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Review |
1 Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9ST, UK
2 Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain
Correspondence
David Jackson
dj10{at}st-andrews.ac.uk
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ABSTRACT |
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Published online ahead of print on 1 August 2008 as DOI 10.1099/vir.0.2008/004606-0.
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Influenza A viruses |
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). Influenza A viruses are important pathogens with worldwide prevalence. Their natural host is waterfowl; however, they also infect humans and other animals such as pigs, horses and various avian species (Webster et al., 1992). It is this zoonotic characteristic that allows the generation of potentially pandemic strains. Three human pandemics occurred during the last century, with the 1918 ‘Spanish’ influenza pandemic resulting in up to 40 million deaths (Simonsen et al., 1997). Influenza viruses also cause seasonal epidemics, which are due to the acquisition of mutations in the viral surface glycoproteins. Disease severity caused by these strains is limited in the general population; however, they can be fatal in elderly patients and those with underlying pulmonary or cardiac diseases (Barker & Mullooly, 1982).
To restrict virus proliferation, virus-infected cells usually mount a potent and diverse antiviral response (Randall & Goodbourn, 2008). Thus, to survive in nature, influenza A viruses have evolved multiple mechanisms to circumvent these defences. Some strategies are strain-specific, such as increased replication speed (Grimm et al., 2007; Kurokawa et al., 1999), or decreased sensitivity to host-cell antiviral effectors (Dittmann et al., 2008). The viral NS1 protein is widely regarded as the common factor by which all influenza A viruses antagonize host immune responses (Egorov et al., 1998; Garcia-Sastre et al., 1998; Kochs et al., 2007b). Indeed, mutant influenza A viruses unable to express NS1 only display high pathogenicity in mice lacking antiviral mediators such as STAT1 (Garcia-Sastre et al., 1998), or the dsRNA-activated protein kinase, PKR (Bergmann et al., 2000; Kochs et al., 2007b). Thus, the available data strongly indicate that the major function of NS1 in current in vivo models is to antagonize IFN-
/β-mediated antiviral responses. However, NS1 is a multifunctional protein that performs a plethora of activities, which may additionally contribute towards efficient virus replication and virulence during infection. These include: (i) temporal regulation of viral RNA synthesis; (ii) control of viral mRNA splicing; (iii) enhancement of viral mRNA translation; (iv) regulation of virus particle morphogenesis; (v) suppression of host immune/apoptotic responses; (vi) activation of phosphoinositide 3-kinase (PI3K); and (vii) involvement in strain-dependent pathogenesis. All of these functions of NS1 rely on its ability to participate in a multitude of protein–protein and protein–RNA interactions (summarized in Figs 1 and 2). Here, we review the various roles of NS1 during the replication cycle of influenza A viruses. We highlight the potential importance of each individual function and discuss how a single protein might have such a multifunctional nature.
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Synthesis and biochemistry of NS1 |
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; Palese & Shaw, 2007). It is encoded on a collinear mRNA derived from vRNA segment eight, which upon splicing results in the synthesis of the nuclear export protein mRNA (NEP, previously termed NS2) (Inglis et al., 1979; Lamb & Choppin, 1979). Both mRNA species share 56 nucleotides at the 5' end, resulting in NS1 and NEP sharing 10 N-terminal amino acids (Lamb & Lai, 1980). In infected cells, the steady-state amount of spliced NEP mRNA is only approximately 10 % that of unspliced NS1 mRNA (Lamb et al., 1980). As described below, regulation of splicing is controlled, in part, by the viral NS1 protein itself (Garaigorta & Ortin, 2007) and may represent a mechanism for autoregulation of protein levels within infected cells.
NS1 has a strain-specific length of 230–237 aa, and an approximate molecular mass of 26 kDa (Palese & Shaw, 2007). However, naturally occurring NS1 proteins with C-terminal truncations (
15–30 aa) are not uncommon (Suarez & Perdue, 1998). Furthermore, sequence analysis shows that, during the 1940s, the 230 aa long NS1 protein of circulating human H1N1 viruses gained a 7 aa C-terminal extension via a single nucleotide mutation (Fig. 4). This extension was subsequently retained in human H1N1, H2N2 and H3N2 viruses until the 1980s, when both co-circulating H1N1 and H3N2 viruses lost the extension via reversion of the original mutation. The significance of the extension and why it was subsequently lost is not entirely clear, although it has recently been functionally implicated in the nuclear and nucleolar localization of NS1 (Melen et al., 2007). Given the variability in NS1 length, the general importance of reported interactions between the NS1 C terminus and various cellular proteins, such as poly(A)-binding protein I (PABPI) and PDZ domain-containing proteins, remains unclear.
