Published online May 28, 2020. doi: 10.3748/wjg.v26.i20.2584
Peer-review started: February 28, 2020
First decision: April 9, 2020
Revised: May 8, 2020
Accepted: May 19, 2020
Article in press: May 19, 2020
Published online: May 28, 2020
Despite the existence of effective preventive vaccines, an estimated 257 million people worldwide live with chronic hepatitus B virus (HBV) infection and more than 880000 people die due to the development of liver cirrhosis and/or hepatocellular carcinoma. Although infection can be controlled with existing treatment, eradication is currently impossible due to the persistence of covalently closed circular DNA in hepatocyte nuclei that acts as a template for viral expression. New therapeutic approaches are needed, and gene therapy has been proposed as one of the most promising options. HBV core protein [encoded by the HBV core gene (HBC)] is a structural protein with functional activity that has a key role in viral replication and disease progression. Accordingly, it could be a potential target for new therapeutic and diagnostic strategies, and its variability could be a valuable prognostic factor for disease progression.
As eradication of HBV infection is currently unachievable, new therapeutic strategies are necessary. Moreover, current treatments cannot interfere with the expression of viral proteins that can favor disease progression. Gene therapy based on silencing RNA is one of the most promising therapeutic approaches currently under investigation. The identification of hyper-conserved regions in key viral genes and proteins (such as HBC) is essential to orchestrate an effective strategy regardless of clinical stage or viral genotype.
This study aimed to identify, by next-generation sequencing, hyper-conserved regions in HBC quasispecies of patients with different clinical stages of chronic HBV infection that could be a valuable target for gene therapy. Considering the essential role of the HBC gene and its encoded protein HBV core protein in HBV infection, changes in gene and protein conservation in specific clinical groups could be determining factors in disease progression and hence serve as prognostic factors for clinical follow-up.
The HBC gene was amplified by a 3-nested PCR protocol and later sequenced by next-generation sequencing (MiSeq, Illumina, United States) in 38 HBV-monoinfected chronic patients [16 with chronic hepatitis B infection without liver damages (CHB group), 5 with liver cirrhosis (LC group) and 17 with hepatocellular carcinoma (HCC group)]. Quasispecies sequences were genotyped by distance-based discriminant analysis, and general and intergroup nucleotide (nt) and amino acid (aa) conservation was determined by sliding window analysis. The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with a genotype-specific consensus sequence.
Three nt (nt 1900-1929, 2249-2284, 2364-2398) and two aa (aa 117-120, 159-167) hyper-conserved regions shared by all the clinical groups were identified. By comparing gene and protein conservation between the different clinical groups, a similar pattern of conservation was observed, although CHB showed five nt less conserved regions (nt 1946-1992, 2060-2095, 2145-2175, 2230-2250, 2270-2293) and LC one aa less conserved region (between aa 140 and 160). Moreover, some group-specific conserved regions were detected at both nt (nt 2306-2334 in CHB and 1935-1976 and 2402-2435 in LC) and aa (aa 98-103 in CHB and 28-30 and 51-54 in LC) levels. No differences in indel frequency were observed between the clinical groups. Contrarily, we identified an aa substitution (P79Q) that was more frequent in HCC [median (interquartile range) frequency of 15.82 (0-78.9) vs 0 (0-0) for the other groups; P < 0.05 vs the CHB group].
We have identified a number of nt and aa regions that were highly conserved in the presence of different viral genotypes and clinical stages. These could be valuable targets for future pangenotypic and panclinical therapeutic and diagnostic strategies. The different clinically related conserved regions and the P79Q aa substitution could potentially be used as prognostic factors for disease progression.
Our findings could guide the creation of a new gene therapy strategy based on RNA silencing. In-depth analysis of group-specific conserved or variable regions and their role in disease progression is needed. Further in vitro studies are required to determine whether the P79Q aa substitution might affect viral replication and to investigate associations between cell damage and onset of HCC.