scholarly journals Adaptive Mutations Resulting in Enhanced Polymerase Activity Contribute to High Virulence of Influenza A Virus in Mice

2009 ◽  
Vol 83 (13) ◽  
pp. 6673-6680 ◽  
Author(s):  
Thierry Rolling ◽  
Iris Koerner ◽  
Petra Zimmermann ◽  
Kristian Holz ◽  
Otto Haller ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Polymerase Activity ◽  
Host Cells ◽  
Mammalian Host ◽  
Avian Host ◽  
Viral Polymerase ◽  
Adaptive Mutations ◽  
High Virulence ◽  
Adaptation Experiment

ABSTRACT High virulence of influenza virus A/Puerto Rico/8/34 in mice carrying the Mx1 resistance gene was recently shown to be determined by the viral surface proteins and the viral polymerase. Here, we demonstrated high-level polymerase activity in mammalian host cells but not avian host cells and investigated which mutations in the polymerase subunits PB1, PB2, and PA are critical for increased polymerase activity and high virus virulence. Mutational analyses demonstrated that an isoleucine-to-valine change at position 504 in PB2 was the most critical and strongly enhanced the activity of the reconstituted polymerase complex. An isoleucine-to-leucine change at position 550 in PA further contributed to increased polymerase activity and high virulence, whereas all other mutations in PB1, PB2, and PA were irrelevant. To determine whether this pattern of acquired mutations represents a preferred viral strategy to gain virulence, two independent new virus adaptation experiments were performed. Surprisingly, the conservative I504V change in PB2 evolved again and was the only mutation present in an aggressive virus variant selected during the first adaptation experiment. In contrast, the virulent virus selected in the second adaptation experiment had a lysine-to-arginine change at position 208 in PB1 and a glutamate-to-glycine change at position 349 in PA. These results demonstrate that a variety of minor amino acid changes in the viral polymerase can contribute to enhanced virulence of influenza A virus. Interestingly, all virulence-enhancing mutations that we identified in this study resulted in substantially increased viral polymerase activity.

scholarly journals PB2 Residue 271 Plays a Key Role in Enhanced Polymerase Activity of Influenza A Viruses in Mammalian Host Cells

2010 ◽  
Vol 84 (9) ◽  
pp. 4395-4406 ◽  
Author(s):  
Kendra A. Bussey ◽  
Tatiana L. Bousse ◽  
Emily A. Desmet ◽  
Baek Kim ◽  
Toru Takimoto
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Virus Titer ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Host Cells ◽  
Mammalian Host ◽  
Influenza A Viruses ◽  
H5n1 Influenza ◽  
Avian Viruses

ABSTRACT The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

scholarly journals Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Cellular Protein ◽  
Human Influenza ◽  
Viral Polymerase

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase and to influence both polymerase activity and interspecies restriction. We report here that either human ANP32A or ANP32B is indispensable for human influenza A virus RNA replication. The contribution of huANP32B is equal to that of huANP32A, and together they play a fundamental role in the activity of human influenza A virus polymerase, while neither human ANP32A nor ANP32B supports the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving avian ANP32A alone to support viral replication. Two amino acid mutations at sites 129 to 130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A and ANP32B as key factors for both virus replication and adaptation.IMPORTANCEThe key host factors involved in the influenza A viral polymerase activity and RNA replication remain largely unknown. We provide evidence here that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza viral RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily nonfunctional in supporting viral replication because of a mutation at sites 129 and 130. These sites play an important role in ANP32A/ANP32B and viral polymerase interaction and therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

scholarly journals Equine Mx1 Restricts Influenza A Virus Replication by Targeting at Distinct Site of its Nucleoprotein

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1114 ◽  
Author(s):  
Urooj Fatima ◽  
Zhenyu Zhang ◽  
Haili Zhang ◽  
Xue-Feng Wang ◽  
Ling Xu ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Substantial Reduction ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Dynamic Force ◽  
Equine Influenza Virus ◽  
Equine Influenza ◽  
Adaptive Mutations ◽  
Antiviral Activities

