Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.     

Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease

Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B(40)) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B(40) to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg(459) decreasing Gly(460) within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B.     

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.     

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.     

Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a.

Expression of dengue virus gene products involves specific proteolytic cleavages of a precursor polyprotein. To study the flanking sequences required for expression of the dengue virus nonstructural glycoprotein NS1, we constructed a series of recombinant vaccinia viruses that contain the coding sequence for NS1 in combination with various lengths of upstream and downstream sequences. The NS1 products expressed by these viruses in infected CV-1 cells were immune precipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show that the 24-residue hydrophobic sequence preceding NS1 was necessary and sufficient for the production of glycosylated NS1 and that this sequence was cleaved from NS1 in the absence of most dengue virus proteins. This finding is consistent with previous proposals that this hydrophobic sequence serves as an N-terminal signal sequence that is cleaved by signal peptidase. The cleavage between the C terminus of NS1 and the downstream protein NS2a occurred when the complete NS2a was present. Recombinant viruses containing NS1 plus 15 or 49% of NS2a produced proteins larger than authentic NS1, indicating that the cleavage between NS1 and NS2a had not occurred. Failure of cleavage was not corrected by coinfection with a recombinant virus capable of cleavage. These results suggest that NS2a may be a cis-acting protease that cleaves itself from NS1, or NS2a may provide sequences for recognition by a specific cellular protease that cleaves at the NS1-NS2a junction.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.     

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.     

In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.     

Flavivirus enzyme-substrate interactions studied with chimeric proteinases: identification of an intragenic locus important for substrate recognition.

The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.     

Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs

It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-alpha/beta) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-alpha/beta synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-alpha/beta induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.     

Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein

We present a novel mechanism by which viruses may inhibit the alpha/beta interferon (IFN-alpha/beta) cascade. The double-stranded RNA (dsRNA) binding protein NS1 of influenza virus is shown to prevent the potent antiviral interferon response by inhibiting the activation of interferon regulatory factor 3 (IRF-3), a key regulator of IFN-alpha/beta gene expression. IRF-3 activation and, as a consequence, IFN-beta mRNA induction are inhibited in wild-type (PR8) influenza virus-infected cells but not in cells infected with an isogenic virus lacking the NS1 gene (delNS1 virus). Furthermore, NS1 is shown to be a general inhibitor of the interferon signaling pathway. Inhibition of IRF-3 activation can be achieved by the expression of wild-type NS1 in trans, not only in delNS1 virus-infected cells but also in cells infected with a heterologous RNA virus (Newcastle disease virus). We propose that inhibition of IRF-3 activation by a dsRNA binding protein significantly contributes to the virulence of influenza A viruses and possibly to that of other viruses.