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Article

Review

J. Microbiol. Biotechnol. 2019; 29(8): 1184-1192

Published online August 28, 2019 https://doi.org/10.4014/jmb.1903.03053

Copyright © The Korean Society for Microbiology and Biotechnology.

Structure and Function of the Influenza A Virus Non-Structural Protein 1

Chang Woo Han 1, Mi Suk Jeong 2 and Se Bok Jang 1*

1Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 46241, Republic of Korea, 2Korea Nanobiotechnology Center, Pusan National University, Busan 46241, Republic of Korea

Correspondence to:Se Bok  Jang
sbjang@pusan.ac.kr

Received: March 25, 2019; Accepted: June 3, 2019

Abstract

The influenza A virus is a highly infectious respiratory pathogen that sickens many people with respiratory disease annually. To prevent outbreaks of this viral infection, an understanding of the characteristics of virus-host interaction and development of an anti-viral agent is urgently needed. The influenza A virus can infect mammalian species including humans, pigs, horses and seals. Furthermore, this virus can switch hosts and form a novel lineage. This so-called zoonotic infection provides an opportunity for virus adaptation to the new host and leads to pandemics. Most influenza A viruses express proteins that antagonize the antiviral defense of the host cell. The non-structural protein 1 (NS1) of the influenza A virus is the most important viral regulatory factor controlling cellular processes to modulate host cell gene expression and double-stranded RNA (dsRNA)-mediated antiviral response. This review focuses on the influenza A virus NS1 protein and outlines current issues including the life cycle of the influenza A virus, structural characterization of the influenza A virus NS1, interaction between NS1 and host immune response factor, and design of inhibitors resistant to the influenza A virus.

Keywords: Influenza A virus, NS1, virus-host interaction, antiviral defense, inhibitor

Fundamental Biology of Influenza Viruses

Influenza viruses cause a seasonal epidemic that can occur on a regular basis, posing a major threat to world health [1]. For the influenza virus to infect an organism, the host interferon (IFN) response must be overcomed first [2]. Interferons and other inborn immune mediators have devastating impacts on the result of influenza virus infections in terms of disease severity, since highly pathogenic avian influenza (HPAI) viruses are often associated with excessive cytokine responses [3]. Synthesis, structure and intracellular localization are essential to investigations of influenza virus infection, host response, antiviral drugs and vaccines [4]. Early influenza virus infections should eliminate the virus by inducing efficient activation of the IFN system. Recent studies have focused on understanding the fundamental biology of influenza viruses to meet these needs.

Influenza viruses belong to the Orthomyxoviridae family and are classified as either type A, B, C, or the recently identified type D [5, 6]. Influenza A viruses (IAVs) contain 8, negative-sense, single-stranded viral RNA (vRNA) gene segments, which encode transcripts for ten basic viral proteins, in addition, various strain-dependent accessory proteins [7]. The 8 genome segments of the influenza A virus have been shown to contain the genetic information for the following eight virus polypeptides: matrix1 (M1), matrix2 (M2), hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), non-structural protein 1 (NS1), non-structural protein 2 (NS2: also described as nuclear export protein, NEP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase basic protein 1-F2 (PB1-F2) [8]. Viral protein, notably the non-structural protein (NS1), is generally regarded as the common factor by which most influenza A viruses antagonize host innate immune responses [9]. NS1 is encoded by viral segment 8, which encodes the viral nuclear export protein NEP. In addition, the influenza A virus NS1 protein interacts with a variety of proteins to inhibit host cell immune responses and promote viral replication [10]. NS1 is important for multiple viral functions, including temporal adjustment of viral RNA synthesis, control of viral mRNA splicing, adjustment of virus particle morphogenesis, and suppression of host apoptotic response through the activation of phosphoinositide 3-kinase (PI3K) [11-13]. It has also been shown to interact with replication intermediates of viral RNA to block these molecules from recognition by cellular pattern recognition receptors (PRRs) [14]. Although the RNA-binding and effector domains of NS1 structures have been identified, the studies related to their specific protein-protein interactions of the full-length Influenza virus A NS1 are still uncertain [15].

