Antiviral responses versus virus-induced cellular shutoff: a game of thrones between influenza A virus NS1 and SARS-CoV-2 Nsp1

Abstract

Following virus recognition of host cell receptors and viral particle/genome internalization, viruses replicate in the host via hijacking essential host cell machinery components to evade the provoked antiviral innate immunity against the invading pathogen. Respiratory viral infections are usually acute with the ability to activate pattern recognition receptors (PRRs) in/on host cells, resulting in the production and release of interferons (IFNs), proinflammatory cytokines, chemokines, and IFN-stimulated genes (ISGs) to reduce virus fitness and mitigate infection. Nevertheless, the game between viruses and the host is a complicated and dynamic process, in which they restrict each other via specific factors to maintain their own advantages and win this game. The primary role of the non-structural protein 1 (NS1 and Nsp1) of influenza A viruses (IAV) and the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respectively, is to control antiviral host-induced innate immune responses. This review provides a comprehensive overview of the genesis, spatial structure, viral and cellular interactors, and the mechanisms underlying the unique biological functions of IAV NS1 and SARS-CoV-2 Nsp1 in infected host cells. We also highlight the role of both non-structural proteins in modulating viral replication and pathogenicity. Eventually, and because of their important role during viral infection, we also describe their promising potential as targets for antiviral therapy and the development of live attenuated vaccines (LAV). Conclusively, both IAV NS1 and SARS-CoV-2 Nsp1 play an important role in virus-host interactions, viral replication, and pathogenesis, and pave the way to develop novel prophylactic and/or therapeutic interventions for the treatment of these important human respiratory viral pathogens.

Keywords: NS1; Nsp1; SARS-CoV-2; antivirals; influenza A virus; innate immunity; live attenuated vaccines; non-structural protein 1.

Figure 1

Schematic illustration of IAV NS segment and SARS-CoV-2 genome organization. (A) Schematic representation of the unspliced and spliced mRNA forms transcribed from the IAV NS vRNA segment. The unspliced mRNA transcript encodes for the NS1 protein while the spliced mRNA transcripts encode the NS2 or NEP and NS3 protein which is typically the NS1 mRNA transcript with an internal deletion. (B) SARS-CoV-2 vRNA genome organization and viral structural (S, E, M and N; green), accessory (ORF 3a, 3b, 6, 7a, 7b, 8, 9b, 9c and 10; gray), and non-structural (Nsp1-16) proteins encoded from ORF1a (orange) and ORF1b (light blue). HCoV Nsp1 is expressed from the N-terminal of ORF1a. NCR, Non-coding region; ORF, Open reading frame; m7G, 7-methylguanosine (m7G) cap structure; AAA, poly (A) tail; Nsp, Non-structural protein; PLpro, papain-like protease; 3CLPro, 3-chymotrypsin-like protease; Mpro, main protease. Figure was created with BioRender.com.

Figure 2

Structural analyses of IAV NS1 and SARS-CoV-2 Nsp1. (A) Functional motifs of IAV NS1 include the RNA-binding domain (RBD) or N-terminal domain (NTD), followed by a short linker peptide (LP), the C-terminal domain (CTD) or effector domain (ED), and the strain specific C-terminal tail (CTT). Amino acid (aa) residues are indicated by numbers in the primary structure of IAV NS1 (bottom). IAV NS1 monomer adapted from the X-ray structure of H5N1 NS1 (PDB ID: 3F5T) (Bornholdt and Prasad, 2008), showing the NTD, LP with a 5 aa deletion (residues 80-84), predominant in IAV H5N1 NS1 since 2003 (Li et al., 2014), and the CTD (top). (B) SARS-CoV-2 Nsp1 consists of a short N-terminal tail (NTT), an N-terminal domain (NTD), and a C-terminal tail (CTT) that contains a linker peptide (LP) and the C-terminal domain (CTD). The aa residues are indicated by numbers in the primary structure of SARS-CoV-2 Nsp1 (bottom). SARS-CoV-2 Nsp1 monomer adapted from the X-ray structure (PDB: 8AOU) is shown on top (Wang et al., 2023). Figure was assembled with BioRender.com.

Figure 3

Nuclear localization signals (NLS), nuclear export signal (NES), and documented post-translational modifications (PTMs) of IAV NS1 protein. Typical IAV NS1 proteins contain two nuclear localization signals (NLS1 and NLS2). The NLS2 is overlapping with a nucleolar localization signal (NoLS) forming the nuclear/nucleolar localization signal (NLS2/NoLS). In addition, IAV NS1 also contains a NES. IAV NS1 protein is usually subjected to crucial PTM in infected cells, including phosphorylation (green), ubiquitination via SUMOylation (red) and ISGylation (orange), and acetylation (blue). The PTMs are indicated according to their position in IAV NS1. Figure was created with BioRender.com.

Figure 4

Schematic representation of cellular/viral interactors of IAV NS1 and SARS-CoV-2 Nsp1. (A) IAV NS1 interacts with four groups of proteins, which are involved in the maturation and nucleo-cytoplasmic export of cellular mRNAs (CPSF30, PABPII, nucleolin, NXF1/TAP and several other proteins of the nuclear export machinery), proteins involved in mRNA transport and translation (hstaufen, eIF4GI and PABPI), proteins involved in the IFN antiviral response and signaling cascades (RIG-I, TRIM-25, PKR, PACT, p85-b subunit of PI3K and PDZ-containing proteins), and eventually viral proteins including NP and viral polymerase components (highlighted in red) (Knipe and Howley, 2013). (B) SARS-CoV-2 Nsp1 shuts off innate immune responses via indirect or direct interaction of its NTD with fundamental cellular proteins, including NXF1, and interaction of its CTD with the 40S and 80S ribosome subunits. SARS-CoV-2 Nsp1 NTD can also selectively mediate the translation of viral mRNA via interaction with the stem-loop 1 (SL1) region of the SARS-CoV-2 5’ untranslated region (UTR, highlighted in red). Figure was created with BioRender.com.

