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

Abstract

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 (IFNB1, IFNL1 and IFNL2/3) 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.

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

“…Since the binding level to the “avian-type” receptor, 3′SLN, was at the detection limit of our assay among all tested viruses (data not shown) [ 12 ], we studied the binding pattern of the H1N1 variants to 3′SLN glycan in comparison with 6′SLN in the presence of NA inhibitor, zanamivir, using Biacore surface plasmon resonance biosensor ( Fig 4B and 4C ). Our data showed that both the CA/04 +MAA and CA/04 +SNA variants had higher association rate constants of binding with 6′SLN as compared to CA/04 and CA/04 +Calu-3 (k on ≈ 7.5 x 10 7 M -1 x s -1 ; S2 Table ).…”

“…Amino acid changes of the same residues (155 and 183) occurred in the pandemic CA/04 strain in four independent studies either by selection in human airway epithelial cells, mice, or by human antibody [ 12 , 28 – 30 ]. This indicates that the emergence of these two HA1 mutations is not due to random fluctuation, but rather reflects their positive selection.…”

“…The Madin-Darby canine kidney (MDCK) cell line and the human lung epithelial cell line (Calu-3) were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained as described elsewhere [ 12 ].…”

“…The extracellular virus yield reduction assay was performed as described previously in 24-well plates containing confluent Calu-3 cells grown on membrane supports (6.5-mm Transwell, Corning Inc., Corning, NY, USA) [ 12 ]. The concentrations of MAA or SNA lectins ranged from 1 to 1000 μg/ml and they were added to the apical compartment of Calu-3 cells for 2 h. After pretreatment, the cells were overlaid with 2× lectin-containing medium (100 μl/well), infected with influenza virus at a multiplicity of infection (MOI) of 0.1 PFU/cell, and incubated for 48 h at 37°C.…”

“…The affinity of each virus for biotinylated 3′- and 6′-sialylglycopolymers was measured in a direct binding assay as described previously [ 12 , 17 ]. Briefly, plates were pre-coated with each virus at 4°C for 16 h, followed by washing with 0.05% Tween 20 in PBS (PBS-T).…”