Functional Evolution of the 2009 Pandemic H1N1 Influenza Virus NS1 and PA in Humans

ABSTRACT In 2009, a pandemic H1N1 influenza A virus (IAV) (pH1N1) emerged in the human population from swine causing a pandemic. Importantly, this virus is still circulating in humans seasonally. To analyze the evolution of pH1N1 in humans, we sequenced viral genes encoding proteins inhibiting general gene expression (nonstructural protein 1 [NS1] and PA-X) from circulating seasonal viruses and compared them to the viruses isolated at the origin of the pandemic. Recent pH1N1 viruses contain amino acid changes in the NS1 protein (E55K, L90I, I123V, E125D, K131E, and N205S), as previously described (A. M. Clark, A. Nogales, L. Martinez-Sobrido, D. J. Topham, and M. L. DeDiego, J Virol 91:e00721-17, 2017, https://doi.org/10.1128/JVI.00721-17), and amino acid changes in the PA-X protein (V100I, N204S, R221Q, and L229S). These amino acid differences between early and more recent pH1N1 isolates are responsible for increased NS1-mediated inhibition of host gene expression and decreased PA-X-mediated shutoff, including innate immune response genes. In addition, currently circulating pH1N1 viruses have acquired amino acid changes in the PA protein (V100I, P224S, N321K, I330V, and R362K). A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from currently circulating viruses is fitter in replication in cultured cells and in mice and is slightly more pathogenic than the original ancestor pH1N1 virus. These results demonstrate the need to monitor the evolution of pH1N1 in humans for mutations in the viral genome that could result in enhanced virulence. Importantly, these results further support our previous findings suggesting that inhibition of global gene expression mediated by NS1 and PA-X proteins is subject to a balance which can determine virus pathogenesis and fitness. IMPORTANCE IAVs emerge in humans from animal reservoirs, causing unpredictable pandemics. One of these pandemics was caused by an H1N1 virus in 2009, and this virus is still circulating seasonally. To analyze host-virus adaptations likely affecting influenza virus pathogenesis, protein amino acid sequences from viruses circulating at the beginning of the pandemic and those circulating currently were compared. Currently circulating viruses have incorporated amino acid changes in two viral proteins (NS1 and PA-X), affecting innate immune responses, and in the PA gene. These amino acid differences led to increased NS1-mediated and decreased PA-X-mediated inhibition of host gene expression. A recombinant pH1N1 virus containing PA, PA-X, and NS1 genes from recently circulating viruses is fitter in replication in tissue culture cells and in mice, and the virus is more pathogenic in vivo. Importantly, these results suggest that a balance in the abilities of NS1 and PA-X to induce host shutoff is beneficial for IAVs.

Download Full-text

Effects of the C-Terminal Truncation in NS1 Protein of the 2009 Pandemic H1N1 Influenza Virus on Host Gene Expression

PLoS ONE ◽

10.1371/journal.pone.0026175 ◽

2011 ◽

Vol 6 (10) ◽

pp. e26175 ◽

Cited By ~ 11

Author(s):

Jiagang Tu ◽

Jing Guo ◽

Anding Zhang ◽

Wenting Zhang ◽

Zongzheng Zhao ◽

...

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

H1n1 Influenza ◽

Host Gene ◽

Pandemic H1n1 ◽

Ns1 Protein ◽

Host Gene Expression ◽

H1n1 Influenza Virus ◽

Pandemic H1n1 Influenza

Download Full-text

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

Journal of Virology ◽

10.1128/jvi.00721-17 ◽

2017 ◽

Vol 91 (17) ◽

Cited By ~ 20

Author(s):

Amelia M. Clark ◽

Aitor Nogales ◽

Luis Martinez-Sobrido ◽

David J. Topham ◽

Marta L. DeDiego

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Amino Acid ◽

Proinflammatory Cytokines ◽

Influenza A ◽

Host Gene ◽

Pandemic H1n1 ◽

Ns1 Protein ◽

Host Gene Expression ◽

Mouse Lungs

ABSTRACT In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up to the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) that allow the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of proinflammatory cytokines, resulting in viral attenuation in vivo. This might represent an adaptation of pH1N1 virus to humans.

Download Full-text

Emergence and characterisation of pandemic H1N1 influenza viruses in Hungarian swine herds

Acta Veterinaria Hungarica ◽

10.1556/avet.2012.059 ◽

2013 ◽

Vol 61 (1) ◽

pp. 125-134 ◽

Cited By ~ 1

Author(s):

Ádám Bálint ◽

István Kiss ◽

Krisztián Bányai ◽

Imre Biksi ◽

Katalin Szentpáli-Gavallér ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Viral Fitness ◽

Pandemic H1n1 ◽

H1n1 Influenza Virus ◽

Pandemic H1n1 Influenza ◽

Molecular Features ◽

Swine Herds

In 2010, two novel porcine H1N1 influenza viruses were isolated from pigs with influenza-like illness in Hungarian swine herds. Sequence and phylogenetic analysis of these strains revealed that they shared molecular features with the pandemic H1N1 influenza virus strains, which emerged globally during 2009. The PB2, HA and NA genes contained unique amino acid changes compared to the available new H1N1 influenza virus sequences of pig origin. Furthermore, the investigated strains could be separated with respect to parallel amino acid substitutions affecting the polymerase genes (PB2, PB1 and PA) and the nucleoprotein (NP) gene, supporting the proposed complementarities between these proteins, all required for the viral fitness. Molecular characterisation of two Hungarian human pandemic H1N1 isolates was also performed, so that we could compare contemporaneous strains of different host species origins. Shared molecular motifs in various genes of animal and human influenza strains suggested that the Hungarian porcine strains could have originated from humans through direct interspecies transmission. This study is among the few that support the natural human-to-pig transmission of the pandemic H1N1 influenza virus.

Download Full-text

The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression

Journal of Virology ◽

10.1128/jvi.00877-17 ◽

2017 ◽

Vol 91 (22) ◽

Cited By ~ 9

Author(s):

Aitor Nogales ◽

Caroline Chauché ◽

Marta L. DeDiego ◽

David J. Topham ◽

Colin R. Parrish ◽

...

Keyword(s):

Gene Expression ◽

Amino Acid ◽

Immune Responses ◽

Influenza Viruses ◽

Innate Immune ◽

Host Gene ◽

Innate Immune Responses ◽

Host Gene Expression ◽

Avian Origin ◽

Canine Influenza

ABSTRACT Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2 viruses. Influenza A virus (IAV) nonstructural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. In contrast, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. IMPORTANCE Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses, of equine and avian origins, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses, and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression, but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, which contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza virus replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals.

Download Full-text