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; Ludwig et al., 1991). A number of NS1 proteins from avian influenza viruses together with those of all human, swine and equine influenza viruses are described as allele A NS1 proteins, whereas those of allele B are exclusively from avian viruses. The level of homology within each allele is 93–100 %; however, between alleles it can be as little as 62 %. When a recombinant human virus containing an allele B NS1 protein was used to infect squirrel monkeys, there was a decrease in the ability of the virus to replicate in the respiratory tract compared with wild-type (wt) virus (Treanor et al., 1989). This suggested that allele A NS1 proteins have a replicative advantage in mammalian hosts. Further analysis revealed that allele A NS1 proteins are under continual selection pressure to mutate, whereas those of allele B are not (Ludwig et al., 1991). It is possible that allele B NS1 proteins represent the archaic version of this protein and that, after entering the human influenza virus population via reassortment events, NS1 has been under a strong selection pressure to mutate, giving rise to the allele A NS1 proteins. The large degree of evolutionary divergence between the two alleles may indicate that there are significant functional constraints on NS1 proteins between host species. The significance of NS1 alleles for the virulence and pathogenicity of certain influenza viruses is not clear; however, the majority of highly pathogenic avian influenza viruses isolated from humans have contained an allele A NS1 protein (Zohari et al., 2008).
Post-translational modification of NS1 proteins may also be a strain-specific virus polymorphism. Indeed, phosphorylation of NS1 has been reported for only some influenza A viruses (Petri et al., 1982), and at least two distinct sites of modification have been proposed based upon biochemical and structural work: Ser-195 and Thr-197 (Bornholdt & Prasad, 2006; Privalsky & Penhoet, 1981). However, phosphorylation of these two residues has yet to be experimentally confirmed. NS1 phosphorylation appears to occur rapidly after translation, within the cell nucleus (Privalsky & Penhoet, 1981). It is still unknown if any physiological role for NS1 phosphorylation exists.
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Structure of the NS1 protein |
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), which in vitro binds with low affinity to several RNA species in a sequence independent manner (Chien et al., 2004; Hatada & Fukuda, 1992; Qian et al., 1995), and a C-terminal ‘effector’ domain (residues 74–230) (Fig. 3b, c), which predominantly mediates interactions with host-cell proteins, but also functionally stabilizes the RNA-binding domain (Wang et al., 2002). Full-length NS1 likely exists as a homodimer, with both the RNA-binding and effector domains contributing to multimerization (Nemeroff et al., 1995).
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-helices (Chien et al., 1997; Liu et al., 1997). Dimerization is essential for binding dsRNA (Wang et al., 1999) and the stoichiometry of dimer : dsRNA is 1 : 1 (Chien et al., 2004). Two identical helices from each NS1 monomer contribute towards dsRNA-binding by forming antiparallel ‘tracks’ on either side of a deep cleft (Liu et al., 1997). The ‘tracks’ consist of conserved basic and hydrophilic residues that appear to form complementary contacts with the polyphosphate backbone of dsRNA (Yin et al., 2007). Residues in NS1 that mediate this interaction, either directly or via improving complex stability, include Thr-5, Pro-31, Asp-34, Arg-35, Arg-38, Lys-41, Gly-45, Arg-46 and Thr-49 (Wang et al., 1999; Yin et al., 2007). It should be noted that alanines are commonly substituted for both Arg-38 and Lys-41 in many experimental studies in order to abrogate the RNA-binding activity of NS1 (Fig. 3a
).
Crystallographic studies revealed that the C-terminal effector domain of both a human and avian NS1 protein (residues 74–230) can independently homodimerize, with each monomer consisting of seven β-strands and three -helices (Bornholdt & Prasad, 2006; Hale et al., 2008a). Within each monomer, the β-strands form a twisted, crescent-like, anti-parallel β-sheet around a long, central -helix. There is currently no structure available for the C-terminal 25 amino acids of NS1, a region which is involved in many strain-specific functions (Fig. 1). It is possible that this stretch of NS1 is intrinsically disordered, and thus may only adopt an ordered structure upon binding the appropriate ligand. Such intrinsic disorder is noteworthy given the C-terminal variability in the lengths of many NS1 proteins.
