The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression

Abstract

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19 and 20) and another within NSP1 (R124) which are required for viral evasion, and thus might present promising drug targets. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of anti-viral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

Parsing the role of NSP1 in SARS-CoV-2 infection

“…Despite numerous studies, it remains unclear how viral transcripts overcome nsp1-mediated translation suppression and specifically how this evasion relates to the translation and degradation activities of nsp1. Our results together with recent studies (Bujanic et al, 2021; Finkel et al, 2021a; Mendez et al, 2021; Vora et al, 2022) show that in the setting of nsp1 overexpression, cellular mRNA translation is strongly inhibited by nsp1. The observation that nsp1-CD, which does not lead to mRNA degradation, inhibits translation to a lesser extent than WT nsp1, suggests that both the translation inhibition and the degradation activities contribute to nsp1’s ability to shutoff cellular protein synthesis.…”

Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets

“…The ligands with the highest affinity were peptides derived from the NF-kappa-B-repressing factor (NKRF 8-23 ; AEGIDIGEMPSYDLVL), the protein kinase LMTK3 (LMTK3 22-36 ; PAHPDGFALGRAPLA), and the Axin-1 (AXIN1 1-15 ; MNIQEQGFPLDLGAS), which bound to Nsp9 with K D values of 30-50 μM ( Table S4) . Of these, NKRF has been found to interact with other SARS-CoV-2 proteins including Nsp1 46 and Nsp10 47 with the latter interaction thought to regulate interleukin-8 production. A peptide from the Neurogenic locus notch homolog protein 4 (NOTCH4 1605-1620 ) displayed lower affinity, and bound to Nsp9 with a K D ~130 μM.…”

“…MNIQEQGFPLDLGAS), which bound to Nsp9 with KD values of 30-50 µM (Table S4). Of these, NKRF has been found to interact with other SARS-CoV-2 proteins including Nsp1 46 and Nsp10 47 with the latter interaction thought to regulate interleukin-8 production. A peptide from the Neurogenic locus notch homolog protein 4 (NOTCH41605-1620) displayed lower affinity, and bound to Nsp9 with a KD ~130 µM.…”

Bottom-up assembly of viral replication cycles

“…non-structural proteins 1 and 2) and host proteins initiate or sustain the formation of phase-separated organelles that serve as viral factories promoting the self-assembly of SARS-CoV-2 virion fragments 46 . Exploiting the natural function of nonstructural SARS-CoV-2 proteins to engineer a synthetic replication cycle is a promising strategy as it may allow simultaneously blocking the translation of host mRNAs and enabling the formation of virion factories 47 . However, while designing a replication module on the basis of the cellular machinery allows one to closely model natural virus replication, this strategy compromises the quantitative and controllable characteristics of the overall system.…”