Non-conservative mutations have been shown to enhance or decrease the virulence of the H1N1 virus by slightly stabilizing or destabilizing the structural domain of the NS1 or NS1-dsRNA, thereby increasing or decreasing the function of the NS1 protein. Other strains of the NS2 protein caused non-conservative mutations in other regions and were slightly impaired in function [16]. In the infected cell, NS1 is localized in the nucleus and NS2 in the cytoplasm; hence, the virus function was slightly inhibited and consequently decreased the pathogenicity of the virus. Thus, these studies justify the structure-function relationships of the viral NS1 and NS2 proteins. Recently, many research groups have been identifying full-length NS1 structures or specific interaction proteins that can inhibit influenza A virus NS1. Therefore, understanding the full-length NS1 protein can facilitate investigation of the interaction between influenza A virus and antiviral response.

Life Cycle of the Influenza A Virus

When the influenza A virus enters the body, the influenza HA molecule recognizes sialic (N-acetylneuraminic) acid on the host cell surface. HA is a type I transmembrane glycoprotein exposed on the virus surface as a homotrimer, in which each monomer consists of two subunits, HA1 and HA2 [17, 18]. In the low pH environment of the endosome, the cleaved HA with a fusogenic stalk domain of HA2 mediates fusion between the viral membrane and the endosomal membrane and permits entrance of viral ribonucleoprotein (vRNP) complex into the host cell. The vRNP complex consists of 8 negative-sense, single-stranded vRNA, nucleoprotein (NP) and the influenza A virus RNA polymerase (consisting of PA, PB1, and PB2) [19, 20] (Fig. 1). Subsequent fusion allows the vRNP complex to be released into the cellular cytoplasm, after which it enters the host nucleus by active transport. The nucleus is the where all influenza virus RNA synthesis takes place. To start the process of transcription, the virus RNA polymerase binds to the highly conserved and near complementary 13 nucleotides at the 5’ end and 12 nucleotides at the 3’ end of all eight segments. However, the influenza virus polymerase does not have inherent capping activity and it synthesizes viral messenger RNAs using the 5’ cap of host pre-mRNAs derived from cellular transcripts by a unique ‘cap-snatching’ mechanism that is mediated by the PB1 and PB2 proteins [21, 22]. Its replication mechanism is primer-independent and proceeds via a complementary RNA (cRNA) replicative intermediate [23]. Mature viral mRNAs are exported to the cytoplasm by the viral proteins M1 and NEP/NS2 and translated like host mRNA [24, 25]. Next, viral HA, NA, and M2 proteins are synthesized on the rough endoplasmic reticulum and matured in the Golgi complex. Finally, the formation of mature virus particles (virions) and budding occur at the plasma membrane [26]. In the later stage of the viral replication cycle, non-structural NS1 protein affects the morphogenesis of viral particles, but it is not a structural component of the viral particles [27].

Figure 1. Life cycle of the influenza A virus. The hemagglutinin (HA) spike on the viral envelope binds to a sialic acid receptor on the surface membrane of the infected cell and is then endocytosed. The virus is exposed to the cytoplasm and the viral ribonucleoprotein (vRNP) complex is released and then transported into the host cell nucleus. The vRNP acts as an independent functional unit that directs the synthesis of two positive-sense RNAs (mRNA and cRNA) by using its negative-sense vRNA as a template. After transcription and processing, positive-sense mRNAs are exported and translated in the cytoplasm. Newly-formed influenza virus RNPs are transported to the cytoplasmic membrane, where they are packaged into budding virions.

