence analysis indicates that linker STLPETTVV region is completely conserved among all HBV genotypes, except genotype E which contains STLPENTVV. The four cysteines residues at position 48, 61, 107 and 183 are not essential for core particle formation, however these residues can further stabilize HBV core particles or HBc dimers (33). The consensus sequence analysis shows that all of these cysteine residues are 100% conserved among all HBV genotypes. The 132Y, 127R, 129P, and 139I are critical for HBc dimer formation (34). Consensus sequence analysis indicate that these residues are completely conserved among all HBV genotypes. The amino acid regions between 98R to 115V and 117E to 145E are 100% conserved among all HBV genotypes, indicating potential of this region for the HBV life cycle and/or viral pathogenesis.
The C-terminal domain (CTD) contains highly basic residues (arginine rich, protamine-like) that resemble to histone tails, which are critical for non-specific nucleic acid binding (35, 36). The CTD is dispensable for capsid assembly and functionally plays important role in pgRNA packaging and reverse transcription (37–39). The CTD phosphorylation is important for specific viral RNA packaging (40–42). The three major phosphorylation sites in CTD are 155S, 162S, 170S, while four minor sites are 160T, 168S, 176S and 178S (42–45). The consensus sequence analysis shows that except genotype H (which contains 155A instead of 155S), all of the aforementioned phosphorylation sites in CTD are 100% conserved among all HBV genotypes. The arginine-rich CTD domain contains distinct nuclear localization and cytoplasmic retention signals (46). Major RNA-recognizing activity of CTD is attributed to 150R to 157R sequence (47). The RRRR following 149V also provide RNA binding (35). Consensus sequence analysis shows that this RRRGRSPRmotif is highly conserved among all HBV genotypes. In HBV genotype A between amino acids 152R and 153G, there exists two additional amino acids DR, therefore generating RRRDRGRSPRmotif. The DNA-recognizing activity of CTD is attributed to three repeated SPRRR motifs within 157 to 177 amino acid sequence. Consensus sequence analysis shows that these motifs are completely conserved among all HBV genotypes, except genotype H which contains 155A. Among 16XaaP motifs in HBc protein, 4D5P, 19L20P, 24F25P, 78D79P, 128T129P, 129P130P, 133R134P,134P135P, and137A138P in NTD,143L144P in linker domain, and160T161P, 162S163P, and 170S171Pmotifs in CTD were 100% conserved among all HBV genotypes.
Recent findings have implicated active roles of HBc and HBx in epigenetic regulation of viral-host interplay (9, 48). The multiplicity of HBx and HBc functions and their capacity to influence cccDNA mini-chromosome for enhanced viral replication, elevates these proteins as excellent targets for antiviral therapeutics. (9, 48). The HBx and HBc consensus sequences were used to predict highly conserved B and T cell binding epitopes. Several highly or semi-conserved B cell binding epitopes were predicted, we selected highly conserved epitopes with 70–100% conservation among all ten HBV genotypes. Similarly, several MHC-I or -II related epitopes exhibited maximum allele-binding affinity indicating as possible T-cell related epitopes. Among HBx related B-cell binding epitopes, due to complete (100%) conservancy among all HBV genotypes, the X-B2 and X-B4 epitopes mightbe consider as better targets for B-cell based vaccine development. Similarly among HBc related B-cell binding epitopes, due to high (80%) conservancy among all HBV genotypes, the C-B6 and C-B7 epitopes might be better targets for B-cell based vaccine development. On the other hand, among HBx related MHC-I specific epitopes the X-M2, X-M5, X-M8, X-M11, X-M12, X-M20, X-M22, X-M25, X-M27 and X-M32; whileamong MHC-II specific epitopes, the X-T4, X-T6 and X-T8 could be adopted for synthetic vaccine against multi-genotypes of HBV. Similarly for HBc related MHC-I specific epitopes the C-M1, C-M2,C-M4, C-M6-11, C-M13,C-M19, C-M24-26,C-M30, C-M34-36,C-M40 and C-M43-45; while among MHC-II specific epitopes, the C-T1-3, C-T9, C-T10, C-T12, C-T14 and C-T16-18 epitopes could be ideal epitopes with high conservancy across all HBV genotypes. The use of conserved epitopes predicted against NS3-4A from global consensus sequences could provide broader protection against multi-isotypes of hepatitis C virus (49). Our study suggests conserved epitopes against HBx and HBc global consensus sequences that may be invoked as potential targets for development of effective vaccine candidates and conserved residues could also be attributed for designing novel site specific anti-HBV agents which can target all major genotypes of HBV. Though present study indicates B-cell or T-cell related antigens on the basis of in-silico analysis, the antigenic potential of aforementioned peptides should be further characterized in HBV infection animal models.