The precise dimeric assembly of the NS1 effector domain has yet to be fully established, as two dimer conformations have recently been proposed: strand–strand (Bornholdt & Prasad, 2006), and helix–helix (Hale et al., 2008a) (Fig. 3b, c). Amino acids involved at both dimer interfaces appear reasonably well-conserved; however, biochemical evidence indicates that Trp-187 (a residue located at the helix–helix interface) is essential for dimerization of an avian NS1 effector domain in solution (Hale et al., 2008a). This suggests that the helix–helix dimer, at least for the avian NS1 protein used, is likely to be biologically relevant (Fig. 3b, c). It should be noted that the published human effector domain structure is from an allele A NS1 protein (Bornholdt & Prasad, 2006), whilst that published for an avian influenza virus is from an allele B NS1 protein (Hale et al., 2008a). Thus, it may be that the structural differences observed in these two studies are NS1 allele-specific. Interestingly, very recent data suggest that a third dimeric state of the NS1 effector domain also probably exists (PDB ID: 2RHK). As a full-length NS1 protein structure has yet to be determined, the actual conformation of the complete NS1 dimer may differ significantly from that already published for the two individual domains. Alternatively, it may be that NS1 has various dimeric states that occur in either a strain- or ligand-specific manner, a mode of action that would clearly contribute to the multifunctional nature of NS1.
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Intracellular localization of NS1 |
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; Newby et al., 2007). Nevertheless, in virus-infected cells NS1 predominantly localizes to the nucleus, but a significant proportion can also be found in the cytoplasm (Greenspan et al., 1988; Newby et al., 2007), particularly at later times post-infection (Garaigorta et al., 2005; Melen et al., 2007). Within the nucleus, NS1 has been shown to localize to ND10 structures (Sato et al., 2003).
Depending on the viral strain, NS1 contains one or two nuclear localization sequences (NLS) (Fig. 1) (Greenspan et al., 1988), which mediate the active nuclear import of NS1 via binding to cellular importin- (Melen et al., 2007). As such, translocation of NS1 into the nucleus is extremely rapid (Privalsky & Penhoet, 1981). NLS1 is highly conserved, monopartite, and involves three residues also involved in binding dsRNA (Arg-35, Arg-38 and Lys-41). In contrast, the bipartite NLS2 comprises specific amino acids (Lys-219, Arg-220, Arg-231 and Arg-232) found at the C-termini of some NS1 proteins (Melen et al., 2007). As NLS2 is absent from the NS1 proteins of a large number of virus strains, it is difficult to ascribe a function to this sequence with regard to viral replication. Concurrent with NLS2 is a functional nucleolar localization signal (NoLS), which includes additional basic residues (Arg-224 and Arg-229) (Melen et al., 2007) (Fig. 1). Interestingly, NS1 has recently been shown to interact with nucleolin (Murayama et al., 2007), a major multifunctional nucleolar protein (Fig. 1). Despite this, the nucleolar function of NS1 is unknown; however, a mutant influenza A virus expressing a truncated NS1 protein unable to localize to nucleoli was not attenuated for replication in tissue culture (Melen et al., 2007).
Cytoplasmic localization of a subpopulation of NS1 is potentially regulated by three mechanisms. It is possible that newly synthesized NS1 is initially sequestered in the cytoplasm by a cellular or viral binding partner that acts by masking the NLS. Alternatively, it has been reported that a latent nuclear export signal (NES) in NS1 causes its nucleo-cytoplasmic transport (Li et al., 1998). The NES lies within residues 138–147, requires leucines at positions 144 and 146, and is normally ‘masked’ by residues 148–161 which lie adjacent to it (Li et al., 1998). Thus, during infection the NES probably requires ‘unmasking’ in the nucleus for cytoplasmic localization of NS1 to occur. Additionally, it is possible that competition between the NLS and NES exists, such that the NES only becomes dominant after the NLS itself has also been masked by a nuclear NS1-binding partner. The molecular events that govern these three putative mechanisms have yet to be established, but it is likely that specific cellular factors play key roles in determining the intracellular localization of NS1. For example, regulation by phosphorylation of NS1 is a possibility, given that mutation of Ser-195, a potential phosphorylation site in NS1 (Bornholdt & Prasad, 2006), appears to contribute to the nuclear retention of NS1 (Garaigorta et al., 2005). Varied intracellular distribution of NS1 during infection may be essential for its ability to perform different functions.
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Role of NS1 in regulating splicing of segment eight mRNAs |
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; Lu et al., 1994). Although the segment eight pre-mRNA is able to form spliceosomes, the subsequent catalytic steps appear to be inhibited by NS1 in a process requiring specific basic residues within the RNA-binding domain (Lu et al., 1994). This has recently been confirmed by experiments in which NS1 was synthesized from functional vRNPs, resulting in the inhibition of segment eight mRNA splicing (Garaigorta & Ortin, 2007). Such inhibition required the N-terminal region of NS1, but appeared independent of RNA-binding. It was also found that NS1 specifically downregulated nuclear export of its own mRNA by a process requiring NS1 RNA-binding activity (Alonso-Caplen & Krug, 1991; Alonso-Caplen et al., 1992; Garaigorta & Ortin, 2007). The biological reasons for this are currently unknown.