Structural Characterization of the NS1 Protein

The non-structural protein 1 (NS1) is a crucial influenza A virus immune regulator that antagonizes antiviral response. The NS1 protein counteracts the production of IFNs and the activities of IFN-induced proteins that restrict influenza virus replication [28]. Furthermore, NS1 is a multifunctional regulatory viral protein that plays a critical role as a post-transcriptional regulatory factor in the life cycle of influenza virus; specifically, binding virion RNA [29, 30]. NS1 protein comprises 215–237 amino acids (aa) relying on the strain, and has a molecular weight of about 26 kDa [31]. NS1 protein consists of 2 distinct functional domains connected by a flexible linker region (LR): an N-terminal RNA-binding domain (RBD, aa 1–73) and a C-terminal effector domain (ED, aa 74–207) (Fig. 2A). The nucleotide sequence (NLS) (aa 35–41) overlaps the sequences needed for dsRNA binding [32]. The NS1 protein can conduct functions in both the nucleus and cytoplasm. The sequences of NS1 proteins from independent virus strains are compared. H3N2 has sequence identities of 66.96% with H5N1 and 91.30% with H2N2. In addition, H3N2 has sequence identities of 84.78% with H1N1 and 76.04% with H7N9. The highest sequence variations appear in the C-terminal region of the ED (Fig. 2B).

Figure 2. Structure of the non-structural protein 1 (NS1) domain. NS1 is composed of N-terminal RNA-binding domain (RBD, amino acids 1–73) and C-terminal effector domain (ED, amino acids 74–207). (B) The sequence of NS1 proteins from different virus strains is largely conserved. (C) Schematic ribbon diagrams of the RNA-binding domain (PDB ID, 2N74) and effector domain (PDB ID, 3EE8).

The NS1 full-length is found as a homodimer in vitro with both the RNA-binding and effector domains bringing to dimerization [33]. The dimeric NS1 binds to the dsRNA duplex with a 1:1 stoichiometry [34]. The RBD is a six-helical symmetrical arrangement with each monomer consisting of three α-helices (Fig. 2C). Each monomer aligns to organize antiparallel strands configured with many conserved hydrophilic and basic amino acids, including the critical amino acids Arg 38 and Lys 41, which interact directly with the dsRNA [35]. In addition, bioinformatics and protein structural studies about the interface between the two monomers suggest that the N-terminal RBD forms a highly stable homodimer. The monomer of the c-terminal ED is comprised of 3 α-helices and 7 β-strands. The β-strands form an antiparallel twisted β-sheet surrounding one part of the central α3 long helix, which is held in place through an extensive network of hydrophobic interactions between the twisted β-sheet and the α-helix. The ED of NS1 from the influenza A virus also forms weak dimers in solution, with a helix-helix interaction mediated by tryptophan 187 [36]. This ED dimerization contributes to cooperativity in binding dsRNA by full-length NS1 proteins, which probably only occurs efficiently via cooperative binding of NS1 multimers.

In addition, the binding sites for the 30-kDa subunit of cleavage and polyadenylation specificity factor (CPSF 30) are near the ED of NS1 protein, and the CPSF 30 binding sites are located in the 144 and 186 amino acid regions. The NS1 protein possesses the sequence 226ARSK229 at its ED that is similar to the sequence 1ARTK4 at the N-terminal of histone H3. Analogous to the histone H3K4 sequence, host enzymes methylate and acetylate the terminal lysine in the 226ARSK229 sequence of the NS1 in vivo and in vitro. The methylated NS1 mimics H3 and binds to a specific transcription factor, polymerase-associated factor 1 complex (PAF1C) and chromodomain helicase DNA-binding protein 1 (CHD1), which results in inhibition of inducible expression of specific genes containing host antiviral genes. Influenza A virus strains circulating in humans differ in the capability of the NS1 protein to inhibit the activation of interferon-beta transcription and IRF3. The C-terminal difference of NS1 ED accounts for various functional differences among the NS1 proteins having different viral strains. The C-terminal region of ED is widely involved in giving to the IFN-b and IRF3 transcriptional phenotypes of the NS1 protein [37]. The homology of the amino acid at this site of the 196 position is closely related to the IFN-b and IRF3 transcriptional phenotypes of the NS1. The NS1 protein, which does not inhibit the activation of IFN-b and IRF3 transcription, contains K at this position, while the NS1 protein that inhibits this activation contains E at this position.

