Transcriptome Analysis of Hepatitis C Virus-Infected Hepatoma Cells Following Stringent Removal of Genomic DNA

Abstract

Hepatitis C Virus (HCV) is a positive strand RNA virus that infects hepatocytes and reprograms cellular gene expression, contributing to chronic liver disease and hepatocellular carcinoma. HCV replicates slowly and induces limited changes in the cellular transcriptome. Here, we improved analysis of low-abundance transcripts, including long non-coding RNAs (lncRNAs), in the context of HCV replication. We stringently removed genomic DNA from total RNA from Huh-7 hepatoma cell lines by a combination of TRIzol, acidic phenol, silica matrix columns and recombinant DNase I. RNA sequencing (RNA-Seq) and targeted validation were used to characterize Lnc-ITM2C-1 splice variants and expression of its neighboring gene, G protein-coupled receptor 55 (GPR55), to investigate their potential roles in HCV replication. Challenging our previous findings, under our improved experimental conditions, Lnc-ITM2C-1 and GPR55 were neither involved in a positive regulation circuit with HCV nor induced detectable transcriptome changes. Secondary analysis of RNA-Seq data according to HCV infection status revealed statistically robust HCV-induced expression of 21 host cell genes involved in pathways like stress response (in particular ATF3), lipid metabolism, cytoskeleton organization and growth regulation. In contrast, we found that expression changed for only a limited subset of previously reported HCV-associated lncRNAs (e.g., EGOT). This highlights the necessity of stringent gDNA removal for analysis of low-abundance transcripts like lncRNAs.

The authors' abstract, as published at the source. Viruses, 2026 · DOI ↗