The mechanism by which NS1 inhibits segment eight mRNA splicing has yet to be fully established. However, it is possible that a novel cellular 70 kDa NS1-binding protein, termed NS1-BP, may be involved. NS1-BP was initially identified as an interaction partner for NS1 in yeast two-hybrid screens (Wolff et al., 1998). Given that NS1-BP predominantly co-localizes with the spliceosome assembly factor SC35, it was suggested that this protein is normally involved in cellular mRNA splicing. During influenza A virus infection, the cytoplasmic fraction of NS1-BP redistributes to the nucleus, and apparently co-localizes with NS1 (Wolff et al., 1998). Similar immunofluorescence experiments have demonstrated that NS1 expression causes redistribution of cellular splicing factors in nuclei of infected cells (Fortes et al., 1995). These reports, together with findings that NS1 can bind and disrupt complexes between specific small nuclear RNAs (snRNAs) (essential components of spliceosomes), highlight likely biological interactions between NS1 and the cellular mRNA splicing machinery (Lu et al., 1994; Qiu et al., 1995; Wang & Krug, 1998).
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Effects of NS1 expression on virus-specific RNA and protein synthesis |
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). This observation was confirmed using viruses expressing NS1 proteins with C-terminal truncations; however, such a phenotype appeared to be strain-specific (Falcon et al., 2004). These reports gave the first indications that NS1 plays a role in controlling viral RNA replication during infection. Substitution of residues 123 and 124 in NS1 was shown to prevent the NS1-mediated binding and inhibition of the dsRNA-activated antiviral protein kinase, PKR (Min et al., 2007). Thus, this mutant induced PKR activation and exhibited reduced viral protein synthesis at late times post-infection. However, the mutant virus was not attenuated in tissue-culture due to enhanced vRNA synthesis at early times post-infection, which resulted in increased viral mRNA transcription and early viral protein synthesis. Given that these effects were apparently independent of PKR, it was speculated that the same residues in wt NS1 normally act to temporally regulate vRNA synthesis during infection. At the mechanistic level, NS1 has previously been reported to interact with the viral polymerase complex (Marion et al., 1997b) and has a high affinity for dsRNA in the form of vRNA-like panhandle structures (Hatada & Fukuda, 1992; Hatada et al., 1997). Indeed, evidence is accumulating for a number of functional interactions between NS1 and replicating RNPs during infection (Twu et al., 2007).
Selective translation of viral mRNAs
It has been reported that, during influenza A virus infection, there is selective translation of viral mRNAs over cellular mRNAs, a process possibly mediated by sequences in the 5'UTR of viral mRNAs (Garfinkel & Katze, 1993). A number of proteins appear to bind the 5'UTR of viral mRNAs, including NS1 (Park & Katze, 1995), and many studies have attempted to determine the effect of NS1 expression on viral protein synthesis. It was reported that NS1 increases translation initiation of viral mRNAs within transfected cells, but does not affect the translation of non-viral mRNAs (de la Luna et al., 1995). It was shown that the 5'UTR sequences of viral mRNAs were responsible for this selective translation. Similarly, Enami et al. (1994) demonstrated that NS1 does not affect viral mRNA transcription, but rather enhances translation in a viral 5'UTR-dependent manner. However, unlike de la Luna et al., these authors were unable to observe an effect of NS1 on translation of mRNAs containing the 5'UTR from vRNA segment eight (Enami et al., 1994). Thus, it is possible that NS1-enhanced viral mRNA translation is vRNA segment-specific.
Studies using temperature-sensitive influenza A viruses with mutations in NS1 demonstrated a reduction in viral protein synthesis (Hatada et al., 1990). It has also been reported that viral protein synthesis in Madin-Darby canine kidney (MDCK) or Madin-Darby bovine kidney cells (MDBK) cells infected with mutant viruses encoding C-terminally truncated NS1 proteins is significantly reduced compared with that in wt virus-infected cells (Egorov et al., 1998; Enami & Enami, 2000). However, these observations may be cell-type specific, as viral protein levels do not differ much between truncated-NS1 and wt virus-infected Vero cells (Egorov et al., 1998; Salvatore et al., 2002). Thus, the normal inhibitory effect of NS1 on host antiviral responses, which are severely impaired in Vero cells, may indirectly contribute towards efficient viral mRNA translation. For example, in IFN-competent cells, IFN induced by viruses encoding truncated NS1 proteins could stimulate activation of antiviral proteins that lead to a reduction in viral protein synthesis.