Interaction between NS1 and Host Immune Response Factors

The influenza A virus NS1 protein is expressed at very high levels in infected host cells. The mRNA that is transcribed from viral RNA segment 8 either encodes NS1, if unspliced, or nuclear export protein after being spliced [38]. The NS1 protein is known to bind to double-stranded RNA via N-terminal RNA-binding domain and it specifically binds to the region of the RNA containing either 5’- or 3’-terminal common genomic sequence [39]. The binding of influenza A virus NS1 protein to dsRNA protects the virus against the antiviral state induced by interferon-α/β (IFN-α/β), primarily by blocking activation of the 2’-5’-oligoadenylate synthetase (OAS)/RNase L pathway (Fig. 3) [40, 41]. Furthermore, the NS1 protein interacts with protein kinase R (PKR) to counteract PKR-induced eukaryotic translation inhibitor factor 2 (eIF2) [42]. Recently, NF90 was found to be involved in host antiviral mechanisms targeting various viruses, [43] regulates PKR activation and stress granule formation in influenza A virus-infected cells and is an interacting protein of influenza A virus NS1 [44]. NF90 displays its antiviral activity by matching the inhibitory role of NS1 on PKR phosphorylation. In addition, the NS1 RBD binds to E3 ubiquitin ligase tripartite motif-containing 25 (TRIM25) and inhibits ubiquitination of retinoic-acid-inducible protein 1 (RIG-I), which reduces the induction of RIG-I-mediated type I IFN [45]. RIG-I and melanoma differentiation-associated proteins (MDA-5) each recognize variant types of viral RNA. RIG-I is most effectively activated by shorter stretches of 5’-triphosphate or 5’-diphosphate dsRNA while MDA-5 is activated by long stretches of dsRNA in a 5’ phosphate-independent manner. Thus, the pathogen-recognition receptors have been seen to recognize variant RNA virus families [46, 47]. Knockdown research studies have demonstrated that IFN induction against influenza virus infection is majorly regulated by RIG-I. Influenza virus-induced IFN expression in the key site of influenza virus replication and airway epithelial cells is RIG-I-dependent [48]. However, MDA-5 may donate to IFN induction by influenza viruses, because small reductions in IFN induction and IFN-stimulated genes responses can be shown in infected MDA-5-deficient cells compared to control cells. In addition, IFNs are still expressed in the next infection with influenza virus or transfections with influenza RNA in chicken cells, which lack RIG-I. Alternatively, IFN induction in this process is adjusted by MDA-5 [49-51]. RNA synthesis of the influenza A virus is catalyzed by a viral polymerase consisting of PA, PB1 and PB2. Recent results have shown that host DDX21 RNA helicase binds to PB1 and inhibits polymerase assembly to limit influenza A virus, thereby reducing viral RNA and protein synthesis [52]. DDX21 binds to the NS1 N-terminal region, which as well joins in other important functions. Viral mutants in which the NS1 protein cannot bind to DDX21 reduce viral protein synthesis in the early and late steps of infection, and when DDX21 is knocked out, the phenotype is converted to the wild type.

Figure 3. The network of the host factors that interacts with the NS1 protein of influenza A virus. The N-terminal RNA-binding domain (RBD) of NS1 binds to the host cellular proteins through double-stranded RNA (dsRNA). RBD binds to the antiviral enzymes, including 2’-5’-oligoadenylate synthetase (2’-5’-OAS)/RNase L and nuclear factor 90 (NF90)-protein kinase R (PKR)/eukaryotic translation inhibitor factor 2 and inhibit the antiviral state. RBD interacts with E3 ubiquitin ligase tripartite motif-containing 25 (TRIM25) and retinoic-acid-inducible protein 1 (RIG-I), limiting the induction of RIG-I-mediated type I IFN (interferon). In addition, the interaction of RBD of the NS1 with RNA helicase DDX21 leads to regulation of viral gene expression. The C-terminal effector domain (ED) of NS1 interacts with the 30-kDa subunit of cellular cleavage (CPSF30)/poly (A)-binding protein II (PABII), eukaryotic translation initiation factor 4GI (eIF4GI), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt). Each of these interactions provide post-transcriptional blocks to cellular mRNA maturation, enhancement of viral mRNA translation, and PIK3/Akt signaling activation.