Interferon-mediated host factors myxovirus (Mx) proteins are key features in regulating influenza A virus (IAV) infections. Viral polymerases are essential for viral replication. The Mx1 protein has been known to interact with viral nucleoprotein (NP) and PB2, resulting in the influence of polymerase activity and providing interspecies restriction. The equine influenza virus has evolved as an independent lineage to influenza viruses from other species. We estimated the differences in antiviral activities between human MxA (huMxA) and equine Mx1 (eqMx1) against a broad range of IAV strains. We found that huMxA has antiviral potential against IAV strains from non-human species, whereas eqMx1 could only inhibit the polymerase activity of non-equine species. Here, we demonstrated that NP is the main target of eqMx1. Subsequently, we found adaptive mutations in the NP of strains A/equine/Jilin/1/1989 (H3N8JL89) and A/chicken/Zhejiang/DTID-ZJU01/2013 (H7N9ZJ13) that confer eqMx1 resistance and sensitivity respectively. A substantial reduction in Mx1 resistance was observed for the two mutations G34S and H52N in H3N8JL89 NP. Thus, eqMx1 is an important dynamic force in IAV nucleoprotein evolution. We, therefore, suggest that the amino acids responsible for Mx1 resistance should be regarded as a robust indicator for the pandemic potential of lately evolving IAVs.

scholarly journals The Influenza A Virus Genotype Determines the Antiviral Function of NF-κB

2016 ◽  
Vol 90 (17) ◽  
pp. 7980-7990 ◽  
Author(s):  
Sharmistha Dam ◽  
Michael Kracht ◽  
Stephan Pleschka ◽  
M. Lienhard Schmitz
Keyword(s):  
Transcription Factor ◽  
Influenza A Virus ◽  
Influenza A ◽  
Genome Engineering ◽  
Reverse Genetics ◽  
Host Cells ◽  
Avian Host ◽  
Essential Components ◽  
Antiviral Function

ABSTRACTThe role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-β) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB.IMPORTANCEThis study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier.

scholarly journals The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 protein

2008 ◽  
Vol 10 (5) ◽  
pp. 1140-1152 ◽  
Author(s):  
Igor Mazur ◽  
Darisuren Anhlan ◽  
David Mitzner ◽  
Ludmilla Wixler ◽  
Ulrich Schubert ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Polymerase Activity ◽  
Virus Protein ◽  
Viral Polymerase

scholarly journals Phosphorylation of influenza A virus NS1 at serine 205 mediates its viral polymerase-enhancing function

2021 ◽  
Author(s):  
Amol Patil ◽  
Darisuren Anhlan ◽  
Verónica Ferrando ◽  
Angeles Mecate-Zambrano ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Cell Line ◽  
Influenza A Virus ◽  
Influenza A ◽  
Phosphorylation Site ◽  
Polymerase Activity ◽  
Swine Flu ◽  
Restriction Factor ◽  
Viral Polymerase ◽  
Human Host ◽  
Potential Host

