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.

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 HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Huan Chen ◽  
Yingjuan Qian ◽  
Xin Chen ◽  
Zhiyang Ruan ◽  
Yuetian Ye ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Molecular Mechanisms ◽  
Polymerase Activity ◽  
Negative Regulator ◽  
Host Protein ◽  
Spectrometric Analysis ◽  
Rna Polymerase Activity

ABSTRACT The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication. IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

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 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 Stress granule-inducing eukaryotic translation initiation factor 4A inhibitors block influenza A virus replication

2017 ◽  
Author(s):  
Patrick D. Slaine ◽  
Mariel Kleer ◽  
Nathan Smith ◽  
Denys A. Khaperskyy ◽  
Craig McCormick
Keyword(s):  
Protein Synthesis ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Translation Initiation Factor ◽  
Host Protein ◽  
Initiation Factor ◽  
Viral Protein Synthesis ◽  
Infected Cells ◽  
Pateamine A

ABSTRACTEukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5’ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection results in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates the feasibility of targeting core host protein synthesis machinery to prevent viral replication.IMPORTANCEInfluenza A virus (IAV) relies on cellular protein synthesis to decode viral messenger RNAs. Pateamine A and silvestrol are natural products that inactivate an essential protein synthesis protein known as eIF4A. Here we show that IAV is sensitive to these eIF4A inhibitor drugs. Treatment of infected cells with pateamine A or silvestrol prevented synthesis of viral proteins, viral genome replication and release of infectious virions. The irreversible eIF4A inhibitor pateamine A sustained long-term blockade of viral replication, whereas viral protein synthesis quickly resumed after silvestrol was removed from infected cells. Prolonged incubation of either infected or uninfected cells with these drugs induced the programmed cell death cascade called apoptosis. Our findings suggest that core components of the host protein synthesis machinery are viable targets for antiviral drug discovery. The most promising drug candidates should selectively block protein synthesis in infected cells without perturbing bystander uninfected cells.

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 Inhibition of Ongoing Influenza A Virus Replication Reveals Different Mechanisms of RIG-I Activation

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
GuanQun Liu ◽  
Yao Lu ◽  
Qiang Liu ◽  
Yan Zhou
Keyword(s):  
Protein Synthesis ◽  
Viral Replication ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Viral Rna ◽  
Chain Elongation ◽  
Type I ◽  
Viral Polymerase ◽  
Viral Protein Synthesis

ABSTRACTPattern recognition receptors provide essential nonself immune surveillance within distinct cellular compartments. Retinoic acid-inducible gene I (RIG-I) is one of the primary cytosolic RNA sensors, with an emerging role in the nucleus. It is involved in the spatiotemporal sensing of influenza A virus (IAV) replication, leading to the induction of type I interferons (IFNs). Nonetheless, the physiological viral ligands activating RIG-I during IAV infection remain underexplored. Other than full-length viral genomes, cellular constraints that impede ongoing viral replication likely potentiate an erroneous viral polymerase generating aberrant viral RNA species with RIG-I-activating potential. Here, we investigate the origins of RIG-I-activating viral RNA under two such constraints. Using chemical inhibitors that inhibit continuous viral protein synthesis, we identify the incoming, but notde novo-synthesized, viral defective interfering (DI) genomes contributing to RIG-I activation. In comparison, deprivation of viral nucleoprotein (NP), the key RNA chain elongation factor for the viral polymerase, leads to the production of aberrant viral RNA species activating RIG-I; however, their nature is likely to be distinct from that of DI RNA. Moreover, RIG-I activation in response to NP deprivation is not adversely affected by expression of the nuclear export protein (NEP), which diminishes the generation of a major subset of aberrant viral RNA but facilitates the accumulation of small viral RNA (svRNA). Overall, our results indicate the existence of fundamentally different mechanisms of RIG-I activation under cellular constraints that impede ongoing IAV replication.IMPORTANCEThe induction of an IFN response by IAV is mainly mediated by the RNA sensor RIG-I. The physiological RIG-I ligands produced during IAV infection are not fully elucidated. Cellular constraints leading to the inhibition of ongoing viral replication likely potentiate an erroneous viral polymerase producing aberrant viral RNA species activating RIG-I. Here, we demonstrate that RIG-I activation during chemical inhibition of continuous viral protein synthesis is attributable to the incoming DI genomes. Erroneous viral replication driven by NP deprivation promotes the generation of RIG-I-activating aberrant viral RNA, but their nature is likely to be distinct from that of DI RNA. Our results thus reveal distinct mechanisms of RIG-I activation by IAV under cellular constraints impeding ongoing viral replication. A better understanding of RIG-I sensing of IAV infection provides insight into the development of novel interventions to combat influenza virus infection.

scholarly journals Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

2002 ◽  
Vol 76 (3) ◽  
pp. 1206-1212 ◽  
Author(s):  
Mirella Salvatore ◽  
Christopher F. Basler ◽  
Jean-Patrick Parisien ◽  
Curt M. Horvath ◽  
Svetlana Bourmakina ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Expression ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Vero Cells ◽  
Cellular Protein ◽  
Host Protein ◽  
Ns1 Protein ◽  
Host Protein Synthesis

ABSTRACT The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-α/β) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein—lacking approximately half of its carboxy-terminal end—showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.

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.

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