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.2011 Jan;85(2):652-62.
doi: 10.1128/JVI.01987-10. Epub 2010 Nov 17.

A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo

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A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo

Gina M Conenello et al. J Virol.2011 Jan.

Abstract

The PB1-F2 protein of influenza A virus can contribute to viral pathogenesis of influenza virus strains. Of note, an N66S amino acid mutation in PB1-F2 has been shown to increase the pathogenesis associated with H5N1 Hong Kong/1997 and H1N1 Brevig Mission/1918 influenza viruses. To identify the mechanism of enhanced immunopathology, we evaluated the host response to two isogenic viruses that differ by a single amino acid at position 66 of the PB1-F2 protein. Various components of the adaptive immune response were ruled out as factors contributing to pathogenesis through knockout mouse studies. Transcriptional profiling of lungs from PB1-F2 66S-infected mice revealed an early delay in innate immune responses. In particular, enhanced activation of type I interferon (IFN) pathway genes, including IFN-β, RIG-I, and numerous interferon-inducible genes, was not observed until day 3 postinfection. The N66S mutant virus caused increased cellularity in the lungs, as a result of monocyte and neutrophil infiltration. Furthermore, numerous cytokines and chemokines related to monocyte and neutrophil migration and maturation were upregulated. The cellular infiltration and increased cytokine expression corresponded to increased PB1-F2 66S titer. These data suggest that PB1-F2 N66S may contribute to the delay of innate immune responses, allowing for unchecked viral growth and ultimately severe immunopathology observed in the lungs.

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Figures

FIG. 1.
FIG. 1.
Morbidity in immune cell knockout mice. Mice were inoculated with 104 PFU of either WH (circles) or WH N66S (squares) virus or PBS-BA-PS (triangles) as a control. Weight was monitored and plotted as percent weight upon inoculation. Error bars indicate standard deviations.
FIG. 2.
FIG. 2.
Interferon-stimulated response genes distinguish WH and WH N66S infection groups at days 1 and 3 postinfection. (A) Functional analysis of differentially expressed genes distinguishing WH and WH N66S infection groups on days 1 and 3 postinfection. The IPA network diagram shows direct functional relationships between genes involved in antimicrobial response, inflammatory response, and infection mechanism. Nodes depict genes in the network, and solid lines represent direct relationships between nodes. For signaling pathways, an arrow pointing from one node to the next signifies an activation event, for example, binding or phosphorylation. Nodes with arrows or connections pointing to themselves indicate self-binding or autoregulation events. (B) Transcription of genes related to antimicrobial response, inflammatory response, and infection mechanism from WH- and WH N66S-infected mice at 1 and 3 dpi. Two-dimensional clustering of differentially expressed genes within the categories of antimicrobial response, inflammatory response, and infection mechanism is represented. The average expression level for each group is shown as the log10 (ratio) expression relative to that for a pool of RNA extracted from lungs of time-matched mock-infected mice (n = 2). Red and green denote increases and decreases, respectively, in expression level relative to that for the uninfected reference. Clustering was performed with the hierarchical unweighted-pair group method using average linkages (UPGMA), with Euclidean distance similarity measure using an average-value ordering function. (C) Transcript levels for interferon response-related genes. Cellular transcript levels at days 1 and 3 postinfection in mouse lungs infected with WH or WH N66S virus, as measured by quantitative real-time PCR. Average log10 (ratio) values were calculated by averagingCT values from three technical replicates in each case. Data represent averages of three biological replicates from two independent experiments. Sdf2 was used as an internal control for each assay. PBS-inoculated lungs at day 3 postinfection were set as the baseline. Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
Cytokine and chemokine levels in infected mice. (A) Gene expression profiles for the examined cytokine and chemokine panel are represented. The average expression level for each group is shown as the log10 (ratio) expression relative to that for a pool of RNA extracted from lungs of time-matched mock-infected mice (n = 2). Red and green denote increases and decreases, respectively, in expression level relative to that for the mock-infected reference. (B) Mice were inoculated with PBS-BA-PS (dotted line as baseline) as a control or 104 PFU of either WH (blue) or WH N66S (red) virus. Mouse lungs were collected on days 1, 3, and 5 postinoculation and homogenized. MIP-1β, RANTES, TNF-α, M-CSF, KC, IFN-γ, G-CSF, MCP-1, and IL-6 levels in lung homogenate were measured using a Millipore Milliplex ELISA kit. IFN-β was measured using a standard ELISA. Each harvested lung homogenate was 1 ml. If the baseline is not visible, the cytokine level for the PBS-BA-PS group was 0. Error bars indicate standard deviations.
FIG. 4.
FIG. 4.
Lung titer and histology in WH- and WH N66S-infected mice. Mice were inoculated with PBS-BA-PS as a control or 104 PFU of either WH or WH N66S virus. Mouse lungs were collected on days 1 to 5 postinoculation. (A) Lung sections were taken on day 5 postinfection and stained with hematoxylin and eosin. Cell infiltration was observed using bright-field microscopy at ×10 magnification of 6-μm sections. (B) Cell counts were taken using cell counting beads and flow cytometry. (C) Lung titers were analyzed and graphed as PFU/lung. Error bars indicate standard deviations.
FIG. 5.
FIG. 5.
Lung cell populations in infected mice. Mice were inoculated with PBS-BA-PS as a control or 104 PFU of either WH or WH N66S virus. Mouse lungs were collected on days 1 to 5 postinoculation. (A and B) Monocytes in the lung cell population were stained using fluorescent antibodies. CD11c+ CD11b+ cells were considered monocytes. (C and D) Neutrophils in the lung cell population were stained using fluorescent antibodies. Ly6C+ cells were considered neutrophils. (E and F) Dendritic cells in the lung cell population were stained using fluorescent antibodies. CD11chigh MHC-II+ CD11b− cells were considered dendritic cells. Percentages of total cells and absolute cell numbers are shown. For panels A through E, PBS samples were averaged from all days and plotted on the graphs on day 1. Error bars indicate standard deviations.
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References

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