Another domain, the C-terminal effector domain of the influenza A virus NS1 protein, interacts with several host factors. Specifically, the NS1 protein provides the critical function of blocking the processing of 3’-terminal cellular or viral pre-mRNAs by binding two cellular proteins: CPSF30 and poly (A)-binding protein II (PABII). As a result of the sequestering of CPSF30 by the NS1 protein, unprocessed cellular pre-mRNAs accumulate in the nucleus, and cellular mRNA production in the cytoplasm is inhibited [53, 54]. In addition, NS1 preferentially enhances translation of the influenza virus mRNA by recruiting eukaryotic translation initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, specifically to its 5’-untranslated region (5’UTR) [55]. Furthermore, the NS1 has been required in control to the phosphatidylinositol 3-kinase (PI3K) activation by binding the PI3K p85β subunit, resulting in the activation of PI3K/protein kinase B (Akt) signaling [36]. Thus, influenza A virus NS1 protein inhibits or activates signaling pathways to insure effective virus replication. Other recent studies have concentrated on the interaction of NS1 mutants with host immune response factors. Mutations of the NS1 protein have been shown to impact on host cell IFN responses and the reduced capability of the NS1 mutant viruses to induce IFN gene. The protein expression is associated with reduced activation of STAT1 and a lower level of ISG expression [10]. The results were demonstrated by mutation at the position 171 in the NS1 resulting in reduced expression of IFN and ISGs by A549 cells.

Inhibitor Development against Influenza A Virus NS1

One of the inhibitors reported, JJ3297, is effected on NS1-induced inhibition of IFN mRNA production, and it depends strictly on the presence of RNase L. The action of JJ3297 is insensitive to other IFN signaling pathway elements such as RIG-I [56]. In another study, several chemical compounds inhibited both influenza virus replication and NS1 function. The compounds (NSC95676, NSC109834, and NSC128164) launched a considerable reduction in viral NS1-specific RNAs (NS1 and M2). However, the infected cells by influenza A NS1 gene knockout viruses (delNS1) did not show the same decrease in overall viral RNA levels. The compounds are active only in the existence of NS1 [57]. Another study emphasized that interference with the interaction between NS1 and CPSF30 is a research target. The second and third zinc fingers (F2F3) domain of CPSF30 binds efficiently to the NS1 protein. Interestingly, cellular expression of a small fragment of CPSF30 sufficient to bind NS1 has been shown to inhibit virus replication and increase production of IFN-β mRNA [58].

Petit et al. have described the binding site and inhibition mechanism of two small-molecule experimental inhibitors of influenza A viruses in detail [59]. They showed that the inhibitors, A9 and A22, interacted with just one of the two separately folded domains of NS1, the NS1 effector domain. In addition, the structures of the antiviral compounds were highly analogous to a fraction of a host CPSF30 protein that the NS1 ED binds to short-circuit the body's immune response. Thus, A9 and A22 exhibited to inhibit influenza viral replication and NS1 function by interrupting with the interaction between CPSF30 and the NS1 effector domain. Other biological studies have shown that Arg 38 of the RBD interacts with dsRNA directly. For example, the influenza A/Udorn/307/72 virus (H3N2) that encodes an R38A NS1 protein is very attenuated, suggesting that small molecule drugs that interrupt NS1-dsRNA binding should block viral replication [60]. Accordingly, the NS1-dsRNA interaction is a prospective target for inhibition of the small molecule drugs.

