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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.

Abstract As a structural protein of SARS‐CoV‐2, the envelope (E) protein not only plays a key role in the formation of viral particles, but also forms ion channels and has pathogenic functions, including triggering cell death and inflammatory responses. The stability of E proteins is controlled by the host ubiquitin‐proteasome system. By screening human deubiquitinases, it is found that ubiquitin‐specific protease 33 (USP33) can enhance the stability of E proteins depending on its deubiquitinase activity, thereby promoting viral replication. In the absence of USP33, E proteins are rapidly degraded, leading to a reduced viral load and inflammation. Using lipid nanoparticle (LNP) encapsulation of siUSP33 by adjusting the lipid components (ionizable cationic lipids), siUSP33 is successfully delivered to mouse lung tissues, rapidly reducing USP33 expression in the lungs and maintaining knockdown for at least 14 days, effectively suppressing viral replication and virulence. This method of delivery allows efficient targeting of the lungs and a response to acute infections without long‐term USP33 deficiency. This research, based on the deubiquitination mechanism of USP33 on the E protein, demonstrates that LNP‐mediated siRNA delivery targeting USP33 plays a role in antiviral and anti‐inflammatory responses, offering a novel strategy for the prevention and treatment of SARS‐CoV‐2.

ABSTRACT 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.

The study explores the inhibition effects of MicroRNAs in osteosarcoma, as a means of suggesting it as treatment for bone cancer. MicroRNAs (miRNAs) are a sort of noncoding RNA molecules that regulates gene expression by targeting mRNAs and play critical roles in tumor development. This study probed the effect of miR-9-5p on osteosarcoma development. Human osteosarcoma cell lines U2-OS, 143B, MG63 and HOS and normal human osteoblast cell line hFOB were cultivated and expression of miR-9-5p and Grb2-associated binding protein 2 (Gab2) measured. The binding of miR-9-5p and Gab2 was confirmed using a bio-information program and dual luciferase reporter gene assay. Loss-of-functions of miR-9-5p and Gab2 were performed to measure their roles in osteosarcoma cell proliferation, invasion, migration and resistance to death. Result showed high miR-9-5p expression and low Gab2 expression in osteosarcoma cells, particularly in U2-OS cells. miR-9-5p directly bound to the 3' untranslated region of Gab2. Down-regulated miR-9-5p induced U2-OS cell proliferation, invasion and the resistance to death, while conversely, silenced Gab2 led to an opposite trend on U2-OS cell growth and metastasis. Moreover, co-effect of inhibited miR-9-5p and silenced Gab2 led to decreased cell proliferation but promoted cell apoptosis compared to inhibited miR-9-5p alone, while it led to enhanced cell proliferation and invasion, but reduced cell apoptosis compared to silenced Gab2 alone. To conclude, this study demonstrated that miR-9-5p could inhibit osteosarcoma cell proliferation, invasion, migration and resistance to death via negatively targeting Gab2. [ABSTRACT FROM AUTHOR]