Figure 5

IAV NS1 and SARS-CoV-2 Nsp1 antagonism of innate immune responses. The induction of innate immune response is initiated by triggering TLRs and RLRs pathways after IAV (red) or SARS-CoV-2 (purple) entrance to susceptible cells. The recognition of PAMPs by PRRs leads to the activation of a signal transduction cascade that involves several modulators, including, among others, TRIF, MyD88, PACT, TRIM25, and Riplet, which leads to the phosphorylation of IRF3/7. Nuclear translocation of phosphorylated IRF3/7 induces the transcription of IFN. Through the Janus kinase (JAK)1 and TYK2 signaling pathway, binding of IFN to the IFNR1/2 receptors leads to the phosphorylation of transcription factors STAT1 and STAT2 which together with IRF9 translocate to the nucleus resulting in the expression of hundreds of ISGs with antiviral activity. Both IAV NS1 (red) and SARS-CoV-2 Nsp1 (purple) can interact with various cellular host factors to inhibit activation of IRF3/7, prevent the maturation and nuclear exportation of cellular mRNA, blocking the loading of cellular mRNA to the ribosomes (Nsp1), or counteracting the antiviral response of different ISGs. This figure was created with Biorender.com.

Figure 6

Effect of CTD truncations in IAV NS1 on IAV pathogenicity and virulence. (A) Schematic representation of the WT and truncated NS1 1-73, 1-99, and 1-126 of EIV, SIV, and CIV. NS1 and NEP ORF are represented as grey and black boxes, respectively. The 3′ and 5′ NCR of the IAV NS vRNA are represented in white boxes. Black solid lines represent stop codons introduced to generate the NS1 truncations. (B) The virulence of each NS1-encoded IAV in different animal species is represented in different colors red (EIV), black (SIV), and violet (CIV). Decreasing the intensity of the color indicates the attenuation of each respective virus. (C) The impact of NS1-encoded IAV on the induction of innate IFN responses is represented where IFN induction/production is lowest in case of WT NS1 and increasing proportionally with the length of the NS1 (i.e., NS1 1-73 < NS1 1-99 < NS1 1-126). This figure was created with BioRender.com.

Figure 7

Schematic representation of novel LAV based on IAV NS1. The different approaches described in this review to generate new LAV for IAV based on alterations on NS1 are shown: (A) IAV NS WT segment: NS1 and NEP ORF are indicated in light green or black boxes, respectively. For additional information see Figure 1A . (B) IAV with truncated NS1 (Yellow): Although many truncated IAV NS1 proteins have been described, only one general example is shown. IAV NS segments with truncated NS1 proteins are affected in counteracting host antiviral immune responses (IFN and cytokines), which results in affecting viral replication and propagation. (C) NS1 deficient IAV: In this LAV approach, only the ORF of NEP is expressed and the mechanism of attenuation is like the one of viruses encoding truncated NS1 proteins (B). (D) IAV with modified NS1 and/or PA-X proteins: In this approach the properties of NS1 and PA-X to inhibit host gene expression are modified, affecting the ability of the virus to counteract innate immune responses. (E) IAV with CD NS: IAV expressing codon deoptimized (CD) NS1 (NS1cd) or both NS1 and NEP (NScd) ORF were generated. NS1 CD and NEP CD ORF are indicated with light blue boxes. In this approach reduced levels of NS1 (NS1cd) or both NS1 and NEP (NScd) result in viral attenuation. (F) IAV with modified M and NS segments: Recombinant IAV with modified vRNA segment 7 (M, brown) alone or in combination with segment 8 (NS), in which the overlapping ORF of the M1 and M2 proteins (Ms), and NS1 and NEP proteins (NSs) are produced from the same transcript using the PTV-1 2A autoproteolytic cleavage site. Infection with IAV encoding Ms results in viral attenuation at high temperatures (37°C or 39°C), showing a temperature sensitive (ts) and attenuated (att) phenotype. Addition of NSs into the Ms increased the att profile of the modified Ms IAV LAV. (G) Rearranged of viral segments 2 (PB1) and 8 (NS): A modified PB1 viral segment encoding PB1 and NEP using the previously described 2A approach results in a reduction in its polymerase activity. The expression of H5 from modified segment 8 containing a truncated NS1 using a FMDV2A autocleavage site results in a bivalent LAV against two different IAV (H9 and H5, red boxes). This figure was created with Biorender.com.

Figure 8

Schematic representation of a LAV approach for SARS-CoV-2. (A) Representation of SARS-CoV-2 encoding Nsp1 WT (light yellow). (B) In the SARS-CoV-2 LAV approach, the polybasic sequence of the viral S protein and the accessory ORF6-8 proteins were removed, together with K164A and H165A mutations in Nsp1 (light blue) to reduce its ability to inhibit host gene expression. This figure was created with Biorender.com.