Influenza A virus (IAV) non-structural protein 1 (NS1) is a protein with multiple functions that are regulated by phosphorylation. Phospho-proteomic screening of H1N1-infected cells identified NS1 to be phosphorylated at serine 205 in intermediate stages of the viral life cycle. Interestingly, S205 is one of six amino acid changes in NS1 of post-pandemic H1N1 viruses currently circulating in humans compared to the original swine-origin 2009 pandemic (H1N1pdm09) virus, suggesting a role in host adaptation. To identify NS1 functions regulated by S205 phosphorylation, we generated both, recombinant PR8 H1N1 NS1 mutants S205G (non-phosphorylatable) and S205N (H1N1pdm09-signature), as well as H1N1pdm09 viruses harboring the reverse mutation NS1 N205S or N205D (phospho-mimetic). Replication of PR8 NS1 mutants was attenuated compared to wild type (WT) in a porcine cell line. However, PR8 NS1 S205N showed a remarkably higher attenuation compared to PR8 NS1 S205G in a human cell line, highlighting a potential host independent advantage of phosphorylatable S205, while an asparagine at this position leads to a potential host-specific attenuation. Interestingly, PR8 NS1 S205G did not show polymerase activity enhancing functions compared to WT, which can be attributed to a diminished interaction with cellular restriction factor DDX21. Analysis of the respective kinase mediating S205 phosphorylation indicated an involvement of CK2. CK2 inhibition significantly reduced replication of WT viruses and decreased NS1-DDX21 interaction as observed for NS1 S205G. In summary, NS1 S205 is required for efficient NS1-DDX21 binding resulting in an enhanced viral polymerase activity, which is likely to be regulated by transient phosphorylation. Importance Influenza A viruses (IAV) still pose a major threat to human health worldwide. As a zoonotic disease, IAV can spontaneously overcome species barriers and even reside in new hosts after efficient adaptation. Investigation of the function of specific adaptational mutations can lead to a deeper understanding of viral replication in specific hosts and can probably help to find new targets for antiviral intervention. In the present study, we analyzed the role of NS1 S205, a phosphorylation site that was re-acquired during circulation of pandemic H1N1pdm09 "Swine flu" in the human host. We found that phosphorylation of human H1N1 NS1 S205 is mediated by cellular kinase CK2 and is needed for efficient interaction with human host restriction factor DDX21 mediating NS1-induced enhancement of viral polymerase activity. Therefore, targeting CK2 activity might be an efficient strategy to limit replication of IAV circulating in the human population.

scholarly journals Fundamental contribution and host range determination of ANP32 protein family in influenza A virus polymerase activity

2019 ◽  
Author(s):  
Haili Zhang ◽  
Zhenyu Zhang ◽  
Yujie Wang ◽  
Meiyue Wang ◽  
Xuefeng Wang ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Rna Replication ◽  
Polymerase Activity ◽  
Viral Polymerase ◽  
Human Influenza Virus ◽  
Virus Rna

ABSTRACTThe polymerase of the influenza virus is part of the key machinery necessary for viral replication. However, the avian influenza virus polymerase is restricted in mammalian cells. The cellular protein ANP32A has been recently found to interact with viral polymerase, and to both influence polymerase activity and interspecies restriction. Here we report that either ANP32A or ANP32B is indispensable for influenza A virus RNA replication. The contribution of ANP32B is equal to that of ANP32A, and together they play a fundamental role in the activity of mammalian influenza A virus polymerase, while neither human ANP32A nor ANP32B support the activity of avian viral polymerase. Interestingly, we found that avian ANP32B was naturally inactive, leaving ANP32A alone to support viral replication. Two amino acid mutations at sites 129-130 in chicken ANP32B lead to the loss of support of viral replication and weak interaction with the viral polymerase complex, and these amino acids are also crucial in the maintenance of viral polymerase activity in other ANP32 proteins. Our findings strongly support ANP32A&B as key factors for both virus replication and adaption.IMPORTANCEThe key host factors involved in the influenza A viral the polymerase activity and RNA replication remain largely unknown. Here we provide evidence that ANP32A and ANP32B from different species are powerful factors in the maintenance of viral polymerase activity. Human ANP32A and ANP32B contribute equally to support human influenza virus RNA replication. However, unlike avian ANP32A, the avian ANP32B is evolutionarily non-functional in supporting viral replication because of a 129-130 site mutation. The 129-130 site plays an important role in ANP32A/B and viral polymerase interaction, therefore determine viral replication, suggesting a novel interface as a potential target for the development of anti-influenza strategies.

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

2010 ◽  
Vol 84 (20) ◽  
pp. 10606-10618 ◽  
Author(s):  
Jihui Ping ◽  
Samar K. Dankar ◽  
Nicole E. Forbes ◽  
Liya Keleta ◽  
Yan Zhou ◽  
...  
Keyword(s):  
Host Range ◽  
Influenza A Virus ◽  
Influenza A ◽  
Lethal Dose ◽  
Polymerase Activity ◽  
Mouse Lung ◽  
Viral Gene ◽  
Adaptive Mutations

ABSTRACT 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.

scholarly journals Phosphorylation of JIP4 at S730 presents anti-viral properties against influenza A virus infection