Marion et al. (1997a) reported that the N-terminal 113 residues of NS1 were required for direct stimulation of viral mRNA translation in transfected COS-1 cells. Although binding of NS1 to the 5'UTR of viral mRNAs may correlate with NS1-mediated enhancement of viral protein synthesis, it is likely that interactions between NS1 and cellular proteins are also required for this effect. During infection, viral mRNAs were shown to be efficiently translated even in the presence of low levels of the cellular eIF4F cap-binding complex (Feigenblum & Schneider, 1993). It was subsequently reported that residues 81–113 of NS1 can interact with eIF4GI, the large subunit of eIF4F (Aragon et al., 2000). Given that mutant NS1 proteins unable to bind eIF4GI are also defective in enhancing viral mRNA translation (Aragon et al., 2000; Marion et al., 1997a), it may be that NS1 normally recruits eIF4GI, and thus eIF4F, to the 5'UTR of viral mRNAs, thereby preferentially increasing viral translation. Furthermore, the N-terminal 81 aa of NS1 have been shown to interact with PABPI, a known interactor of eIF4GI, independently of RNA (Burgui et al., 2003) and mapping studies suggested that a heterotrimeric NS1–PABPI–eIF4GI complex might be possible (Aragon et al., 2000; Burgui et al., 2003). In addition, NS1 can interact with and cause the redistribution of hStaufen, a dsRNA- and tubulin-binding protein related to PKR (Falcon et al., 1999). As hStaufen normally contributes towards microtubular transport of cellular mRNAs to sites of enhanced translation, such as polysomes, it may be that interaction with NS1 promotes efficient viral mRNA translation. In support of this, a proportion of both NS1 and hStaufen have previously been found to co-fractionate with cytoplasmic polysomes in influenza A virus-infected cells (Falcon et al., 1999; Krug & Etkind, 1973). Thus, to increase viral protein synthesis, NS1 appears to interact with viral 5'UTRs, hStaufen, eIF4GI and PABPI to recruit viral mRNAs (at the expense of cellular mRNAs) to multi-protein translation-initiation complexes (Figs 1 and 2). It is still not clear if the observed binding of NS1 to poly(A) sequences (Qiu & Krug, 1994) has any role to play in viral mRNA translation.
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NS1 and the host innate immune response |
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or IFN-β, are soluble cytokines that are synthesized and secreted by cells in response to virus infection, and act in both an autocrine and paracrine manner to upregulate the expression of >300 IFN-stimulated antiviral genes (Randall & Goodbourn, 2008). Although a major function of NS1 is to antagonize host innate immune responses, as detailed below, the mechanisms and targets for NS1 are varied and strain-specific (Hayman et al., 2006; Kochs et al., 2007a; Twu et al., 2007).
NS1 is essential for antagonizing IFN-/β-dependent responses
The generation of influenza A viruses unable to express NS1 (delNS1), or that express truncated forms of NS1, revealed the crucial role for this protein in counteracting the host IFN response (Egorov et al., 1998; Garcia-Sastre et al., 1998; Kochs et al., 2007b). DelNS1 viruses induce large amounts of IFN in infected cells, and are consequently attenuated in IFN-/β-competent systems. Not surprisingly, delNS1 viruses replicate more efficiently in IFN-/β-deficient tissues such as Vero cells; however, virus titres are approximately 10–100-fold lower than for wt (Garcia-Sastre et al., 1998; Kochs et al., 2007b). This may be due to effects of cytokines other than IFN-/β and cytokine-independent or IRF3-dependent responses. The lack of other ‘IFN-independent’ functions of NS1 also probably contributes to this attenuated phenotype.
NS1 limits IFN-β production
A number of studies have attempted to demonstrate how NS1 acts to limit the production of IFN-β. Although such reports have often seemed contradictory, it is now apparent that the IFN-antagonistic properties of different NS1 proteins are strain-specific (Geiss et al., 2002; Hayman et al., 2006; Kochs et al., 2007a). Current evidence indicates that NS1 proteins may have acquired the ability to limit IFN-β induction by both pre-transcriptional (cytoplasmic) and/or post-transcriptional (nuclear) processes. Thus, it has been proposed that the existence and evolution of two such synergistic anti-IFN mechanisms could increase the capacity of some influenza A viruses to adapt to new hosts (Kochs et al., 2007a). In this regard, it is also possible that certain virus strains may have lost one or other of these mechanisms, either naturally or during laboratory passage. For example, the NS1 protein of A/Puerto Rico/8/34 (PR8) clearly limits pre-transcriptional events associated with IFN-β induction, but unlike many other NS1 proteins is apparently unable to block post-transcriptional processing of IFN-β pre-mRNAs (Hayman et al., 2006; Kochs et al., 2007a). The two strategies by which NS1 proteins appear to intercede with the IFN-induction pathway are outlined below.
(i) Pre-transcriptional limitation of IFN-β induction by NS1.
