Identification of Adaptive Mutations in the Influenza A Virus Non-Structural 1 Gene That Increase Cytoplasmic Localization and Differentially Regulate Host Gene Expression

<div><p>The NS1 protein of influenza A virus (IAV) is a multifunctional virulence factor. We have previously characterized gain-of-function mutations in the NS1 protein arising from the experimental adaptation of the human isolate A/Hong Kong/1/1968(H3N2) (HK) to the mouse. The majority of these mouse adapted NS1 mutations were demonstrated to increase virulence, viral fitness, and interferon antagonism, but differ in binding to the post-transcriptional processing factor cleavage and polyadenylation specificity factor 30 (CPSF30). Because nuclear trafficking is a major genetic determinant of influenza virus host adaptation, we assessed subcellular localization and host gene expression of NS1 adaptive mutations. Recombinant HK viruses with adaptive mutations in the NS1 gene were assessed for NS1 protein subcellular localization in mouse and human cells using confocal microscopy and cellular fractionation. In human cells the HK wild-type (HK-wt) virus NS1 protein partitioned equivalently between the cytoplasm and nucleus but was defective in cytoplasmic localization in mouse cells. Several adaptive mutations increased the proportion of NS1 in the cytoplasm of mouse cells with the greatest effects for mutations M106I and D125G. The host gene expression profile of the adaptive mutants was determined by microarray analysis of infected mouse cells to show either high or low extents of host-gene regulation (HGR or LGR) phenotypes. While host genes were predominantly down regulated for the HGR group of mutants (D2N, V23A, F103L, M106I+L98S, L98S, M106V, and M106V+M124I), the LGR phenotype mutants (D125G, M106I, V180A, V226I, and R227K) were characterized by a predominant up regulation of host genes. CPSF30 binding affinity of NS1 mutants did not predict effects on host gene expression. To our knowledge this is the first report of roles of adaptive NS1 mutations that impact intracellular localization and regulation of host gene expression.</p></div

Adaptive properties of rHK viruses expressing NS1 mutations selected upon mouse adaptation.

<p>Heat map illustrating adaptive phenotypes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084673#pone-0084673-g003" target="_blank">Figures 3</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084673#pone-0084673-g005" target="_blank">5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084673#pone-0084673-g007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084673#pone-0084673-g009" target="_blank">9</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084673#pone.0084673-Forbes1" target="_blank">[21]</a>); relative to HK-wt values, where 1 is indicated as white* and the red scale depicts phenotypes greater than HK-wt (increasing shades of red from 1 to 10 (with saturated red for ≥10), and shades of blue scale from 1 to 0 depicts phenotypes lesser than HK-wt values, where data was calculated as a fold change relative to HK-wt value. For virus yield values, maximum virus yield obtained in the growth curve was used to calculate fold change relative to HK-wt virus maximum virus yield. *With the exception of % mortality in the mouse where 0% mortality is indicated as blue and any mutation inducing mortality in the mouse indicated as red.</p

Protein map of NS1 indicating the location of mutations selected upon HK virus mouse adaptation.

<p>Linear map of NS1 protein indicating the location of NS1 mutations selected upon adaption of A/Hong Kong/1/1968 (H3N2) to the mouse, as depicted by black lines. NS1 nuclear localization sites (NLS1 and 2), nuclear export signal (NES), and its adjacent negative regulator of NES are indicated on the protein map as well as the amino and carboxyl protein regions associated with sites of mutations inducing high or low host gene regulation phenotypes (HGR or LGR, respectively).</p

MA NS1 mutants alter subcellular localization of the NS1 protein in infected mouse cells.

<p>Mouse M1 cells were infected at MOI = 3 with rHK NS mutant viruses or the HK-wt virus, or mock infected with PBS. Following 16 hpi, cells were fixed and stained using a polyclonal anti-NS1 primary antibody and Cy3-conjugated secondary antibody as well as DAPI to localize the nucleus. Representative images are shown (taken using a 63× oil immersion objective).</p

MA NS1 mutations increase genomic viral RNA synthesis in infected mouse cells.

<p>Mouse M1 cells were infected in triplicate at an MOI of 2 with rHK NS mutant viruses or the HK-wt virus, and total RNA isolated at 8 hpi was reverse transcribed using primers specific for genomic viral RNA segments. Real-time PCR (qPCR) was performed to quantify NP, M1 and NS1 genomic RNA levels. Results were normalized by β-actin levels, and presented as values relative to HK-wt genomic RNA levels. Data represent the means ± SD (two tailed student’s paired t-test) for NP, NS1 and M1 transcript relative levels (indicated by bracket) or two-tailed student’s t-test for individual mRNA samples (n = 3) relative values (i p<0.05; ii p<0.01; iii p<0.001; iv p<0.0001; v p<0.00001).</p

MA NS1 mutants M106I+L98S and D125G temporally increase cytoplasmic NS1 localization in infected mouse cells.

