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Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30.

Dankar SK, Miranda E, Forbes NE, Pelchat M, Tavassoli A, Selman M, Ping J, Jia J, Brown EG - Virol. J. (2013)

Bottom Line: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields.Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT

Background: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.

Methods: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.

Results: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.

Conclusions: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

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Virulence is increased by each of the 103L and 106I NS1 mutations in the H5N1-NS gene on human and mouse adapted virus backbones as well as for 103S and 106I PR8 NS1 gene mutations in PR8 virus. a and b. Groups of 3 CD-1 mice were infected intranasally with 5 × 106 pfu of rHK viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. c and d. Groups of 3 CD-1 mice were infected intranasally with 1 × 104 pfu with the different rPR8 viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. e and f, Groups of 5 BALB/c mice were infected intranasally with 1 × 104 pfu with wt rPR8 or mutants that differed due to the indicated mutations at positions 103 and 106. The percent of body weight loss was calculated as the mice body weight loss was recorded daily throughout the whole course of the experiment. Values are shown as average +/− standard deviation (*p<0.05, **p<0.01, *** p<0.001; two-tailed student’s t-test).
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Figure 1: Virulence is increased by each of the 103L and 106I NS1 mutations in the H5N1-NS gene on human and mouse adapted virus backbones as well as for 103S and 106I PR8 NS1 gene mutations in PR8 virus. a and b. Groups of 3 CD-1 mice were infected intranasally with 5 × 106 pfu of rHK viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. c and d. Groups of 3 CD-1 mice were infected intranasally with 1 × 104 pfu with the different rPR8 viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. e and f, Groups of 5 BALB/c mice were infected intranasally with 1 × 104 pfu with wt rPR8 or mutants that differed due to the indicated mutations at positions 103 and 106. The percent of body weight loss was calculated as the mice body weight loss was recorded daily throughout the whole course of the experiment. Values are shown as average +/− standard deviation (*p<0.05, **p<0.01, *** p<0.001; two-tailed student’s t-test).

Mentions: To assess the virulence phenotypes associated with the individual 103L and 106I sites the H5N1 NS-wt genome segment that possesses the 103L and 106I mutations as its wt reference sequence or mutants with L103F or I106M mutations alone or in combination were rescued into a human and mouse-adapted backbones, A/HK/1/68 (H3N2) (HK) and A/PR/8/34 (H1N1) respectively. The relative virulence of these viruses on the HK and the PR8 backbones were assessed in groups of 3 and 7 Swiss-Webster (CD-1) mice following intranasal infection with 5.0 × 106 pfu and 1.0 × 104 pfu respectively. The rHK-H5N1-NS-wt (103L+106I) and the three mutants (rHK-H5N1-NS-L103F+106I, rHK-H5N1-NS-103L+I106M and rHK-H5N1-NS-L103F+I106M) did not result in mortality in CD-1 mice (all had LD50 >107.2 pfu) (Figure 1b). Daily body weight monitoring showed the greatest loss for the rHK-H5N1-NS-wt (103L+106I) virus (7% at day 5 pi). Each of the 3 mutant caused significantly less body weight loss on comparison of days 3–6 for H5N1-NS mutants L103F+106I, 103L+I106M and L103F+I106M (P≤0.01, P≤0.05, and P≤0.001 by paired t-test respectively); and single time points showed significant differences for 103L+I106M and L103F+I106M (P≤0.05 by single sample t-test, n = 3) (Figure 1a). Thus each of the individual 103L and 106I mutations contributed to disease severity which was cumulative in combination.


Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30.

Dankar SK, Miranda E, Forbes NE, Pelchat M, Tavassoli A, Selman M, Ping J, Jia J, Brown EG - Virol. J. (2013)

Virulence is increased by each of the 103L and 106I NS1 mutations in the H5N1-NS gene on human and mouse adapted virus backbones as well as for 103S and 106I PR8 NS1 gene mutations in PR8 virus. a and b. Groups of 3 CD-1 mice were infected intranasally with 5 × 106 pfu of rHK viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. c and d. Groups of 3 CD-1 mice were infected intranasally with 1 × 104 pfu with the different rPR8 viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. e and f, Groups of 5 BALB/c mice were infected intranasally with 1 × 104 pfu with wt rPR8 or mutants that differed due to the indicated mutations at positions 103 and 106. The percent of body weight loss was calculated as the mice body weight loss was recorded daily throughout the whole course of the experiment. Values are shown as average +/− standard deviation (*p<0.05, **p<0.01, *** p<0.001; two-tailed student’s t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3733596&req=5

Figure 1: Virulence is increased by each of the 103L and 106I NS1 mutations in the H5N1-NS gene on human and mouse adapted virus backbones as well as for 103S and 106I PR8 NS1 gene mutations in PR8 virus. a and b. Groups of 3 CD-1 mice were infected intranasally with 5 × 106 pfu of rHK viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. c and d. Groups of 3 CD-1 mice were infected intranasally with 1 × 104 pfu with the different rPR8 viruses possessing wt (103L + 106I) or mutant H5N1 NS1 genes that differed due to the indicated mutations at positions 103 and 106. e and f, Groups of 5 BALB/c mice were infected intranasally with 1 × 104 pfu with wt rPR8 or mutants that differed due to the indicated mutations at positions 103 and 106. The percent of body weight loss was calculated as the mice body weight loss was recorded daily throughout the whole course of the experiment. Values are shown as average +/− standard deviation (*p<0.05, **p<0.01, *** p<0.001; two-tailed student’s t-test).
Mentions: To assess the virulence phenotypes associated with the individual 103L and 106I sites the H5N1 NS-wt genome segment that possesses the 103L and 106I mutations as its wt reference sequence or mutants with L103F or I106M mutations alone or in combination were rescued into a human and mouse-adapted backbones, A/HK/1/68 (H3N2) (HK) and A/PR/8/34 (H1N1) respectively. The relative virulence of these viruses on the HK and the PR8 backbones were assessed in groups of 3 and 7 Swiss-Webster (CD-1) mice following intranasal infection with 5.0 × 106 pfu and 1.0 × 104 pfu respectively. The rHK-H5N1-NS-wt (103L+106I) and the three mutants (rHK-H5N1-NS-L103F+106I, rHK-H5N1-NS-103L+I106M and rHK-H5N1-NS-L103F+I106M) did not result in mortality in CD-1 mice (all had LD50 >107.2 pfu) (Figure 1b). Daily body weight monitoring showed the greatest loss for the rHK-H5N1-NS-wt (103L+106I) virus (7% at day 5 pi). Each of the 3 mutant caused significantly less body weight loss on comparison of days 3–6 for H5N1-NS mutants L103F+106I, 103L+I106M and L103F+I106M (P≤0.01, P≤0.05, and P≤0.001 by paired t-test respectively); and single time points showed significant differences for 103L+I106M and L103F+I106M (P≤0.05 by single sample t-test, n = 3) (Figure 1a). Thus each of the individual 103L and 106I mutations contributed to disease severity which was cumulative in combination.

Bottom Line: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields.Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada.

ABSTRACT

Background: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.

Methods: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.

Results: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.

Conclusions: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

Show MeSH
Related in: MedlinePlus