In-silico evaluation of phytochemicals against HBX protein of Hepatitis B virus and devising a CRISPR/Cas9 based strategy against the HBx gene in HBV genome

Abstract

Approximately 250 million people in the world are HBV infected. Antivirals including nucleotide analogues suppress viral replication and transcription which acts only on viral replication and transcription but are unable to eliminate virus from infected host cells. HBV genome resides in the nuclei of infected cells, making it persistent. The reason behind this persistence is the stability of a viral genome form, “covalently closed circular DNA”. This remains episomal in the host cell nucleus and serves as a template for the viral transcription. Almost all viral proteins play specific roles in promoting viral induced hepatocellular carcinoma (HCC). Hepatitis B virus X protein is involved in the unwinding of cccDNA and recruits transcription remodeling factors for regulating the cccDNA epigenetically. In this study, we screened a library of phytochemicals using in-silico approach. The HBX protein structure was docked with the library and those having better scores were selected. The protein-ligand interactions were determined on MOE software. Molecular dynamic simulations were performed using Schrödinger tool. CRISPR/Cas9 strategy was designed to target HBx at gene level. In silico small-guide RNA was designed and cloned in the selected mammalian expression vector. The expression vector was then propagated in bacteria using heat-shock and electroporation method. The results showed that three of the phytochemicals showed high binding affinities than the thresholds with the HBX protein structure. They showed no violation to Lipinski’s rule of 5. Arbutin and Boschnaloside have showed promising results against HBX protein. In-silico designing of sgRNA has no off-target effects and aims to target specifically HBx gene. CRISPR/Cas9 technique may found its way for the elimination of cccDNA from the host cell nucleus. In conclusion, cccDNA possibly be eliminated by targeting HBX protein.