Impact of length of replication competent genome of hepatitis B virus over the differential antigenic secretion

Abstract

Hepatitis B virus (HBV) genome consists of circular partially double stranded DNA of 3.2 kb size which gets converted into covalently closed circular DNA (cccDNA) during its life cycle. It then acts as a template for formation of pregenomicRNA (pgRNA) of 3.5 kb. Absence of appropriate animal models prompted a need to establish a better in vitro culture system to uncover the propagation and survival mechanisms of the virus. There is scarcity of data to represent the significance of varying length of replication competent viral genome on the secretion of viral secretory proteins/antigens and in turn on the overall effects on the accomplishment of the viral life cycle. The present study was undertaken to ascertain a suitable replication competent construct in which the viral life cycle of HBV with varying clinical relevance can be studied efficiently. Two constructs (pHBV 1.3 and pHBV 1X) of different sizes were used to transfect hepatoma cells and consequently the secretory antigens were monitored. In vector free approach (pHBV 1X), 3.2 kb viral DNA is directly transfected in the culture system whereas in vector mediated approach more than full length of viral genome is cloned in a vector (pHBV 1.3X) and transfected to obtain a 3.5 kb pgRNA intermediate. HBV secretes two important antigens; HBsAg and HBeAg. HBsAg is a hallmark of infection and is the first to be secreted in the blood stream whereas HBeAg is a secretory protein and remains associated with the viral replication. The construct pHBV 1.3X referring to as more than full length, by virtue of being capable of undergoing transcription without the synthesis of cccDNA intermediate (unlike the clinical situation where an intermediate step of cccDNA synthesis is an essential component to initiate the viral life cycle) appears to be better system for studying viral life cycle in in vitro culture system. The reasons could be assigned to the fact that as low as 100 ng of viral DNA was shown to quantify the replicative phenotypes with this construct. The better efficiency of this construct at prima facie, appears to be mediated through the significantly higher levels of pgRNA transcript during the viral life cycle.

Biological Characterization of Hepatitis B virus Genotypes: Their Role in Viral Replication and Antigen Expression

“…Even though this is a more robust system, promoting high levels of HBV replication and protein expression, the enhancer I/II and the Core promoter regions that regulate the transcription of pgRNA and Precore mRNA, are duplicated. In addition, more than unit-length HBV constructs are directly transcribed in the cell nucleus regardless of the formation of cccDNA ( Amir et al, 2019 ). Therefore, these features may alter the biological properties of the constructs, which is unsuitable for evaluating differences in HBV replication among genotypes.…”

“…However, in these constructs, the complete HBx open reading frame and the enhancer I/II regions are duplicated. In addition, more than unit-length HBV constructs are directly transcribed in the cell nucleus bypassing the formation of cccDNA replicative intermediate ( Amir et al, 2019 ). Hence, the utilization of these constructs may not be ideal to study differences in HBV replication and protein expression among genotypes.…”

A novel piperazine derivative that targets hepatitis B surface antigen effectively inhibits tenofovir resistant hepatitis B virus

“…The wild-type and lamivudine resistant mutant (rtM204I) 1.3 × HBV plasmids (genotype D) were kindly provided by Dr. Syed Naqui Kazim (Jamia Millia Islamia, India) 73 . The tenofovir resistant CYEI quadruple mutant (genotype C) 4 was a kind gift from Prof. Kyun-Hwan (Konkuk University, South Korea).…”

MicroRNA‑1271‑5p alleviates the malignant development of hepatitis B virus‑mediated liver cancer via binding to AQP5

“…Compared with that of the general population, chronic hepatitis B virus (HBV) carriers have a higher risk of contracting HCC, and HBV is the most direct cause of HCC (3). The HBV DNA molecule, which is a circular double-stranded DNA containing part of a single-stranded region, transforms into covalently closed circular (ccc)DNA during its life cycle (4). As a marker of HBV infection, hepatitis B e-antigen (HBeAg) positivity is associated with high viral loads, which further leads to high expression levels of hepatitis B surface antigen (HBsAg) (5).…”