Much progress has been achieved in studies of the influenza virus. However, the influenza virus continues to be a major health threat, especially with respect to the NS1 protein, which is a potent toxic factor that antagonizes host immune response. Recently, several studies have also seen that NS1 affects the morphogenesis of the virus particle. For this reason, research groups are searching for small molecules or peptides that can inhibit NS1 [56, 57]. Although some inhibitors have been developed, there are still many problems that need to be addressed. Every year, influenza viruses kill 250,000 to 500,000 people all over the world. The virus is known to quickly evolve to bring about pandemic strains that few people have immunity to resist. Viral resistance has hindered the effectiveness of a few initial antiviral agents that were worked up to cure influenza; therefore, there is a great need for novel antiviral compounds. In this review, the crucial role of the influenza A virus NS1 protein in negative regulation of innate antiviral responses is explained and the need for better insight into the function of NS1 protein is suggested. A more complete understanding of the structure and function of this influenza A virus NS1 protein will be applicable to the development of new anti-influenza therapies.

Acknowledgments

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2018R1D1A1B07043701) to S.B.J. and (2016R1D1A1B02011142) to M.S.J.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Life cycle of the influenza A virus. The hemagglutinin (HA) spike on the viral envelope binds to a sialic acid receptor on the surface membrane of the infected cell and is then endocytosed. The virus is exposed to the cytoplasm and the viral ribonucleoprotein (vRNP) complex is released and then transported into the host cell nucleus. The vRNP acts as an independent functional unit that directs the synthesis of two positive-sense RNAs (mRNA and cRNA) by using its negative-sense vRNA as a template. After transcription and processing, positive-sense mRNAs are exported and translated in the cytoplasm. Newly-formed influenza virus RNPs are transported to the cytoplasmic membrane, where they are packaged into budding virions.
Journal of Microbiology and Biotechnology 2019; 29: 1184-1192https://doi.org/10.4014/jmb.1903.03053

Fig 2.

Figure 2.Structure of the non-structural protein 1 (NS1) domain. NS1 is composed of N-terminal RNA-binding domain (RBD, amino acids 1–73) and C-terminal effector domain (ED, amino acids 74–207). (B) The sequence of NS1 proteins from different virus strains is largely conserved. (C) Schematic ribbon diagrams of the RNA-binding domain (PDB ID, 2N74) and effector domain (PDB ID, 3EE8).
Journal of Microbiology and Biotechnology 2019; 29: 1184-1192https://doi.org/10.4014/jmb.1903.03053

Fig 3.

Figure 3.The network of the host factors that interacts with the NS1 protein of influenza A virus. The N-terminal RNA-binding domain (RBD) of NS1 binds to the host cellular proteins through double-stranded RNA (dsRNA). RBD binds to the antiviral enzymes, including 2’-5’-oligoadenylate synthetase (2’-5’-OAS)/RNase L and nuclear factor 90 (NF90)-protein kinase R (PKR)/eukaryotic translation inhibitor factor 2 and inhibit the antiviral state. RBD interacts with E3 ubiquitin ligase tripartite motif-containing 25 (TRIM25) and retinoic-acid-inducible protein 1 (RIG-I), limiting the induction of RIG-I-mediated type I IFN (interferon). In addition, the interaction of RBD of the NS1 with RNA helicase DDX21 leads to regulation of viral gene expression. The C-terminal effector domain (ED) of NS1 interacts with the 30-kDa subunit of cellular cleavage (CPSF30)/poly (A)-binding protein II (PABII), eukaryotic translation initiation factor 4GI (eIF4GI), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt). Each of these interactions provide post-transcriptional blocks to cellular mRNA maturation, enhancement of viral mRNA translation, and PIK3/Akt signaling activation.
Journal of Microbiology and Biotechnology 2019; 29: 1184-1192https://doi.org/10.4014/jmb.1903.03053

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