Studies using PR8/NS1 demonstrated that this protein prevents dsRNA- and virus-mediated activation of the IRF-3, NF
B and c-Jun/ATF-2 transcription factors, which are otherwise essential for IFN-β induction (Ludwig et al., 2002; Talon et al., 2000a; Wang et al., 2000). Such inhibition was shown to occur pre-transcriptionally, and to require two residues in NS1 that strongly contribute to RNA-binding: Arg-38 and Lys-41 (Talon et al., 2000a) (Figs 1 and 3a). It was originally postulated that PR8/NS1 may act by sequestering aberrant viral dsRNA away from host-encoded sensors (Talon et al., 2000a). However, dsRNA has yet to be detected in influenza A virus-infected cells (Weber et al., 2006), and it is now evident that unique components of the influenza virus ssRNA genome can be directly recognized by the cytoplasmic pathogen sensor, RIG-I (Pichlmair et al., 2006). As such, recent work now indicates that PR8/NS1 may mediate its pre-transcriptional block on IFN-β induction by forming a complex with RIG-I (Guo et al., 2007; Mibayashi et al., 2007; Opitz et al., 2007; Pichlmair et al., 2006). Consistent with initial observations (Talon et al., 2000a), co-precipitation of RIG-I with PR8/NS1 is largely dependent upon Arg-38 and Lys-41 in PR8/NS1 (Pichlmair et al., 2006), suggesting that these two residues are involved in a potential protein–protein interaction, or that RNA acts as an intermediary component (Fig. 2). Indeed, direct binding of PR8/NS1 to RIG-I has yet to be demonstrated (Mibayashi et al., 2007), and the presence of 5'-triphosphorylated ssRNA clearly enhances stability of PR8/NS1–RIG-I complexes (Pichlmair et al., 2006). Intriguingly, PR8/NS1 has also been reported to block the function of both a constitutively active RIG-I construct lacking its RNA-binding helicase domain, and IPS-1, a downstream effector of RIG-I (Mibayashi et al., 2007). These data indicate that PR8/NS1-mediated inhibition of the RIG-I/IPS-1 signalling pathway probably occurs by a complex molecular mechanism that has yet to be fully established.
(ii) Post-transcriptional limitation of IFN-β induction by NS1.
It is not clear if co-precipitation of RIG-I is a feature exhibited by all influenza A virus NS1 proteins. Indeed, comparative studies between the NS1 proteins of PR8 and A/Texas/36/91 (Tx/NS1) revealed that Tx/NS1 interacts relatively poorly with RIG-I, and is partially limited in its ability to prevent IRF-3 dimerization/activation (Kochs et al., 2007a). Despite this, Tx/NS1 completely blocks IFN-β mRNA synthesis during infection (Kochs et al., 2007a). The ability of NS1 to prevent the nuclear post-transcriptional processing of RNA polymerase II transcripts appears to be a common additional strategy that many influenza A virus strains use to limit IFN-β production (Fortes et al., 1994; Hayman et al., 2006, 2007; Kochs et al., 2007a; Lu et al., 1994; Nemeroff et al., 1998; Noah et al., 2003; Qiu & Krug, 1994; Shimizu et al., 1999; Twu et al., 2007).
General inhibition of nucleo-cytoplasmic transport of all poly(A)-containing mRNAs was one of the first functions ascribed to NS1 (Fortes et al., 1994; Qiu & Krug, 1994). At the time, it was speculated that global nuclear retention of cellular mRNAs by NS1 might provide a pool of cap-donors for the viral polymerase complex, thus increasing priming of viral mRNA transcription. However, it is now apparent that blocking cellular mRNA processing and transport may be an effective means to limit a number of host-cell processes, including the innate antiviral response. Given that NS1 does not prevent nuclear export of RNAs lacking poly(A) sequences, it was suggested that direct binding of NS1 to the 3' poly(A) tail of mRNAs was the mechanism by which this inhibition occurred (Qiu & Krug, 1994). However, viral mRNAs are not prevented from leaving the nucleus of infected cells, despite them having a poly(A) tail. Therefore, interactions between NS1 and proteins directly involved in mRNA maturation and nucleo-cytoplasmic transport may play the greater and more specific role in cellular mRNA export inhibition.
