Generation of influenza A viruses with decreased sensitivity to IFN-λ1.

<p>A/California/04/09 virus was passaged in Calu-3 cells in the presence (red line) or absence (blue line) of increasing concentrations of IFN-λ1 (green line). *<i>P</i> < 0.05, °<i>P</i> < 0.01, compared to the values for the CA/04^–IFN-λ1 virus.</p

Replication of the A/California/04/09, CA/04^+IFN-λ1 and CA/04^–IFN-λ1 viruses in Calu-3 cells.

<p>The results are expressed as log₁₀PFU/ml from three independent experiments. °<i>P</i> < 0.01, compared to the values for the wild-type virus.</p

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses - Fig 6

<p><b>(A, B, C) Influenza-induced IFN gene expression levels in Calu-3 cells.</b> Cells were infected with the indicated recombinant viruses (MOI = 1) and the levels of IFNs were quantified by qPCR at 12, 48, and 72 hpi. Values were determined by comparison to standard curves for each gene and the results are expressed as RNA copy numbers. *<i>P</i> < 0.05; °<i>P</i> < 0.01, compared to the values for the CA/04 virus. <b>(D, E) IFN-λ1 and –λ2/3 protein production in Calu-3 cells.</b> Cells were infected with viruses at a MOI of 5. Supernatants were collected at 24, 48, and 72 hpi, and the levels of secreted IFN-λ1 protein were determined by ELISA. °<i>P</i> < 0.01, compared to the values for the CA/04 virus.</p

Growth characteristics of wild-type and two passaged H1N1 influenza viruses.

<p>Growth characteristics of wild-type and two passaged H1N1 influenza viruses.</p

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses

<div><p>Influenza A viruses pose a constant potential threat to human health. In view of the innate antiviral activity of interferons (IFNs) and their potential use as anti-influenza agents, it is important to know whether viral resistance to these antiviral proteins can arise. To examine the likelihood of emergence of IFN-λ1-resistant H1N1 variants, we serially passaged the A/California/04/09 (H1N1) strain in a human lung epithelial cell line (Calu-3) in the presence of increasing concentrations of recombinant IFN-λ1 protein. To monitor changes associated with adaptation of this virus to growth in Calu-3 cells, we also passaged the wild-type virus in the absence of IFN-λ1. Under IFN-λ1 selective pressure, the parental virus developed two neuraminidase (NA) mutations, S79L and K331N, which significantly reduced NA enzyme activity (↓1.4-fold) and sensitivity to IFN-λ1 (↓˃20-fold), respectively. These changes were not associated with a reduction in viral replication levels. Mutants carrying either K331N alone or S79L and K331N together induced weaker phosphorylation of IFN regulatory factor 3 (IRF3), and, as a consequence, much lower expression of the IFN genes (<i>IFNB1</i>, <i>IFNL1</i> and <i>IFNL2/3</i>) and proteins (IFN-λ1 and IFN-λ2/3). The lower levels of IFN expression correlated with weaker induction of tyrosine-phosphorylated STAT1 and reduced RIG-I protein levels. Our findings demonstrate that influenza viruses can develop increased resistance to the antiviral activity of type III interferons.</p></div

Nucleotide and amino acid substitutions identified in selected H1N1 influenza viruses.

<p>Nucleotide and amino acid substitutions identified in selected H1N1 influenza viruses.</p

Growth characteristics and NA enzymatic properties of recombinant H1N1 influenza viruses.

<p>Growth characteristics and NA enzymatic properties of recombinant H1N1 influenza viruses.</p

Generation and characterization of interferon-lambda 1-resistant H1N1 influenza A viruses - Fig 4

<p><b>(A) Receptor specificity of A/California/04/09, CA/04</b>^{<b>+IFN-λ1</b>}, <b>and CA/04</b>^{<b>–IFN-λ1</b>}<b>viruses.</b> *<i>P</i> < 0.05, compared to the values for the wild-type virus. <b>(B) Polymerase activity of RNP complexes of wild-type and CA/04</b>^{<b>–IFN-λ1</b>} <b>containing PA V14I mutation.</b> The values represent the means ± standard deviations of activity of each RNP complex relative to that of CA/04 virus. °<i>P</i> < 0.01, compared to the values for the CA/04 virus. <b>(C) Replication efficiency of CA/04, CA/04-M1</b>^{<b><i>A183G</i></b>}, <b>and CA/04-M2</b>^<b>E70K</b> <b>viruses in Calu-3 cells.</b> Representative results expressed as log₁₀PFU/ml from three independent experiments are shown. *<i>P</i> < 0.05, compared to the values for the CA/04 virus. <b>(D) Antiviral activity of IFN-λ1 against CA/04, CA/04-M1</b>^{<b><i>A183G</i></b>}, <b>and CA/04-M2</b>^<b>E70K</b> <b>viruses as measured by cell ELISA.</b></p

The use of plant lectins to regulate H1N1 influenza A virus receptor binding activity

<div><p>We applied an <i>in vitro</i> selection approach using two different plant lectins that bind to α2,3- or α2,6-linked sialic acids to determine which genetic changes of the A/California/04/09 (H1N1) virus alter hemagglutinin (HA) receptor binding toward α2,3- or α2,6-linked glycans. Consecutive passages of the A/California/04/09 virus with or without lectins in human lung epithelial Calu-3 cells led to development of three HA1 amino acid substitutions, N129D, G155E, and S183P, and one mutation in the neuraminidase (NA), G201E. The S183P mutation significantly increased binding to several α2,6 SA-linked glycans, including YDS, 6′SL(N), and 6-Su-6′SLN, compared to the wild-type virus (↑3.6-fold, <i>P</i> < 0.05). Two other HA1 mutations, N129D and G155E, were sufficient to significantly increase binding to α2,6-linked glycans, 6′SLN and 6-Su-6′SLN, compared to S183P (↑4.1-fold, <i>P</i> < 0.05). These HA1 mutations also increased binding affinity for 3′SLN glycan compared to the wild-type virus as measured by Biacore surface plasmon resonance method. In addition, the HA1 N129D and HA1 G155E substitutions were identified as antigenic mutations. Furthermore, the G201E mutation in NA reduced the NA enzyme activity (↓2.3-fold). These findings demonstrate that the A/California/04/09 (H1N1) virus can acquire enhanced receptor affinity for both α2,3- and α2,6-linked sialic receptors under lectin-induced selective pressure. Such changes in binding affinity are conferred by selection of beneficial HA1 mutations that affect receptor specificity, antigenicity, and/or functional compatibility with the NA protein.</p></div

Antiviral activity of MAA (A), SNA (B), and combination of both lectins (C) against the CA/04, CA/04^+MAA, CA/04^+SNA, and CA/04^+Calu-3 viruses as determined by virus reduction assay in Calu-3 cells.

<p>Antiviral activity of MAA (A), SNA (B), and combination of both lectins (C) against the CA/04, CA/04^+MAA, CA/04^+SNA, and CA/04^+Calu-3 viruses as determined by virus reduction assay in Calu-3 cells.</p