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Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.

ABSTRACT: The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.

SUBMITTER: Perez-Cidoncha M

PROVIDER: S-EPMC4041880 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.

Pérez-Cidoncha Maite M Killip Marian J MJ Asensio Víctor J VJ Fernández Yolanda Y Bengoechea José A JA Randall Richard E RE Ortín Juan J

PloS one 20140602 6

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to ...[more]

PMID: 24887174

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Project description:To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon ? (IFN-?) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-? induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-? induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.The F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.

Dataset Information

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.

Publications

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype.

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