Influenza virus A/Udorn/72 (Ud) has been extensively used to model the nuclear inhibition of cellular pre-mRNA processing by NS1. The C-terminal effector domain of Ud/NS1 binds directly to two zinc-finger regions in the 30 kDa subunit of cleavage and polyadenylation specificity factor (CPSF30) (Nemeroff et al., 1998; Noah et al., 2003; Twu et al., 2006) and interacts with poly(A)-binding protein II (PABPII) (Chen et al., 1999). Binding to PABPII requires residues 223–237 of Ud/NS1 (Li et al., 2001), whilst binding to CPSF30 appears to require Phe-103, Met-106, Leu-144 and residues 184–188 of Ud/NS1 (Kochs et al., 2007a; Li et al., 2001; Noah et al., 2003; Twu et al., 2006, 2007) (Figs 1 and 2). Glu-96 may also be functionally significant (Shimizu et al., 1999). PR8/NS1, which is unable to block the processing of RNA polymerase II transcripts, is unable to interact with CPSF30 due to amino acid substitutions at residues 103 and 106 (Kochs et al., 2007a). The Ud/NS1–CPSF30 complex is thought to prevent CPSF30 from binding cellular pre-mRNAs, thereby inhibiting normal cleavage and polyadenylation of the 3'-end of host-cell mRNAs (Nemeroff et al., 1998). As polyadenylation of influenza A virus mRNAs is independent of cellular 3'-end processing factors (Palese & Shaw, 2007), viral mRNAs are not affected by CPSF30 inhibition. Furthermore, the interaction of Ud/NS1 with PABPII may specifically block the nuclear export of fully processed mRNAs that partially escape 3'-end formation inhibition (Chen et al., 1999). A recent study has also proposed a third method by which NS1 proteins may cause nuclear retention of host-cell mRNAs: NS1 appears to form an inhibitory complex with components of the cellular mRNA nuclear export machinery, specifically NXF1, p15, Rae1, E1B-AP5 and Nup98 (Satterly et al., 2007) (Fig. 1). Viral mRNAs must overcome this global block on nucleo-cytoplasmic transport; however, it is still unclear how this occurs.
Although the multiple strategies by which NS1 inhibits host-cell mRNA processing seem somewhat mutually redundant, there may be unidentified benefits to the virus of efficiently targeting this cellular process. For example, some NS1 proteins have been reported to limit host-cell gene expression in response to IFN- and tumour necrosis factor (TNF)- (Geiss et al., 2002; Hayman et al., 2006, 2007; Kochs et al., 2007a; Seo et al., 2002), thus rendering the virus less sensitive to the antiviral effects of these cytokines. Ud viruses expressing NS1 proteins unable to bind CPSF30 have been shown to induce large amounts of both IFN-β and cytokine-independent antiviral mRNAs (Noah et al., 2003). Consequently, these mutant viruses are attenuated in both IFN-/β-competent and IFN-/β-deficient cells (Noah et al., 2003; Twu et al., 2006). Therefore the functional consequences of inhibiting host-cell mRNA processing extend far beyond sole antagonism of IFN-β production.
NS1 limits the activity of PKR and OAS
NS1 can directly block the function of two cytoplasmic antiviral proteins: 2'-5'-oligoadenylate synthetase (OAS) (Min & Krug, 2006), and the dsRNA-dependent serine/threonine protein kinase R (PKR) (Min et al., 2007) (Fig. 2). Both OAS/RNase L and PKR are key regulators of viral transcription/translation processes, but play additional roles in other innate defences such as IFN-β induction and the host apoptotic response (Garcia et al., 2006; Silverman, 2007).
OAS is activated by dsRNA, a putative by-product of viral replication, and polymerizes ATP into 2'-5' oligoadenylate chains. These chains cause dimerization and activation of the latent RNase, RNase L, which inhibits virus replication by degradation of RNA (Silverman, 2007). Data indicate that a predominant function of the NS1 RNA-binding domain is to out-compete OAS for interaction with dsRNA, thereby inhibiting this host antiviral strategy (Min & Krug, 2006). Given the role of RNase L in augmenting the production of IFN-β (Silverman, 2007), it is possible that NS1-mediated OAS inactivation also contributes to suppression of IFN-β synthesis (Donelan et al., 2003; Talon et al., 2000a).
dsRNA also binds and activates PKR, thereby releasing PKR auto-inhibition. A major substrate for activated PKR is the eukaryotic translation initiation factor 2 (eIF2), the phosphorylation of which leads to a reduction in both cellular and viral protein synthesis (Garcia et al., 2006). In vitro experiments initially indicated that NS1 may also compete with PKR for binding dsRNA (Hatada et al., 1999; Lu et al., 1995). However, an RNA-binding defective NS1 protein efficiently limited PKR activation in response to dsRNA or PACT, a protein activator of PKR (Li et al., 2006a). Furthermore, NS1 has been shown to interact with PKR in a dsRNA-independent manner, which required NS1 residues 123–127 (Li et al., 2006a; Min et al., 2007; Tan & Katze, 1998). Based on domain mapping studies, it has been proposed that NS1 binds to a linker region in PKR, and thereby prevents a conformational change that is normally required for release of PKR auto-inhibition (Li et al., 2006a). Such a mechanism would allow NS1 to circumvent both dsRNA- and PACT-mediated inhibition of translation by PKR. However, it remains to be determined if an observed NS1–PACT interaction has any functional consequences (Li et al., 2006a).