<p>Mouse M1 cells were infected at MOI = 3 with the rHK NS mutant viruses, the HK-wt virus, or prototype mouse-adapted viruses A/PR/8/34 (H1N1) (PR8) or A/WSN/33 (H1N1) (WSN). Following 4, 8, 16, and 24 hpi, cells were fixed and stained using a polyclonal anti-NS1 antibody and Cy3-conjugated secondary as well as DAPI to localize the nucleus. (A) Representative images are shown, taken at 63× using oil immersion. (B) Data represent the average percentage of cells positive for the NS1 antigen detected in the cytoplasm ± SD (analysis of n = 5 randomly selected images) for 4–24 hpi (*p<0.05, ***p<0.001, ****p<0.0001, *****p<0.00001; two-tailed student’s t-test compared to HK-wt values).</p

Mouse-adaptive NS1 mutations regulate mouse gene expression by two different mechanisms.

<p>Mouse M1 cells were infected in triplicate at an MOI of 2 with rHK NS mutants or HK-wt virus, and total RNA isolated at 8 hpi was analyzed by microarray using GeneChip Mouse Exon 1.0 ST Array (Affymetrix, Santa Clara, CA, USA). Microarray gene expression data were normalized and analyzed by Flexarray 1.6.1. Genes were considered as up or down regulated relative to mock infected cells if they were ≤1 or ≥1 log2 fold different (≤ or ≥2 fold differences) expression level (p≤0.05; ANOVA). (A) The number of differentially regulated host genes that were significantly up or down regulated ≤2 or ≥2 fold at the p≤0.05 by ANOVA is plotted for each mutant. The mutants formed two groups with either “low gene regulation” (LGR) or “high t gene regulation” (HGR) phenotypes. (B) Heat map of differentially regulated genes in mouse cells infected with HK-wt or rHK NS1 mutant viruses relative to mock infected M1 cells. Genes (total = 5274) were included for hierarchical clustering analysis among mutants if they were differentially regulated (≤2⁻¹or ≥2¹ fold differences) and significantly different from mock infected cells for one or more of the mutants, and gene regulation signatures were also analyzed for hierarchical clustering among viruses. The scale depicts up (red) and down (blue) regulated genes according to the log2 scale shown; equal expression is indicated in white (log 2⁰ = 1).</p

Increased cytoplasmic distribution of NS1 is a host-dependent phenotype of MA NS1 mutant viruses.

<p>(A) Human A549 cells were infected at MOI = 3 with the rHK NS mutant viruses, the HK-wt virus, or the WSN or PR8 viruses. Following 16 hpi, the cells were fixed and stained using a polyclonal anti-NS1 antibody and Cy3-conjugated secondary antibody as well as DAPI to localize the nucleus. Representative images are shown, taken at 63× using oil immersion. (B) Human A549 cells were infected with rHK NS mutant viruses, the HK-wt virus, or the WSN or PR8 viruses, or mock infected with PBS. At 16 hpi cells were lysed and differentially centrifuged to obtain whole cell (w), nuclear (n) and cytosolic (c) fractions. Cell fractions were resuspended in SDS buffer then were separated by SDS PAGE electrophoresis, followed by Western blot analysis to detect the NS1 protein as well as loading markers tubulin and histone H3. (B) Representative blot of 2–3 independent experiments, which were (C) analysed by densitometry where the amount of NS1 or NS3 protein in the nuclear and cytoplasmic fractions of a cell infected with a given virus was normalized to histone H3 and tubulin levels of the whole cell lysate, respectively. Cytoplasmic distribution was calculated by dividing cytoplasmic NS1 by nuclear NS1. Data represent the means ± SE (*p<0.05, two-tailed student’s t-test).</p

MA NS1 mutations alter subcellular localization of the NS1 protein in infected M1 cells.

<p>Mouse M1 cells were infected (MOI = 2) with rHK NS1 mutant viruses, the HK-wt virus, or the PR8, or WSN viruses, or mock infected with PBS. At 16 hpi the cells were lysed and differentially centrifuged to obtain whole cell (w), nuclear (n) and cytosolic (c) fractions. Cell fractions were resuspended in SDS buffer then were separated by SDS PAGE electrophoresis, followed by Western blot analysis to detect the NS1 protein as well as loading markers tubulin and histone H3. (A) Representative blot of 2–3 independent experiments, which were (B) analysed by densitometry where the amount of NS1 or NS3 protein in the nuclear and cytoplasmic fractions of a cell infected with a given virus was normalized to histone H3 and tubulin levels of the whole cell lysate, respectively. Cytoplasmic distribution was calculated by dividing cytoplasmic NS1 by nuclear NS1. Data represent the means ± SE (*p<0.05, **p<0.01; two-tailed student’s t-test).</p

Inhibition of Influenza A PR8 virus by ATA, AH and NAA.

<p>Confluent MDCK cells in 6 well plates were infected with 0.001 MOI of influenza A PR8 virus and incubated for 48 h with 100 µg/ml of AH, ATA, NAA or DMSO alone. Culture media and cell lysates were collected and viral titers were determined by plaque assay.</p