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

ABSTRACT: 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.

SUBMITTER: Solorzano A

PROVIDER: S-EPMC1143661 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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

Solórzano Alicia A Webby Richard J RJ Lager Kelly M KM Janke Bruce H BH García-Sastre Adolfo A Richt Jürgen A JA

Journal of virology 20050601 12

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 e ...[more]

PMID: 15919908

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Project description:Influenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties.IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

Project description:Recently, two new influenza A-like viruses have been discovered in bats, A/little yellow-shouldered bat/Guatemala/060/2010 (HL17NL10) and A/flat-faced bat/Peru/033/2010 (HL18NL11). The hemagglutinin (HA)-like (HL) and neuraminidase (NA)-like (NL) proteins of these viruses lack hemagglutination and neuraminidase activities, despite their sequence and structural homologies with the HA and NA proteins of conventional influenza A viruses. We have now investigated whether the NS1 proteins of the HL17NL10 and HL18NL11 viruses can functionally replace the NS1 protein of a conventional influenza A virus. For this purpose, we generated recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing the NS1 protein of the PR8 wild-type, HL17NL10, and HL18NL11 viruses. These viruses (r/NS1PR8, r/NS1HL17, and r/NS1HL18, respectively) were tested for replication in bat and nonbat mammalian cells and in mice. Our results demonstrate that the r/NS1HL17 and r/NS1HL18 viruses are attenuated in vitro and in vivo However, the bat NS1 recombinant viruses showed a phenotype similar to that of the r/NS1PR8 virus in STAT1-/- human A549 cells and mice, both in vitro and in vivo systems being unable to respond to interferon (IFN). Interestingly, multiple mouse passages of the r/NS1HL17 and r/NS1HL18 viruses resulted in selection of mutant viruses containing single amino acid mutations in the viral PB2 protein. In contrast to the parental viruses, virulence and IFN antagonism were restored in the selected PB2 mutants. Our results indicate that the NS1 protein of bat influenza A-like viruses is less efficient than the NS1 protein of its conventional influenza A virus NS1 counterpart in antagonizing the IFN response and that this deficiency can be overcome by the influenza virus PB2 protein.IMPORTANCE Significant gaps in our understanding of the basic features of the recently discovered bat influenza A-like viruses HL17NL10 and HL18NL11 remain. The basic biology of these unique viruses displays both similarities to and differences from the basic biology of conventional influenza A viruses. Here, we show that recombinant influenza A viruses containing the NS1 protein from HL17NL10 and HL18NL11 are attenuated. This attenuation was mediated by their inability to antagonize the type I IFN response. However, this deficiency could be compensated for by single amino acid replacements in the PB2 gene. Our results unravel a functional divergence between the NS1 proteins of bat influenza A-like and conventional influenza A viruses and demonstrate an interplay between the viral PB2 and NS1 proteins to antagonize IFN.

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

Publications

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

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