Abstract
Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8, the C-terminus with His 8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In T-REx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.
Highlights
SARS-CoV-2 is a large positive-sense, single-stranded RNA virus that encodes four structural proteins, several accessory proteins, and sixteen nonstructural proteins (Figure 1)
In the context of SARS-CoV-2, crosslinking and analysis of cDNA (CRAC) is expected to identify host RNAs that are targeted by viral factors, while total RNA-associated proteome purification (TRAPP) will reveal how host cell RNA-protein interactions are globally remodeled in response to specific viral proteins
There has been a large amount of research activity surrounding COVID-19 but, understandably, this has predominately focused on epidemiology and the development of anti-viral strategies
Summary
SARS-CoV-2 is a large positive-sense, single-stranded RNA virus that encodes four structural proteins, several accessory proteins, and sixteen nonstructural proteins (nsp1-16) (Figure 1). The latter are mainly engaged in enzymatic activities important for translation and replication of the RNA genome. Nonstructural proteins target host RNA metabolism in order to manipulate cellular gene expression or facilitate immune evasion (Gordon et al, 2020; Thoms et al, 2020; Yuen et al, 2020). Understanding these pathways in greater detail will be an important step in the development of antiviral therapies. In the context of SARS-CoV-2, CRAC is expected to identify host RNAs that are targeted by viral factors, while TRAPP will reveal how host cell RNA-protein interactions are globally remodeled in response to specific viral proteins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
