Synergistic impact of mutations in Hepatitis B Virus genome contribute to its occult phenotype in chronic Hepatitis C Virus carriers

We characterized occult HBV (OHBV) from hepatitis B surface antigen (HBsAg)-negative chronic HCV carriers of Eastern India to explore the impact of genomic variability of HBV in causing undetectability of HBsAg and low viremia that define the occult phenomenon. Screening of sera samples revealed the presence of OHBV in 17.8% of HCV-infected patients. Determination of full-length OHBV sequences and comparison with that from HBsAg-positive carriers led to the detection of distinct substitutions/mutations in PreS2, S, P and X ORFs and in X-promoter and Enhancer-II of OHBV. These mutations were introduced in wild-type HBV and their effects were evaluated by transfection in Huh7 cells. In vitro assays demonstrated that S-substitutions resulted in antigenically modified HBsAg that escaped detection by immunoassays whereas those in ORF-P caused significant decline in viral replication. Impairment in Enhancer-II and X-promoter activities were noted due to occult-associated mutations that generated reduced pregenomic RNA and intracellular HBV-DNA. Additionally, Enhancer-II mutations altered the small to large surface protein ratio and diminished extracellular HBV-DNA and HBsAg secretion. Further, mutations in PreS2, X and enhancer-II increased Grp78-promoter activity, suggesting that OHBV could trigger endoplasmic reticulum stress. Thus viral mutations contribute synergistically towards the genesis of occult phenotype and disease progression.