Highly Pathogenic H5N1 Influenza A Virus Strains Provoke Heterogeneous IFN-α/β Responses That Distinctively Affect Viral Propagation in Human Cells

Abstract

The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-α/β barrier involve mutations in multiple H5N1 genes.

Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus

“…The NS1-deficient Pan/99 variant has previously been shown to trigger secretion of Ͼ10-fold higher levels of IFN-␤ than those with the wild type, and similar findings exist for other strain backgrounds (40,51,52). It was therefore not unexpected that ISG expression was strongly increased in ⌬NS1 virus-infected cells compared to wild-type-infected cells, but it came as a surprise that the populations of noninfected cells with an ISG signature were of comparable sizes for both viruses (Fig.…”

“…The recombinant viruses PR/8 NS1 S103 I106 and PR/8 NS1 S103F I106M were kindly provided by Georg Kochs (Freiburg, Germany) (41). An established reverse genetic system was employed to generate Pan99-derived recombinant wild-type and NS1 mutant IAVs (40 Confocal laser scanning microscopy. For indirect immunofluorescence staining, cells were seeded on coverslips on the day before infection.…”

PB2-588I Enhances 2009 H1N1 Pandemic Influenza Virus Virulence by Increasing Viral Replication and Exacerbating PB2 Inhibition of Beta Interferon Expression

“…The decreased ability of the divergent NS1 protein to antagonize the human type I IFN system explained why some H5N1 strains replicated well in human hosts (37). The NS1 mutations in an H3N2 subtype of the virus significantly enhanced IFN-␤ antagonism and reduced IFN-␤ production in mammalian cells (38).…”

Quantitative Proteomic Approach Identifies Vpr Binding Protein as Novel Host Factor Supporting Influenza A Virus Infections in Human Cells

“…The following antibodies were used in this study: anti-CrkL (Merck Millipore), anti-VprBP (Abcam), antiRsl1D1 (Sigma-Aldrich), anti-SAMHD1 (Sigma-Aldrich), anti-NP (Serotec), anti-M1 (Serotec), anti-NS1 (27) (Fig. 2).…”

“…1D). As a control, the recombinant influenza Pan ⌬NS1 mutant virus was used to determine strong influenza virus-dependent IFN-␤ induction in the absence of antagonistic NS1 protein (27). Growth curve analyses in Vero cells that do not express intact IFN-␣/␤ genes showed similar differences in the replication of the Pan and Mal viruses suggesting that the poor propagation of the avian virus in human cells is not caused by the type I IFN response (Fig.…”