The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a significant threat to global public health and safety. SARS-CoV-2 is an enveloped RNA virus consisting of four structural proteins, the coronavirus (S) glycoprotein, the membrane (M) protein, the ion channel envelope (E) protein, and the nucleocapsid protein (NP). NP is involved in the transcription, replication and packaging of the viral genome through a liquid-liquid phase separation (LLPS) mechanism. It has been found that human coronavirus NP is produced at high levels in infected cells and is involved in virosome assembly. Therefore, there is an urgent need to identify the molecular mechanisms that form the basis of the essential viral action of NP for the development of COVID-19 therapies.

Fig. 1. LLPS in SARS-CoV-2 infection. (Wang B, et al., 2021)

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A tightly packed complex of NP and genomic RNA forms the core of the virus and assembles within the virion factory to form dynamic compartments within the host cell associated with human stress particles. CD BioSciences provides professional services to analyze the potential for multivalent RNA-binding nucleocapsid proteins from SARS-CoV-2 to coalesce with RNA via LLPS. We focus on characterizing the formation of biomolecular condensates of NP with viral genomic RNA in vitro and in mammalian cells.

• Analysis of SARS-CoV-2-NP LLPS In Vitro
  Our laboratory has established assays for studying LLPS-prone proteins, in which the phase separation of SARS-CoV-2-NP is determined by adding TEV protease to induce LLPS. In addition, our technical team can perform experiments under various solution conditions to test the basic biochemical characteristics of the interactions mediating NP phase separation. The specific procedure is as follows:
  (1) Samples containing MBP-labeled full-length NPs were prepared.
  (2) TEV protease was added to the above samples.
  (3) Phase separation of SARS-CoV-2-NP was tested by measuring the turbidity of the solution and confirming phase separation by microscopy.

• Analysis of NP and SARS-CoV-2 Genomic RNA Interaction

• The SARS-CoV-2 RNA genome was assembled into a DNA plasmid by reverse transcription.
• The above plasmids were transcribed in vitro to generate fragments of the viral RNA genome
• We provide computer simulations to predict that these RNA fragments can form intramolecular and intermolecular base-pairing interactions and contain a wide range of secondary structure elements.
• We provide advanced cross-linked mass spectrometry (CLMS) to identify interactions between the different structural domains of full-length N proteins in the absence of RNA.
• Based on a high-throughput microscopy platform, we can identify small molecules that may interfere with NP phase separation, over 1,200 compounds in the drug library.

• Analysis of SARS-CoV-2-NP LLPS in Mammalian Cells
  To test whether N proteins also undergo phase separation in mammalian cells, we can express NP-GFP or fluorescent probes in a variety of cells to test for phase separation of NP in mammalian cells.

Importantly, our technical team is developing recombinant vaccines based on the SARS-CoV-2 nucleocapsid protein for antiviral immunity, and investigating antiviral strategies that allow the NP gene to be used as a potential drug target. In addition, we are committed to using NP as an important diagnostic marker for COVID-19.

CD BioSciences aims to provide you with a comprehensive analysis of the nucleocapsid protein LLPS of SARS-CoV-2 virus, and to provide a feasible and novel strategy for designing therapeutics for Covid-19 by inhibiting RNA-induced phase separation of SARS-CoV-2-NP. Our services are widely used in preclinical research on COVID-19 biology, diagnosis and treatment. If you are interested in our services, please feel free to contact us.