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Hepatitis B virus (HBV) infection is a major health burden worldwide, and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral trement. HBV core protein (HBc) has emerged as a promising antiviral target, as it plays important roles in critical steps of the viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains under debate. In this study, different approaches were used to address this question. In synthesized HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen (HBsAg) production in a hepatoma cell line and primary human hepatocytes. Reconstitution of HBc did not alter the expression of cccDNA-derived HBV markers. Similar results were obtained from an in vivo mouse model harboring cccDNA. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays revealed transcription regulation of HBc-deficient cccDNA chromatin similar to that of wild-type cccDNA. Furthermore, treatment with capsid assembly modulators (CAMs) dramatically reduced extracellular HBV DNA but could not alter viral RNA and HBsAg. Our results demonstrate that HBc neither affects histone modifications and transcription factor binding of cccDNA nor directly influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, they do not suppress cccDNA transcriptional activity. Thus, therapeutics targeting capsid or HBc should not be expected to sufficiently reduce cccDNA transcription. [ABSTRACT FROM AUTHOR]

Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-β promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFNβ promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. [ABSTRACT FROM AUTHOR]