TRIM21 promotes ubiquitination of SARS-CoV-2 nucleocapsid protein to regulate innate immunity.

The innate immune response is the first line of host defense against viral infections, but its role in immunity against SARS-CoV-2 remains unclear. By using immunoprecipitation coupled with mass spectroscopy, we observed that the E3 ubiquitin ligase TRIM21 interacted with the SARS-CoV-2 nucleocapsid (N) protein and ubiquitinated it at Lys375 . Upon determining the topology of the TRIM21-mediated polyubiquitination chain on N protein, we then found that polyubiquitination led to tagging of the N protein for degradation by the host cell proteasome. Furthermore, TRIM21 also ubiquitinated the N proteins of SARS-CoV-2 variants of concern, including Alpha, Beta, Gamma, Delta, and Omicron together with SARS-CoV and MERS-CoV variants. Herein, we propose that ubiquitylation and degradation of the SARS-CoV-2 N protein inhibited SARS-CoV-2 viral particle assembly, by which it probably involved in preventing cytokine storm. Eventually, our study has fully revealed the association between the host innate immune system and SARS-CoV-2 N protein, which may aid in developing novel SARS-CoV-2 treatment strategies.

Immunomolecular assay based on selective virion capture by spike antibody and viral nucleic acid amplification for detecting intact SARS-CoV-2 particles.

BACKGROUND: Effective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application due to factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immuno molecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions. RESULTS: After constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 102 transduction units/mL of viral titer with high confidence (~ 95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0 ~ 2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper compared with aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions. Complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced. CONCLUSION: Convenient, inexpensive, and accurate complete virus detection can be applied to many fields, including monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions, including Hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments.