Abstract There are more than 250 million of chronic HBV carriers worldwide. Chronic HBV infection is the main cause of hepatocellular carcinoma (HCC). There is no cure for HBV. Interferon therapy gives temporary benefits only to a subset of patients. Regardless that FDA-approved nucleos(t)ide analogs can greatly inhibit HBV replication, chronic HBV infection is not well under control. None of the drugs works in all patients, and is able to achieve the loss of the surface antigen (HBsAg, or HBV envelope proteins) in most cases. HBV frequently rebounds after treatment is stopped. Mechanism of HBV infection is not fully understood, which affects treatment options. This proposal will analyze the relationship between HBV integrant-derived RNAs (id-RNAs) coding for HBsAg and HBV life cycle. HBV DNA integration into host genome is not considered important for HBV life cycle, and id-RNAs are not well studied. Analyzing matching liver/HCC tissues from chronic HBV carrier humans using RT-PCR, we found that regardless of HBV replication: id-RNAs accumulated in all tissues and represented 45- 100% of all HBV RNAs found in 60% of tissues (40% livers/80% HCCs); RNAs polyadenylated via cryptic HBV poly(A) signal (could be integrant-derived or produced by HBV replication) were found in 60% livers/40% HCCs, and were the most abundant HBV RNAs in 20% livers/20% HCCs; and replication-derived RNAs (rd- RNAs) polyadenylated using conventional HBV poly(A) signal were the least abundant species in 70% of tissues (40% livers/100% HCCs). Furthermore, analysis of selected tissues by RNA sequencing also showed id-RNAs' abundance in most of the analyzed samples. The data suggest that id-RNAs may produce significant fraction of HBsAg in cells bearing HBV integrants. If the same cells also support HBV replication, then a great fraction of formed HBV virions likely will bear id-RNA-derived HBsAg (id-HBsAg) as a major component of their envelopes. Thus, infectivity of these HBV virions, and their ability to support virus spread will mostly depend on the properties of id-HBsAg, and not on those of HBV replication-derived HBsAg (rd-HBsAg). Thus, id-RNAs could regulate the maintenance of chronic HBV infection, which may call for revision of the current model of HBV infection that suggests that HBV life cycle is independent of the integration. It also became apparent that anti-HBV drugs fail to achieve loss of serum HBsAg, because major amounts of HBsAg can be produced from id-RNAs regardless of HBV replication. In light of our data, Aim 1 using a large set of liver/HCC tissues will find if id-RNAs' abundance is common during chronic HBV infection. Aims 2 and 3 will (i) find if large fraction of id- RNAs do not bear alterations in HBsAg-coding sequences, and thus can serve as sizeable source of functional HBsAg regardless of HBV replication, (ii) analyze how 3'-end untranslated regions of id-RNAs (including the host inserts) affect HBsAg synthesis, and (iii) find if id-HBsAg can ef...