Abstract Hepatitis delta virus (HDV) is a natural sub-viral agent of hepatitis B virus (HBV), which uses HBV envelope proteins (i.e., surface antigen, HBsAg) to form the virions and infect hepatocytes via HBV receptor. Out of 257 million people chronically infected with HBV worldwide about 20 million are also chronically infected with HDV. HDV remains a significant human pathogen. HDV infection causes additional liver pathogenesis, and can accelerate liver disease, can cause more rapid and frequent cirrhosis, and can elevate risk of hepatocellular carcinoma (HCC). Anti-HBV drugs do not block HDV infection. No drugs directly targeting HDV exist in clinic. Mechanism of HDV infection is far from being fully understood, which affects the treatment options. HDV-HBV interactions are complex and under-studied. The regulation of HDV life cycle by the drug-resistant mutants of HBV remains under-studied as well. Therefore, this proposal will examine how the drug resistance mutations in HBV genome selected during the treatment with FDA-approved anti-HBV drugs, lamivudine, adefovir, telbivudine, entecavir or tenofovir, can influence HDV infection. The primary mutations that occur in the open reading frame (ORF) of HBV polymerase can lead to the corresponding drug resistance mutations (DRMs) in the overlapping ORF for HBsAg. DRMs were found in the large cytosolic loop (LCL), major antigenic loop (MAL), HDV-binding site (HDV-BS), and second, third and fourth transmembrane domains (TMDs II-IV) of HBsAg. MAL is critical for infectivity of HBV and HDV, and it is a major regulator of the antigenicity of HBV and HDV virions (i.e., recognition of the virions by anti-HBsAg antibodies). HDV-BS mediates HBsAg interactions with the large delta antigen during HDV assembly. Thus, DRMs are expected to regulate the mechanisms of the HDV assembly and infectivity, and could affect HDV-host interactions. Our previous work suggested that the infectivity of the virions, virus spread and super-infection (i) could determine if initial HDV infection will be cleared or it will become persistent (i.e., chronic) infection, and (ii) also could be critical for the maintenance of the chronic state of HDV infection. We also found that amino acids of HBsAg that regulate the assembly of HDV virions can influence their infectivity as well. Furthermore, the efficiency of HDV assembly can greatly affect HDV spread and super-infection. Current understanding of the mechanisms how DRMs regulate the assembly and infectivity of HDV, and recognition of HDV by anti-HBsAg antibodies is very limited. Therefore, Aim 1 will analyze in detail how HBsAg bearing DRMs can regulate the assembly of HDV and affect the recognition of HDV virions by anti-HBsAg antibodies of the hepatitis B immune globulin (HBIg). Furthermore, Aim 2 will examine the effects of DRMs on the infectivity of HDV virions. Overall, the proposal will considerably advance our understanding of the mechanisms of how the drug-resistant mutant...