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Hepatitis B virus (HBV) is one of the major etiological pathogens for liver cirrhosis and hepatocellular carcinoma. Chronic HBV infection is a key factor in these severe liver diseases. During infection, HBV forms a nuclear viral episome in the form of covalently closed circular DNA (cccDNA). Current therapies are not able to efficiently eliminate cccDNA from infected hepatocytes. cccDNA is a master template for viral replication that is formed by the conversion of its precursor, relaxed circular DNA (rcDNA). However, the host factors critical for cccDNA formation remain to be determined. Here, we assessed whether one potential host factor, flap structure-specific endonuclease 1 (FEN1), is involved in cleavage of the flap-like structure in rcDNA. In a cell culture HBV model (Hep38.7-Tet), expression and activity of FEN1 were reduced by siRNA, shRNA, CRISPR/Cas9-mediated genome editing, and a FEN1 inhibitor. These reductions in FEN1 expression and activity did not affect nucleocapsid DNA (NC-DNA) production, but did reduce cccDNA levels in Hep38.7-Tet cells. Exogenous overexpression of wild-type FEN1 rescued the reduced cccDNA production in FEN1-depleted Hep38.7-Tet cells. Anti-FEN1 immunoprecipitation revealed the binding of FEN1 to HBV DNA. An in vitro FEN activity assay demonstrated cleavage of 5'-flap from a synthesized HBV DNA substrate. Furthermore, cccDNA was generated in vitro when purified rcDNA was incubated with recombinant FEN1, DNA polymerase, and DNA ligase. Importantly, FEN1 was required for the in vitro cccDNA formation assay. These results demonstrate that FEN1 is involved in HBV cccDNA formation in cell culture system, and that FEN1, DNA polymerase, and ligase activities are sufficient to convert rcDNA into cccDNA in vitro.

Hepatitis B virus (HBV) is the major causative factor of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. We previously demonstrated that a proinflammatory cytokine IL-1β reduced the level of HBV RNA. However, the mechanism underlying IL-1β-mediated viral RNA reduction remains incompletely understood. In this study, we report that immune regulator Monocyte chemotactic protein-1-induced protein 1 (MCPIP1) can reduce HBV RNA in hepatocytes. MCPIP1 expression level was higher in the liver tissue of HBV-infected patients and mice. Overexpression of MCPIP1 decreased HBV RNA, whereas ablating MCPIP1 in vitro enhanced HBV production. The domains responsible for RNase activity or oligomerization, were required for MCPIP1-mediated viral RNA reduction. The epsilon structure of HBV RNA was important for its antiviral activity and cleaved by MCPIP1 in the cell-free system. Lastly, knocking out MCPIP1 attenuated the anti-HBV effect of IL-1β, suggesting that MCPIP1 is required for IL-1β-mediated HBV RNA reduction. Overall, these results suggest that MCPIP1 may be involved in the antiviral effect downstream of IL-1β.

Mitochondrial DNA (mtDNA) mutations are found in many types of cancers and suspected to be involved in carcinogenesis, although the mechanism has not been elucidated. In this study, we report that consecutive C-to-T mutations (hypermutations), a unique feature of mutations induced by APOBECs, are found in mtDNA from cervical dysplasia and oropharyngeal cancers. In vitro , we found that APOBEC3A (A3A) and 3B (A3B) expression, as well as mtDNA hypermutation, were induced in a cervical dysplastic cell line W12 when cultured in a differentiating condition. The ectopic expression of A3A or A3B was sufficient to hypermutate mtDNA. Fractionation of W12 cell lysates and immunocytochemical analysis revealed that A3A and A3B could be contained in mitochondrion. These results suggest that mtDNA hypermutation is induced upon keratinocyte differentiation, and shed light on its molecular mechanism, which involves A3s. The possible involvement of mtDNA hypermutations in carcinogenesis is also discussed.

An Epstein‐Barr virus (EBV)—encoded latent membrane protein 1 (LMP1) is a principal oncogene that plays a pivotal role in EBV‐associated malignant tumors including nasopharyngeal cancer (NPC). Recent genomic landscape studies revealed that NPC also contained many genomic mutations, suggesting the role of LMP1 as a driver gene for the induction of these genomic mutations. Nonetheless, its exact mechanism has not been investigated. In this study, we report that LMP1 alters the expression profile of APOBEC3s(A3s), host deaminases that introduce consecutive C‐to‐U mutations (hypermutation). In vitro, LMP1 induces APOBEC3B (A3B) and 3F(A3F), in a nasopharyngeal cell line, AdAH. Overexpression of LMP1, A3B, or A3F induces mtDNA hypermutation, which is also detectable from NPC specimens. Expression of LMP1 and A3B in NPC was correlated with neck metastasis. These results provide evidence as to which LMP1 induces A3s and mtDNA hypermutation, and how LMP1 facilitates metastasis is also discussed. EBV LMP1 induces host cytidine deaminases, APOBECs, in nasopharyngeal cells. TES2 domain is important for LMP1‐induced LMP1 hypermutates host mitochondrial genome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Understanding the influence of mutations in the SARS-CoV-2 gene on clinical outcomes and related factors is critical for treatment and prevention. Here, we analyzed 209,551 high-coverage complete virus sequences and 321 RNA-seq samples to mine the mutations associated with clinical outcome in the SARS-CoV-2 genome. Several important hotspot variants were found to be associated with severe clinical outcomes. Q57H variant in ORF3a protein were found to be associated with higher mortality rate, and was high proportion in severe cases (39.36%) and 501Y.V2 strains (100%) but poorly proportional to asymptomatic cases (10.04%). T265I could change nsp2 structure and mitochondrial permeability, and evidently higher in severe cases (20.12%) and 501Y.V2 strains (100%) but lower in asymptomatic cases (1.43%). Additionally, R203K and G204R could decrease the flexibility and immunogenic property of N protein with high frequency among severe cases, VUI 202012/01 and 484K.V2 strains. Interestingly, the SARS-CoV-2 genome was more susceptible to mutation because of the high frequency of nt14408 mutation (which located in RNA polymerase) and the high expression levels of ADAR and APOBEC in severe clinical outcomes. In conclusion, several important mutation hotspots in the SARS-CoV-2 genome associated with clinical outcomes was found in our study, and that might correlate with different SARS-CoV-2 mortality rates. Competing Interest Statement The authors have declared no competing interest.