SARS-CoV-2 Non-Structural Proteins

Nsp1: Host Shutoff Factor

The viral nonstructural protein 1 (Nsp1) is the only membrane-associated protein that anchors the replication complex to the cellular membranes. Nsp1 inhibits host translation by interacting with the 40S ribosomal subunit. The Nsp1-40S ribosome complex further induces an endonucleolytic cleavage near the 5’UTR of host mRNAs, targeting them for degradation. Viral mRNAs are not susceptible to Nsp1-mediated endonucleolytic RNA cleavage thanks to the presence of a 5′-end leader sequence and are therefore protected from degradation. By suppressing host gene expression, Nsp1 facilitates efficient viral gene expression in infected cells and evasion from host immune response (Lapointe et al., 2021).

Nsp3: Papain-Like Protease (PLpro)

SARS-CoV-2 papain-like protease (PLpro) is a cysteine protease that acts on three cleavage sites of the viral polyprotein to release mature non-structural proteins (NSP) 1, 2 and 3 (Kandeel et al., 2020). PLpro also strips ubiquitin and ISG15 from host-cell proteins to help coronaviruses to evade the host innate immune responses. PLpro is therefore involved in inhibiting the production of cytokines and chemokines, that are responsible for the activation of the host innate immune response against viral infection. Consequently, PLpro is an important molecular target in the design of various different SARS-CoV-2 antivirals.

Nsp5: 3C-Like Protease (3CLpro)

SARS-CoV-2 3C-like proteinase (3CLpro) is the 5th non-structural protein (NSP5) that uses cysteine activity to cleave the C-terminus of the large replicase polyprotein, 1ab at no fewer than 11 sites (Zhang et al., 2020). The functional importance of 3CLpro in the viral life cycle makes this protease an attractive target for the development of drugs directed against SARS-CoV-2.

Nsp7: RdRp Subunit

Nsp7 and Nsp8 stabilize regions of Nsp12 (RNA-dependent RNA polymerase; RdRp) involved in RNA binding and are essential for a highly active Nsp12 polymerase complex. Nsp7 has been suggested to confer RNA-binding properties to the trimeric polymerase complex (Subissi et al., 2014).

Nsp8: RdRp Subunit

Nsp7 and Nsp8 stabilize regions of Nsp12 (RNA-dependent RNA polymerase; RdRp) involved in RNA binding and are essential for a highly active Nsp12 polymerase complex. Nsp8 has been shown to act as an oligo(U)-templated polyadenylyltransferase and a robust (mono/oligo) adenylate transferase (Tvarogova et al., 2019). Amino acid substitutions in RdRp (P323L) have been shown to cause conformational changes near the Nsp8 binding site that may affect virus replication and transcription (Alosaimi et al., 2021).

Nsp10/16: Methyltransferase (active)

Many eukaryotic viruses have evolved 2′-O-methyltransferases (2′-O-MTase) to modify their viral mRNAs and carry a cap-1 structure (m7GpppNm) at the 5′ end. This 5’ cap structure is important for viral mRNA stability, protein translation and viral immune escape. SARS-CoV possess Nsp16 that has 2′-O-MTase activity. Nsp16 requires Nsp10 for activation which is a conserved mechanism in corona viruses. Mutations have been identified in SARS-CoV-2 Nsp10 and Nsp16, which alter Nsp10/Nsp16 secondary structure, and protein-modelling studies have revealed that such mutations can affect the proteins dynamicity and stability (Azad et al., 2020). Therefore, inhibitors targeting the Nsp10/Nsp16 2′-O-MTase are potential targets for developing anti-coronavirus drugs (Chen et al., 2011).

Nsp12: RdRp Catalytic Subunit

RNA-dependent RNA polymerase (RdRp) is an enzyme that is crucial to life cycle of RNA viruses including coronaviruses, catalysing the synthesis of viral RNA in complex with Nsp7 and Nsp8 (Gao et al., 2020). Given its central role in the coronavirus life cycle, RdRp is a prime target for nucleotide analog inhibitors, such as remdesivir.

Nsp14: Methyltransferase (active)

Coronaviruses codes for a bifunctional non-structural protein 14 (NSP14) that is important for viral replication and transcription. Nsp14 contains an N-terminal exo-ribonuclease (ExoN) domain for proof reading during viral replication and a C-terminal N-7 methyltransferase (N7-MTase) domain for mRNA capping. Nsp14-ExoN is required for native recombination, and inactivation of ExoN results in decreased recombination frequency and altered recombination products (Gribble et al., 2021). Nsp14 associates with several viral proteins. It can bind to Nsp10 and the resulting complex exhibits enhanced ExoN activity in vitro. It can also bind to the polymerase complex (Nsp12/Nsp8/Nsp7) forming a complex that retains all associated enzymatic activities. Nsp14 is known to be a good target for potential small molecule inhibitors against SARS-CoV-2 (Umar et al., 2021).