Ciclopirox inhibits SARS-CoV-2 replication by promoting the degradation of the nucleocapsid protein.

The nucleocapsid protein (NP) plays a crucial role in SARS-CoV-2 replication and is the most abundant structural protein with a long half-life. Despite its vital role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assembly and host inflammatory response, it remains an unexplored target for drug development. In this study, we identified a small-molecule compound (ciclopirox) that promotes NP degradation using an FDA-approved library and a drug-screening cell model. Ciclopirox significantly inhibited SARS-CoV-2 replication both in vitro and in vivo by inducing NP degradation. Ciclopirox induced abnormal NP aggregation through indirect interaction, leading to the formation of condensates with higher viscosity and lower mobility. These condensates were subsequently degraded via the autophagy-lysosomal pathway, ultimately resulting in a shortened NP half-life and reduced NP expression. Our results suggest that NP is a potential drug target, and that ciclopirox holds substantial promise for further development to combat SARS-CoV-2 replication.

TRIM6 facilitates SARS-CoV-2 proliferation by catalyzing the K29-typed ubiquitination of NP to enhance the ability to bind viral genomes.

The Nucleocapsid Protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not only the core structural protein required for viral packaging, but also participates in the regulation of viral replication, and its post-translational modifications such as phosphorylation have been shown to be an important strategy for regulating virus proliferation. Our previous work identified NP could be ubiquitinated, as confirmed by two independent studies. But the function of NP ubiquitination is currently unknown. In this study, we first pinpointed TRIM6 as the E3 ubiquitin ligase responsible for NP ubiquitination, binding to NP's CTD via its RING and B-box-CCD domains. TRIM6 promotes the K29-typed polyubiquitination of NP at K102, K347, and K361 residues, increasing its binding to viral genomic RNA. Consistently, functional experiments such as the use of the reverse genetic tool trVLP model and gene knockout of TRIM6 further confirmed that blocking the ubiquitination of NP by TRIM6 significantly inhibited the proliferation of SARS-CoV-2. Notably, the NP of coronavirus is relatively conserved, and the NP of SARS-CoV can also be ubiquitinated by TRIM6, indicating that NP could be a broad-spectrum anti-coronavirus target. These findings shed light on the intricate interaction between SARS-CoV-2 and the host, potentially opening new opportunities for COVID-19 therapeutic development.

The E3 ligase TRIM22 restricts SARS-CoV-2 replication by promoting proteasomal degradation of NSP8.

Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is mediated by a complex of non-structural proteins (NSPs), of which NSP7 and NSP8 serve as subunits and play a key role in promoting the activity of RNA-dependent RNA polymerase (RdRp) of NSP12. However, the stability of subunits of the RdRp complex has rarely been reported. Here, we found that NSP8 was degraded by the proteasome in host cells, and identified tripartite motif containing 22 (TRIM22) as its E3 ligase. The interferon (IFN) signaling pathway was activated upon viral invasion into host cells, and TRIM22 expression increased. TRIM22 interacted with NSP8 and ubiquitinated it at Lys97 via K48-type ubiquitination. TRIM22 overexpression significantly reduced viral RNA and protein levels. Knockdown of TRIM22 enhanced viral replication. This study provides a new explanation for treating patients suffering from SARS-CoV-2 with IFNs and new possibilities for drug development targeting the interaction between NSP8 and TRIM22.IMPORTANCENon-structural proteins (NSPs) play a crucial role in the replication of severe acute respiratory syndrome coronavirus 2, facilitating virus amplification and propagation. In this study, we conducted a comprehensive investigation into the stability of all subunits comprising the RNA-dependent RNA polymerase complex. Notably, our results reveal for the first time that NSP8 is a relatively unstable protein, which is found to be readily recognized and degraded by the proteasome. This degradation process is mediated by the host E3 ligase tripartite motif containing 22 (TRIM22), which is also a member of the interferon stimulated gene (ISG) family. Our study elucidates a novel mechanism of antiviral effect of TRIM22, which utilizes its own E3 ubiquitin ligase activity to hinder viral replication by inducing ubiquitination and subsequent degradation of NSP8. These findings provide new ideas for the development of novel therapeutic strategies. In addition, the conserved property of NSP8 raises the possibility of developing broad antiviral drugs targeting the TRIM22-NSP8 interaction.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

A TurboID-based proximity labelling approach for identifying the DNA-binding proteins.

