Order and disorder bound together in SARS-CoV-2 Nsp1 suppress host translation

Abstract

In this issue of Structure, Wang et al. investigate the interplay between folded and disordered regions of the SARS-CoV-2 non-structural protein 1 (Nsp1) that promotes the suppression of host protein translation. Their investigation will lead to novel avenues to therapeutically target critical viral functions necessary for host immune-response suppression.

In this issue of Structure, Wang et al. investigate the interplay between folded and disordered regions of the SARS-CoV-2 non-structural protein 1 (Nsp1) that promotes the suppression of host protein translation. Their investigation will lead to novel avenues to therapeutically target critical viral functions necessary for host immune-response suppression.

Main text

Since the World Health Organization declared the outbreak of SARS-CoV-2 a pandemic in March of 2020, over 650 million infections have been reported, and 6.6 million deaths are officially attributed to the viral infection.¹ The speed and scale of the outbreak has induced an unprecedented response from the global scientific community, whose collective efforts rapidly designed, tested, and implemented safe and effective vaccines that have likely prevented millions of additional deaths. The development of vaccines on such a compressed timeline would not have been possible without the rapid acquisition and dissemination of basic biochemical knowledge of SARS-CoV-2 mechanisms of infection, host immune suppression, replication, and spreading of the virus upon infection of host cells. Structural biology has contributed considerably to these efforts with numerous cryo-electron microscopy, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy studies extensively characterizing almost all the relevant structural and non-structural proteins (Nsp). Collectively, these studies have uncovered the atomic details of the molecular mechanisms that the viral proteins use to hijack the translational machinery of host cells.²

The leader protein Nsp1, transcribed directly from the viral genomic RNA, plays key roles in suppressing host translation machinery and degradation of host mRNA.³ Encoded by ORF1a, Nsp1 is composed of a structured N-terminal domain (NTD) formed by six barrel-like β-strands flanked by two α-helices, a 3₁₀ helix, and a disordered C-terminal domain (CTD). Contributing to viral evasion of the host immune response, Nsp1 shuts down native translation by interacting with the 40S ribosomal subunit and blocking the mRNA entry channel (Figure 1 ). However, by mechanisms that are not fully understood, viral mRNA manages to escape translation inhibition. Hypotheses attempting to rationalize this apparent dichotomy have suggested that interactions between the NTD of Nsp1 and the viral mRNA 5′untranslated region (UTR) mediate selectivity for viral over host mRNA.⁴ Alternatively, other observations indicate that the viral 5′UTR does not show affinity for free Nsp1, and deletion of either the NTD or the CTD cause the viral load to decrease, suggesting the importance of both domains for the activity of the viral replication machinery.^{5 , 6}

Figure 1.

Nsp1 folded and disordered regions work in tandem to inhibit host translation

The solution state NMR structure of full-length Nsp1 reveals that the disordered C-terminal domain interacts with the RNA binding site in the folded domain. Upon Nsp1 association with the 40S subunit of the host ribosome, the disordered region inserts into the mRNA entry tunnel, freeing Nsp1 to bind viral mRNA.

In this issue of Structure, Wang et al. (2023) address this question by solving the high-resolution solution NMR structure of full-length Nsp1 (FL-Nsp1). Using a combination of restraints derived from NOEs, hydrogen bonds, residual dipolar couplings (RDCs), and chemical-shift-derived dihedral angles, the structure reveals a globular NTD (residues 10–127) flanked by a short nine residue N-terminal tail, and a large disordered CTD (residues 128–180).⁷ The structure presented here is the first full-length Nsp1 that resolves both the folded NTD, which is consistent with previously reported structures of truncated Nsp1, and the disordered CTD.⁷ While the structure of Nsp1 from SARS-CoV-2 compares well with SARS-CoV-1 Nsp1, major differences are found notably in loops 1 and 2 between β strands β3 and β4 and β4 and β5, respectively, which Lipari-Szabo model-free analysis characterized as flexible. Backbone dynamics analysis also confirmed that the CTD remains intrinsically disordered and thus highly flexible in solution except for a region of approximately 20 residues centered around N160 that have restricted dynamics. This is the same stretch of residues that was previous identified by the authors as having α-helical-like character based on chemical shift analysis.⁸ Indeed, the authors observe long-range NOEs indicating contact between CTD residue W161 and NTD residues R43 and K125, the latter being part of an important RNA binding patch comprising conserved residues LRKxGxKG. In addition to these NOEs, the authors observe more contacts between the NTD and the CTD through NOEs between sidechains of residues V89 and A131, and L177 and V35. Ambiguous NOEs corroborate interactions between the two domains.

