Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

<p>Abstract</p> <p>Background</p> <p>To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.</p> <p>Results</p> <p>To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth <it>in vitro </it>(mouse and canine cells) and <it>in vivo </it>(BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.</p> <p>Conclusions</p> <p>The F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.</p

Multifunctional Adaptive NS1 Mutations Are Selected upon Human Influenza Virus Evolution in the Mouse

The role of the NS1 protein in modulating influenza A virulence and host range was assessed by adapting A/Hong Kong/1/1968 (H3N2) (HK-wt) to increased virulence in the mouse. Sequencing the NS genome segment of mouse-adapted variants revealed 11 mutations in the NS1 gene and 4 in the overlapping NEP gene. Using the HK-wt virus and reverse genetics to incorporate mutant NS gene segments, we demonstrated that all NS1 mutations were adaptive and enhanced virus replication (up to 100 fold) in mouse cells and/or lungs. All but one NS1 mutant was associated with increased virulence measured by survival and weight loss in the mouse. Ten of twelve NS1 mutants significantly enhanced IFN-β antagonism to reduce the level of IFN β production relative to HK-wt in infected mouse lungs at 1 day post infection, where 9 mutants induced viral yields in the lung that were equivalent to or significantly greater than HK-wt (up to 16 fold increase). Eight of 12 NS1 mutants had reduced or lost the ability to bind the 30 kDa cleavage and polyadenylation specificity factor (CPSF30) thus demonstrating a lack of correlation with reduced IFN β production. Mutant NS1 genes resulted in increased viral mRNA transcription (10 of 12 mutants), and protein production (6 of 12 mutants) in mouse cells. Increased transcription activity was demonstrated in the influenza mini-genome assay for 7 of 11 NS1 mutants. Although we have shown gain-of-function properties for all mutant NS genes, the contribution of the NEP mutations to phenotypic changes remains to be assessed. This study demonstrates that NS1 is a multifunctional virulence factor subject to adaptive evolution

Experimental Evolution of Human Influenza Virus H3 Hemagglutinin in the Mouse Lung Identifies Adaptive Regions in HA1 and HA2▿

The genetic basis for virulence and host switching in influenza A viruses (FLUAV) is largely unknown. Because the hemagglutinin (HA) protein is a determinant of these properties, HA evolution was mapped in an experimental model of mouse lung adaptation. Variants of prototype A/Hong Kong/1/68 (H3N2) (wild-type [wt] HK) human virus were selected in both longitudinal and parallel studies of lung adaptation. Mapping of HA mutations found in 11 independently derived mouse-adapted populations of wt HK identified 27 mutations that clustered within two distinct regions in or near the globular frameworks of the HA1 and HA2 subunits. The adaptive mutations demonstrated multiple instances of convergent evolution involving four amino acid positions (162, 210, and 218 in HA1 and 154 in HA2). By use of reverse genetics, convergent HA mutations were shown to affect cell tropism by enhancing infection and replication in primary mouse tracheal epithelial cells in vitro and mouse lung tissue in vivo. Adaptive HA mutations were multifunctional, affecting both median pH of fusion and receptor specificity. Specific mutations within both adaptive regions were shown to increase virulence in a mouse lung model. The occurrence of mutations in the HA1 and HA2 adaptive regions of natural FLUAV host range and virulent variants of avian and mammalian viruses is discussed. This study has identified adaptive sites and regions within the HA1 and HA2 subunits that may guide future studies of viral adaptation and evolution in nature

PB2 and Hemagglutinin Mutations Are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus▿

