Hepatitis B Virus X Gene Differentially Modulates Subgenotype F1b and F4 Replication

Abstract

Hepatitis B virus (HBV) is classified into ten genotypes and numerous subgenotypes (sgt). In particular, sgt F1b and sgt F4, native of Latin America, have been associated with differences in clinical and virological characteristics. Hepatitis B virus X protein (HBx) is a multifunctional regulatory protein associated with the modulation of viral transcription and replication. In this work, we analyzed the role of the X gene and the encoded X protein in sgtF1b and sgtF4 replication. Transfection with HBx deficient genomes revealed remarkable differences in the replicative capacity of sgtF1b and sgtF4 mutants. The silencing of HBx increased sgtF1b X(-) transcription and replication by more than 2.5 fold compared to the wild type variant, while it decreased sgtF4 X(-) transcription and replication by more than 3 fold. Trans-complementation of HBx restore sgtF1b and sgtF4 wild type transcription and replication levels. In addition, transfection with chimeric variants, carrying wild type (F1b/XF4 and F4/XF1b) or mutated (F1b/X(-)F4 and F4/X(-)F1b) X gene of one sgt in the backbone of the other sgt, showed that the nucleotide sequence of the X gene, that includes regulatory elements that modulate pgRNA transcription, was responsible for the disparity observed between sgtF1b X(-) and sgtF4 X(-). These results showed that sgtF1b and sgtF4 X gene play a central role in regulating HBV transcription and replication, which eventually lead to a common purpose, to reach wild type replication levels of sgtF1b and sgtF4 viruses.

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

“…In contrast, other studies have shown higher replication rates for genotypes A2, C2, and D3 compared to genotype B2 ( Sozzi et al, 2016 ) or higher rates for genotype C compared to genotypes B2 and A2 ( Sugiyama et al, 2006 ) following transfection of HuH-7 cells with 1.3- or 1.24-fold copy of the HBV genome, respectively. These controversial results might be a consequence of the different experimental systems used in the studies, as well as the subgenotypes utilized, since dissimilar biological characteristics have been described even between subgenotypes of the same genotype ( Bhoola and Kramvis, 2016 ; Khatun et al, 2018 ; Elizalde et al, 2019 ).…”

“…As previously described, linear HBV monomers were excised from the plasmids by restriction enzyme digestion with 5U of Bsp QI (New England Biolabs, Beverly, MA, United States) at 50°C. The 3.2kb fragments were gel purified with PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s instructions, and the DNA was quantified by spectrometry ( Elizalde et al, 2019 ). In order to mimic viral variability, a mix of 10 to 20 clones (pUC19-full-length HBV genomes) of each genotype were used in all experiments.…”

“…The following primers were used for the amplification: sense 5’-ATGGAGACCACCGTGAACGC-3′ (nt 1608–1627) and antisense 5′-AGGCACAGCTTG GTGGCTTG-3′ (nt 1887–1868). To avoid amplification of the input linear HBV DNA used for transfection, primers that specifically amplify relaxed circular HBV DNA were used ( Elizalde et al, 2019 ). Serial dilutions of an HBV replication-competent plasmid (pCH-9/3091) were used as quantification standards.…”