Biotin proximity labeling is a technique based on the TurboID enzyme that can be used to capture weak or dynamic interactions that had previously not been used to map proteins interacting with a specific DNA sequence. Here, we present a protocol for identifying specific DNA-sequence-binding proteins. We describe steps for biotin labeling of DNA-binding proteins, protein enrichment and sodium dodecyl sulfate polyacrylamide gel electrophoresis separation, and proteomic analysis. For complete details on the use and execution of this protocol, please refer to Wei et al. (2022).1.

Human liver cancer organoids: Biological applications, current challenges, and prospects in hepatoma therapy.

Liver cancer and disease are among the most socially challenging global health concerns. Although organ transplantation, surgical resection and anticancer drugs are the main methods for the treatment of liver cancer, there are still no proven cures owing to the lack of donor livers and tumor heterogeneity. Recently, advances in tumor organoid technology have attracted considerable attention as they can simulate the spatial constructs and pathophysiological characteristics of tumorigenesis and metastasis in a more realistic manner. Organoids may further contribute to the development of tailored therapies. Combining organoids with other emerging techniques, such as CRISPR-HOT, organ-on-a-chip, and 3D bioprinting, may further develop organoids and address their bottlenecks to create more practical models that generalize different tissue or organ interactions in tumor progression. In this review, we summarize the various methods in which liver organoids may be generated and describe their biological and clinical applications, present challenges, and prospects for their integration with emerging technologies. These rapidly developing liver organoids may become the focus of in vitro clinical model development and therapeutic research for liver diseases in the near future.

DDX56 transcriptionally activates MIST1 to facilitate tumorigenesis of HCC through PTEN-AKT signaling.

Rationale: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver that is the leading cause of cancer-related mortality worldwide. However, genetic alterations and mechanisms underlying HCC development remain unclear. Methods: Tissue specimens were used to evaluate the expression of DEAD-Box 56 (DDX56) to determine its prognostic value. Colony formation, CCK8, and EdU-labelling assays were performed to assess the effects of DDX56 on HCC proliferation. The in vivo role of DDX56 was evaluated using mouse orthotopic liver xenograft and subcutaneous xenograft tumor models. Dual-luciferase reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays were performed to examine the effect of DDX56 on the MIST1 promoter. Results: DDX56 expression in HCC tissues was elevated and this increase was strongly correlated with poor prognoses for HCC patients. Functionally, DDX56 promoted HCC cell proliferation both in vitro and in vivo, while mechanistically interacting with MECOM to promote HCC proliferation by mono-methylating H3K9 (H3K9me1) on the MIST1 promoter, leading to enhanced MIST1 transcription and subsequent regulation of the PTEN/AKT signaling pathway, which promotes HCC proliferation. More importantly, the PTEN agonist, Oroxin B (OB), blocked the DDX56-mediated PTEN-AKT signaling pathway, suggesting that treating HCC patients with OB may be beneficial as a therapeutic intervention. Furthermore, we observed that ZEB1 bound to DDX56 and transcriptionally activated DDX56, leading to HCC tumorigenesis. Conclusions: Our results indicated that the ZEB1-DDX56-MIST1 axis played a vital role in sustaining the malignant progression of HCC and identified DDX56 as a potential therapeutic target in HCC tumorigenesis.

Caspase sensors based on NanoLuc.

Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.

Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling.

The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.

Strategies to improve the fluorescent signal of the tripartite sfGFP system.

Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein-protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1-9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1-9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th ß-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.

Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay.

The capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replication in vitro by decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.

Fluorescent protein tagged hepatitis B virus capsid protein with long glycine-serine linker that supports nucleocapsid formation.

Fusion core proteins of Hepatitis B virus can be used to study core protein functions or capsid trafficking. A problem in constructing fusion core proteins is functional impairment of the individual domains in these fusion proteins, might due to structural interference. We reported a method to construct fusion proteins of Hepatitis B virus core protein (HBc) in which the functions of fused domains were partially kept. This method follows two principles: (1) fuse heterogeneous proteins at the N terminus of HBc; (2) use long Glycine-serine linkers between the two domains. Using EGFP and RFP as examples, we showed that long flexible G4S linkers can effectively separate the two domains in function. Among these fusion proteins constructed, GFP-G4S186-HBc and RFP-G4S47-HBc showed the best efficiency in rescuing the replication of an HBV replicon deficient in the core protein expression, though both of the two fusion proteins failed to support the formation of the relaxed circular DNA. These fluorescent protein-tagged HBcs might help study related to HBc or capsids tracking in cells.