2021 ◽  
Author(s):  
Juliana Del Sarto ◽  
Vanessa Gerlt ◽  
Marcel Edgar Friedrich ◽  
Darisuren Anhlan ◽  
Viktor Wixler ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Direct Reduction ◽  
Polymerase Activity ◽  
Host Protein ◽  
Protein Accumulation ◽  
Viral Polymerase ◽  
Mrna Accumulation ◽  
Interacting Protein ◽  
Altered Expression

AbstractInfluenza A virus (IAV) is the causative agent of flu disease that results in annual epidemics and occasional pandemics. IAV alters several signaling pathways of the cellular host response in order to promote its replication. Therefore, our group investigates different host cell pathways modified in IAV infection as promising targets for long-lasting therapeutic approaches. Here, we show that c-Jun NH2-terminal kinase (JNK)-interacting protein (JIP) 4 is dynamically phosphorylated in IAV infection. Lack of JIP4 resulted in higher virus titers with significant differences in viral protein and mRNA accumulation as early as within the first replication cycle. In accordance, decreased IAV titers and protein accumulation was observed during overexpression of JIP4. Strikingly, the anti-viral function of JIP4 does neither originate from a modulation of JNK or p38 MAPK pathways, nor from altered expression of interferons or interferon-stimulated genes, but rather from a direct reduction of viral polymerase activity. Furthermore, interference of JIP4 with IAV replication is linked to phosphorylation of the serine at position 730, that is sufficient to impede with the viral polymerase and is mediated by the Raf/MEK/ERK pathway. Collectively, we provide evidence that JIP4, a host protein modulated in IAV infection, exhibits anti-viral properties that are dynamically controlled by its phosphorylation at S730.

scholarly journals Phosphorylation of JIP4 at S730 presents anti-viral properties against influenza A virus infection

2021 ◽  
Author(s):  
Juliana Del Sarto ◽  
Vanessa Gerlt ◽  
Marcel Edgar Friedrich ◽  
Darisuren Anhlan ◽  
Viktor Wixler ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Polymerase Activity ◽  
Host Protein ◽  
Protein Accumulation ◽  
Viral Polymerase ◽  
Mrna Accumulation ◽  
Altered Expression ◽  
Interferon Stimulated Genes

Influenza A virus (IAV) is the causative agent of flu disease that results in annual epidemics and occasional pandemics. IAV alters several signaling pathways of the cellular host response in order to promote its replication. Therefore, some of these pathways can serve as targets for novel anti-viral agents. Here, we show that c-Jun NH2-terminal kinase (JNK)-interacting protein (JIP) 4 is dynamically phosphorylated in IAV infection. Lack of JIP4 resulted in higher virus titers with significant differences in viral protein and mRNA accumulation as early as within the first replication cycle. In accordance, decreased IAV titers and protein accumulation was observed during overexpression of JIP4. Strikingly, the anti-viral function of JIP4 does neither originate from a modulation of JNK or p38 MAPK pathways, nor from altered expression of interferons or interferon-stimulated genes, but rather from a direct reduction of viral polymerase activity. Furthermore, interference of JIP4 with IAV replication seems to be linked to phosphorylation of the serine at position 730 that is sufficient to impede with the viral polymerase. Collectively, we provide evidence that JIP4, a host protein modulated in IAV infection, exhibits anti-viral properties that are dynamically controlled by its phosphorylation at S730. Importance Influenza A virus (IAV) infection is a world health concern and current treatment options encounter high rates of resistance. Our group investigates host pathways modified in IAV infection as promising new targets. Host protein JIP4 is dynamically phosphorylated in IAV infection. JIP4 absence resulted in higher virus titers, viral protein and mRNA accumulation within the first replication cycle. Accordingly, decreased IAV titers and protein accumulation was observed during JIP4 overexpression. Strikingly, the anti-viral function of JIP4 does neither originate from a modulation of JNK or p38 MAPK pathways, nor from altered expression of interferons or interferon-stimulated genes, but rather from a reduction in viral polymerase activity. Interference of JIP4 with IAV replication is linked to phosphorylation of serine 730. We provide evidence that JIP4, a host protein modulated in IAV infection, exhibits anti-viral properties that are dynamically controlled by its phosphorylation at S730.

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