Serial mouse lung passage of a human influenza A virus, A/Hong Kong/1/68 (H3N2) (HK-wt), produced a mouse-adapted variant, MA, with nine mutations that was >103.8-fold more virulent. In this study, we demonstrate that MA mutations of the PB2 (D701N) and hemagglutinin (HA) (G218W in HA1 and T156N in HA2) genes were the most adaptive genetic determinants for increased growth and virulence in the mouse model. Recombinant viruses expressing each of the mutated MA genome segments on the HK-wt backbone showed significantly increased disease severity, whereas only the mouse-adapted PB2 gene increased virulence, as determined by the 50% lethal dose ([LD50] >101.4-fold). The converse comparisons of recombinant MA viruses expressing each of the HK-wt genome segments showed the greatest decrease in virulence due to the HA gene (102-fold), with lesser decreases due to the M1, NS1, NA, and PB1 genes (100.3- to 100.8-fold), and undetectable effects on the LD50 for the PB2 and NP genes. The HK PB2 gene did, however, attenuate MA infection, as measured by weight loss and time to death. Replication of adaptive mutations in vivo and in vitro showed both viral gene backbone and host range effects. Minigenome transcription assays showed that PB1 and PB2 mutations increased polymerase activity and that the PB2 D701N mutation was comparable in effect to the mammalian adaptive PB2 E627K mutation. Our results demonstrate that host range and virulence are controlled by multiple genes, with major roles for mutations in PB2 and HA

Mouse adapted NS1 mutations increase pathology and virus spread in the mouse lung.

<p>Groups of 4 CD-1 mice were infected with 1×10⁵ PFU dose of selected rHK NS mutant or rHK-wt viruses and two sets of lungs were collected at both 2 and 6 dpi. IF: Virus was detected by immunofluorescence; frozen lung sections were stained with anti-HK primary antibody, Cy3- conjugated secondary antibody (red), and nuclei were stained with Hoeschst (blue). Images were taken using a 20× objective lens. White arrows indicate foci of staining at 6 dpi. H&E: Lung pathology was assessed by H&E staining. Images were taken using a 10× objective lens.</p

Adaptive properties of rHK viruses expressing mouse-adapted NS mutations⁵.

1<p>Assessed at respective dpi with maximum weight loss for each respective virus.</p>2<p>Fold change values of lung IFN-β levels 1 day pi.</p>3<p>Relative maximum titre obtained 72 hpi (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g006" target="_blank">Fig. 6</a>).</p>4<p>Fold change of overall mRNA expression (NP + M1 + NS1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g007" target="_blank">Fig. 7</a>) or overall viral protein expression (M1 + NS1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031839#pone-0031839-g009" target="_blank">Fig. 9c–d</a>), respectively.</p>5<p>All fold values are relative to rHK-wt phenotype; where 0 represents below the limit of detection by the respective assay, with the excetion of % mortality, which is expressed as values independent to rHK-wt. (nd, not determined).</p

Mouse adapted NS1 mutations increase virulence in CD-1 mice.

<p>Groups of 5 female CD-1 mice were infected intranasally with 5×10⁶ PFU dose of rHK NS MA or HK-wt virus. NS1 mutant M106V + M124I was inoculated at dose of 2.5×10⁶ PFU due to insufficient viral stock titre. Survival (A) and body weight loss (B) were monitored for 14 dpi. Percent body weight is expressed as the mean value of 5 (or total number alive) mice (*p<0.05, **p<0.01, ***p<0.001; two-tailed unequal variance paired student's t-test for days 1–6). Experimental endpoint was defined as >30% body weight loss and respiratory distress. Graphical analysis of data was broken into three mutant sets, with rHK-wt included in each, due to the number of mutants surveyed.</p

Mouse-adapted NS1 mutations enhance virus growth in untreated and in IFN-β primed mouse cells <i>in vitro</i>.

<p>Mouse M1 cells were untreated (left panel) or pre-treated with mouse IFN-β (1000 U/mL) for 24 hours (right panel), then infected in triplicate at an MOI of 0.02 with rHK NS mutants or rHK-wt virus, and supernatant collected 12, 24, 48, and 72 hpi was quantified for viral titre by plaque assay on MDCK cells. Graphical analysis of data was broken into three mutant sets, with rHK-wt included in each, due to the number of mutants surveyed. Data represent the means ± SD (*p<0.05, **p<0.01, ***p<0.001; two-tailed student's paired t-test for 12–72 hpi, indicated by horizontal bracket above time range).</p