NS1 and the host RNAi pathway
RNA interference (RNAi) is an RNA-guided cellular mechanism for downregulating expression of specific genes. Involvement of RNAi in the innate antiviral responses of mammalian cells is still controversial, but NS1 has already been proposed to antagonize such a putative host-cell defence (Li et al., 2004). Overexpression of NS1 inhibited the induction of RNAi in heterologous Drosophila and plant cell systems (Bucher et al., 2004; Delgadillo et al., 2004; Li et al., 2004). However, a similar inhibitory effect has yet to be observed in mammalian cells (Kok & Jin, 2006). Thus, despite recent data suggesting that components of the mammalian RNAi pathway participate in innate anti-influenza virus responses (Matskevich & Moelling, 2007), a functional role for NS1 in RNAi-antagonism during virus infection awaits clarification.
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NS1 and the host adaptive immune response |
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and TNF-β. In a mouse model, the NS1 protein of a human H5N1 influenza virus reduced systemic and pulmonary pro-inflammatory cytokines and prevented TNF--mediated bone marrow lymphocyte depletion (Hyland et al., 2006). Furthermore, in human-derived primary DCs, PR8/NS1 was shown to limit induction of several genes involved in DC maturation and migration (Fernandez-Sesma et al., 2006). Consequently, infected DCs were unable to mature, and failed to stimulate the secretion of IFN- from helper T-cells. The limitation of gene-expression in DCs is specific only for certain genes, and mechanistically appears unrelated to suppression of IFN-β production by PR8/NS1 (Fernandez-Sesma et al., 2006). Given recent studies demonstrating that protection against influenza virus infection requires reactivation of memory T-cells by antigens presented on bone marrow-derived DCs (Castiglioni et al., 2008), the prevention of DC maturation by NS1 may limit virus-clearance by the host. Thus, it will be essential to verify such profound immunosuppressive effects of NS1 in the context of virus infection using a relevant in vivo model. |
NS1 and the host apoptotic response |
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; Kurokawa et al., 1999; Zhirnov & Klenk, 2007; Zhirnov et al., 2002). However, cellular pro-apoptotic factors also promote the efficient propagation of influenza viruses, and certain viral proteins, such as NA and PB1-F2, have pro-apoptotic functions (Palese & Shaw, 2007). Thus, the overall temporal regulation of both pro- and anti-apoptotic mechanisms may be critical for the virus. Limiting apoptosis early during infection could promote events such as genome replication, whilst enhancing apoptosis later may lead to increased release of progeny virions. Apoptosis after viral replication may also increase the phagocytic clearance of infected cells, which might otherwise stimulate cell-mediated cytotoxic responses.
The role of NS1 in apoptosis has not been fully established, as NS1 is reported to have both pro- and anti-apoptotic functions (Ehrhardt et al., 2007; Lam et al., 2008; Schultz-Cherry et al., 2001; Shin et al., 2007b; Stasakova et al., 2005; Zhirnov et al., 2002). Such conflicting data may be a consequence of the specific experimental protocol, cell-type or virus strain used. Alternatively, an intriguing hypothesis is that NS1 contributes temporally to both ‘early’ suppression of apoptosis and ‘late’ induction of cell death.
During virus infection, NS1 clearly displays anti-apoptotic functions which are linked to its ability to limit the production and downstream effects of IFN (Zhirnov et al., 2002). Thus, in IFN-competent MDCK cells, PR8 delNS1 virus induced higher levels of apoptosis than wt PR8 (Zhirnov et al., 2002). However, in Vero cells, which lack IFN-/β genes, both viruses induced similar levels of apoptosis, but at a much slower rate than that observed in MDCK cells (Zhirnov et al., 2002). It is not known if Vero cells are defective in pathways and genes other than IFN-/β, therefore one can only speculate that IFN-/β-antagonism by NS1 is the most important factor in limiting apoptosis. As catalytically active PKR is reported to play a role in apoptosis during influenza virus infection (Takizawa et al., 1996), the direct binding and inhibition of PKR by NS1 could also lead to cell-death suppression. The same may be true for NS1-mediated inhibition of pro-apoptotic OAS/RNase L (Min & Krug, 2006), or the JNK/AP-1 stress pathway (Ludwig et al., 2002). As described below, activation of the host-cell PI3K pathway has recently been described as an additional direct method by which NS1 may limit induction of apoptosis (Ehrhardt et al., 2007; Shin et al., 2007a; Zhirnov & Klen
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ABSTRACT
Influenza A viruses
