Characterization of 5'-human-HBV-3' RNA species transcribed from integrated HBV DNA - Abstract About 296 million people worldwide are chronically infected with hepatitis B virus (HBV). Chronic HBV infection is the main risk factor of hepatocellular carcinoma (HCC). There is no cure for HBV, and FDA-approved anti- HBV drugs failed to achieve much desired loss of serum HBV surface antigen (HBsAg) in most cases. Mechanisms of HBV infection/pathogenesis are not fully understood, which affects therapy options. During HBV replication, in 5-20% of cases, the RNA primer is not translocated during (+) strand DNA synthesis, priming in situ occurs, and the reverse transcription produces not the usual relaxed circular DNA (rcDNA) but double stranded DNA linear genome (DSL) that can be randomly integrated into cellular DNA brakes using help of host DNA repair enzymes. Over time many hepatocytes acquire HBV integrants. A progeny virus is not produced from the integrants, but different RNA species are transcribed from the integrants. They are under- studied. Among them are 5'-human-HBV-3' hybrid RNAs that are initiated from cellular promoter upstream of the integrant and then crossed into the integrated HBV sequence. We found such RNAs in liver/HCC tissues and sera harvested from chronic HBV carriers and in cell lines that were generated from HBV-related HCCs and bear HBV integrants. Not much is known about these RNAs. Our recent data gained using a small number of samples and doing long-read RNA sequencing (long-read RNA-seq) suggested that such RNAs (i) are common; (ii) can accumulate to sizeable levels; (iii) are 5'-human-HBV-3' RNAs and are not 5'-human-HBV- human-3' RNAs; (iv) have the length of about 360 to 3800 nucleotides; (v) often come from not full-length DSL lacking considerable left hand side area; and (vi) are often polyadenylated via cryptic HBV polyadenylation signal (PAS). This study will examine these RNAs in detail. In Aim 1, we will use liver/HCC tissues from chronic HBV carriers and cell lines derived from HBV-related HCCs and bearing HBV integrants, and using long-read RNA-seq will examine the sequence features of the 5'-human-HBV-3' RNAs, such as the sequences of human and HBV parts, positions of polyadenylation in HBV sequence and related PAS, human-HBV junctions; will establish the origins of these RNAs as if they come from full-length of truncated DSL; will identify HBV-HBV recombinant and spliced sequences, multiple transcripts derived from the same integrant, etc.; and generate summary diagram depicting detailed common features of such RNAs. In Aim 2, using long-read RNA-seq data, we will design specific RT-qPCRs and determine the levels for 5'-human-HBV-3' RNAs in comparison to the HBV replication-derived RNAs. In Aim 3, we will use long-read RNA-seq data from Aim 1 and make the vectors expressing 5'-human-HBV-3' RNAs, and, using transfection of Huh7 or HepG2 cells, examine these RNAs' (i) accumulation and stability, (ii) ability to regulate the levels of HBV genome replication, and (iii) potential to alter the host genes' expression. Overall, the proposed study will advance our understanding of the natural 5'- human-HBV-3' RNAs' sequences organization, their biological functions and role in HBV life cycle.