Interestingly, chemical shift perturbation (CSP) and electro-mobility shift assays (EMSA) did not reveal binding of FL-Nsp1 to the SARS-CoV-2 mRNA 5′UTR, despite a preprint reporting that the NTD binds viral RNA.⁹ This discrepancy is proposed to be caused by blocking of the basic, positively charged RNA binding site, including the LRKxGxKG motif, by the acidic, negatively charged CTD that may serve in a sort of protecting role. The authors propose different hypotheses for this interaction, the NTD protects the unstructured CTD tail from degradation before it binds into mRNA entry tunnel of the ribosome inhibiting translation. Alternately, the NTD-to-CTD interaction may prevent non-specific binding between the NTD and viral RNA until the CTD binds to the 40S ribosomal subunit, which would agree with a previous model in which Nsp1 tethering the ribosome is concomitant to its recognition of RNA.⁶ Moreover, Wang et al. interpret the structural and dynamic data to address previous observations that found interactions between the NTD and viral RNA lead to its translation, whereas binding to host mRNA promotes its degradation. They propose that the intrinsically disordered linker region between the CTD and the NTD acts as a switch, contacting loop 2 and undergoing conformational changes that discriminate between host mRNA and viral 5′UTR RNA. This interpretation would also imply that the RNA binding mode of Nsp1 is dependent on both sequence and secondary or tertiary structural elements of the RNA.

This work is an example of how the dynamic information inherent to NMR measurements significantly contributes to the understanding of biological mechanisms at the atomic level. The authors present a high-resolution NMR structure of Nsp1 that includes both the ordered and disordered regions, which are found to interact with each other, information that is missing from typical reductionist structural approaches. This study is yet another case in the growing pile of evidence that illustrates the importance of including intrinsically disordered regions of proteins in structural studies and further reinforces the integral role the interplay between order and disorder has for regulating biological functions. The extent of intrinsic disorder in viral proteomes has been well documented but an understanding of how these flexible and dynamic regions contribute to viral pathogenesis has been poorly characterized. Despite a dearth of structural insights relative to folded proteins, disordered regions in proteins have outsized roles in viral biology, mediating interactions with host cell molecules and promoting viral pathogenesis through inhibition of host immune responses, modulating mRNA translation or processing, or promoting host protein dysfunction through degradation or aggregation mechanisms.¹⁰ The lack of fixed structural elements and subsequent conformational pliability in intrinsically disordered regions enables them to participate in specific yet transient interactions with multiple, diverse biomolecular targets including other intrinsically disordered regions, folded domains of proteins, and structured nucleic acids. Therefore, understanding the dynamics and structural features of viral intrinsic disorder such as in Nsp1 is a further step in deciphering the complex nature of protein plasticity in molecular mechanisms. Finally, the work by Wang et al. illustrates the importance of understanding the structural details of disorder-order interactions as they may serve as viable site for therapeutic targeting. For example, in the case of Nsp1, the authors propose that perturbing the interaction between the NTD and the CTD of Nsp1 through some highly competing peptide to the acidic patch of the NTD could impact its RNA-binding function and thus disrupt SARS-CoV-2 immune evasion mechanisms.