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Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and presents a significant threat to human health. Despite its prevalence, the underlying regulatory mechanisms of HCC remain unclear. In this study, we integrated RNA-seq datasets, proteome dataset and survival analysis and unveiled Stratifin (SFN) as a potential prognostic biomarker for HCC. SFN knockdown inhibited HCC progression in cell cultures and mouse models. Conversely, ectopic expression of Sfn in primary mouse HCC model accelerated HCC progression. Mechanistically, SFN acted as an adaptor protein, activating AKT1 signaling by fostering the interaction between PDK1 and AKT1, with the R56 and R129 sites on SFN proving to be crucial for this binding. In the syngeneic implantation model, the R56A/R129A mutant of SFN inhibited Akt signaling activation and impeded HCC growth. Additionally, peptide inhibitors designed based on the binding motif of AKT1 to SFN significantly inhibited HCC progression. In summary, our findings establish that SFN promotes HCC progression by activating AKT signaling through the R56 and R129 binding sites. This discovery opens new avenues for a promising therapeutic strategy for the treatment of HCC.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide, posing a major global health threat. Export of HBV RNAs from the nucleus to the cytoplasm is indispensable for viral protein translation and genome replication, however the mechanisms regulating this critical process remain largely elusive. Here, we identify a key host factor embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) that binds HBV RNAs and controls their nuclear export. Using an unbiased quantitative proteomics screen, we demonstrate direct binding of ELAVL1 to the HBV pregenomic RNA (pgRNA). ELAVL1 knockdown inhibits HBV RNAs posttranscriptional regulation and suppresses viral replication. Further mechanistic studies reveal ELAVL1 recruits the nuclear export receptor CRM1 through ANP32A and ANP32B to transport HBV RNAs to the cytoplasm via specific AU-rich elements, which can be targeted by a compound CMLD-2. Moreover, ELAVL1 protects HBV RNAs from DIS3+RRP6+ RNA exosome mediated nuclear RNA degradation. Notably, we find HBV core protein is dispensable for HBV RNA-CRM1 interaction and nuclear export. Our results unveil ELAVL1 as a crucial host factor that regulates HBV RNAs stability and trafficking. By orchestrating viral RNA nuclear export, ELAVL1 is indispensable for the HBV life cycle. Our study highlights a virus-host interaction that may be exploited as a new therapeutic target against chronic hepatitis B.

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and liver cancer, despite strong prevention and treatment efforts. The study of the epigenetic modification of HBV has become a research hotspot, including the N6-methyladenosine (m6A) modification of HBV RNA, which plays complex roles in the HBV life cycle. In addition to m6A modification, 5-methylcytosine (m5C) is another major modification of eukaryotic mRNA. In this study, we explored the roles of m5C methyltransferase and demethyltransferase in the HBV life cycle. The results showed that m5C methyltransferase NSUN2 deficiency could negatively regulate the expression of HBV while m5C demethyltransferase TET2 deficiency positively regulates the expression of HBV. Subsequently, we combined both in vitro bisulfite sequencing and high-throughput bisulfite sequencing methods to determine the distribution and stoichiometry of m5C modification in HBV RNA. Two sites: C2017 and C131 with the highest-ranking methylation rates were identified, and mutations at these two sites could lead to the decreased expression and replication of HBV, while the mutation of the "fake" m5C site had no effect. Mechanistically, NSUN2-mediated m5C modification promotes the stability of HBV RNA. In addition, compared with wild-type HepG2-NTCP cells and primary human hepatocytes, the replication level of HBV after NSUN2 knockdown decreased, and the ability of the mutant virus to infect and replicate in wild-type HepG2-NTCP cells and PHHs was substantially impaired. Similar results were found in the experiments using C57BL/6JGpt-Nsun2+/- mice. Interestingly, we also found that HBV expression and core protein promoted the endogenous expression of NSUN2, which implied a positive feedback loop. In summary, our study provides an accurate and high-resolution m5C profile of HBV RNA and reveals that NSUN2-mediated m5C modification of HBV RNA positively regulates HBV replication by maintaining RNA stability.

Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.

Background and Aims: The persistence of viral covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment against hepatitis B virus (HBV). Basic and translational studies are largely hampered due to the lack of feasible small animal models to support HBV cccDNA formation. The aim of this study is to establish a novel mouse model harboring cccDNA. Methods: An adeno-associated virus (AAV) vector carrying a replication-deficient HBV1.04-fold genome (AAV-HBV1.04) was constructed. The linear HBV genome starts from nucleotide 403 and ends at 538, which results in the splitting of HBV surface and polymerase genes. Different HBV replication markers were evaluated for AAV-HBV1.04 plasmid–transfected cells, the AAV-HBV1.04 viral vector–transduced cells, and mice injected with the AAV-HBV1.04 viral vector. Results: Compared with the previously reported AAV-HBV1.2 construct, direct transfection of AAV-HBV1.04 plasmid failed to produce hepatitis B surface antigen and progeny virus. Interestingly, AAV-HBV1.04 viral vector transduction could result in the formation of cccDNA and the production of all HBV replication markers in vitro and in vivo. The formation of cccDNA could be blocked by ATR (ataxia-telangiectasia and Rad3-related protein) inhibitors but not HBV reverse transcription inhibitor or capsid inhibitors. The AAV-HBV1.04 mouse supported long-term HBV replication and responded to antiviral treatments. Conclusions: This AAV-HBV1.04 mouse model can support HBV cccDNA formation through ATR-mediated DNA damage response. The de novo formed cccDNA but not the parental AAV vector can lead to the production of hepatitis B surface antigen and HBV progeny. This model will provide a unique platform for studying HBV cccDNA and developing novel antivirals against HBV infection.

ABSTRACT Since the outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, it has rapidly spread around the world. Persons with asymptomatic disease exhibit viral shedding, resulting in transmission, which presents disease control challenges. However, the clinical characteristics of these asymptomatic individuals remain elusive. We collected samples of 25 asymptomatic and 27 symptomatic COVID-19 patients. Viral titers of throat swabs were determined by quantitative reverse transcription-PCR (qRT-PCR). COVID-19 IgG and IgM were examined. Complete blood counts were determined, and serum biochemistry panels were performed. Cytokines, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were evaluated. T cell, B cell, and NK cell counts were measured using flow cytometry. Although similar viral loads were detected, asymptomatic patients had significantly faster virus turnover than symptomatic patients. Additionally, asymptomatic patients had higher counts of lymphocytes, T cells, B cells, and NK cells. While liver damage was observed in symptomatic patients, as indicated by elevated liver enzymes and decreased liver-synthesized proteins in the blood, asymptomatic patients showed normal liver measurements. Lactate dehydrogenase, a COVID-19 risk factor, was significantly lower in asymptomatic patients. These results suggest that asymptomatic COVID-19 patients had normal clinical indicators and faster viral clearance than symptomatic patients. Lymphocytes may play a role in their asymptomatic phenotype. Since asymptomatic patients may be a greater risk of virus transmission than symptomatic patients, public health interventions and a broader range of testing may be necessary for the control of COVID-19. IMPORTANCE Asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a potential problem for pandemic control through public health strategies. Our results demonstrate that asymptomatic COVID-19 patients have better outcomes than symptomatic patients. This may have been due to more active cellular immune responses and normal liver function. Since asymptomatic patients have no clinical symptoms which can easily prevent timely diagnosis and treatment, they may cause a greater risk of virus transmission than symptomatic patients, which poses a major challenge to infection control. Evidence suggests that nonpharmaceutical public health interventions, like social distancing and face mask ordinances, play important roles in the control of COVID-19. Looking forward, it may be necessary to proceed cautiously while reopening businesses in areas of epidemicity to prevent potential waves of COVID-19 in the future.

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]

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used in vitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in in vitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation. [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]