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 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 Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of the Host Antiviral and Immune Responses

2015 ◽  
Vol 89 (12) ◽  
pp. 6442-6452 ◽  
Author(s):  
Tsuyoshi Hayashi ◽  
Leslie A. MacDonald ◽  
Toru Takimoto
Keyword(s):  
Protein Synthesis ◽  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A Virus ◽  
Influenza A ◽  
Host Protein ◽  
Viral Growth ◽  
Host Shutoff ◽  
Host Protein Synthesis ◽  
Host Antiviral Response

ABSTRACTInfluenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting, PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus, A/California/04/09 (H1N1, Cal), containing mutations at the frameshift motif in the polymerase PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host β-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited beta interferon (IFN-β) expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of viral growth and greater expression of IFN-β mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected mice than in Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune responses to influenza virus.IMPORTANCEVirus-induced host protein shutoff is considered to be a major factor allowing viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity bothin vitroandin vivo. We also demonstrated that, while the growth of Cal PA-XFS was attenuated in the lungs of infected animals, this mutant induced a stronger humoral response than Cal WT. Our findings clearly highlight the importance of PA-X in counteracting the host innate and acquired immune responses to influenza virus, an important global pathogen. This work demonstrates that inhibition of PA-X expression in influenza virus vaccine strains may provide a novel way of safely attenuating viral growth while inducing a more robust immune response.

scholarly journals Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Shuai Cao ◽  
Pragyesh Dhungel ◽  
Zhilong Yang
Keyword(s):  
Protein Synthesis ◽  
Cell Biology ◽  
Influenza A ◽  
Viral Infections ◽  
Cellular Protein ◽  
Host Protein ◽  
Virus Infections ◽  
Cellular Energy ◽  
Host Shutoff ◽  
Host Protein Synthesis

ABSTRACT Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular-energy-generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology.

scholarly journals Adaptation of an H7N7 equine influenza A virus in mice

2007 ◽  
Vol 88 (2) ◽  
pp. 547-553 ◽  
Author(s):  
Kyoko Shinya ◽  
Shinji Watanabe ◽  
Toshihiro Ito ◽  
Noriyuki Kasai ◽  
Yoshihiro Kawaoka
Keyword(s):  
Protein Expression ◽  
Influenza A Virus ◽  
Virus Replication ◽  
Viral Protein ◽  
Influenza A ◽  
Animal Species ◽  
Influenza A Viruses ◽  
Equine Influenza ◽  
Viral Protein Expression ◽  
Wild Waterfowl

Wild waterfowl are a reservoir for influenza A viruses, which can be transmitted from these birds to other animal species. Occasionally, influenza A viruses are transmitted to other animal species from animals other than wild waterfowl, e.g. an equine influenza virus has been transmitted to dogs and caused outbreaks. To understand the molecular mechanism by which influenza A viruses adapt to a new animal species, the molecular changes involved in the adaptation of an H7N7 equine influenza A virus were studied in mice. Mutations in the mouse-adapted virus mapped to one amino acid change in the PA protein, one in PB2 and two in PB1. Of these mutations, the Glu-to-Lys substitution at position 627 of PB2 (PB2-E627K) increased virulence appreciably. To understand the mechanism of this increased virulence, a recombinant virus expressing a reporter green fluorescent protein was constructed, thus enabling the effect of this mutation on viral protein expression to be tested in the context of virus replication in situ. It was found that the PB2-E627K substitution in this equine virus contributed to increased viral protein expression and virus replication in mouse cells and enhanced brain invasiveness in mice. These results demonstrate that the importance of the PB2-E627K substitution for mouse adaptation, which was identified previously in human H5N1 isolates, extends to equine influenza A virus.

scholarly journals NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

2016 ◽  
Vol 90 (21) ◽  
pp. 9693-9711 ◽  
Author(s):  
Marta L. DeDiego ◽  
Aitor Nogales ◽  
Kris Lambert-Emo ◽  
Luis Martinez-Sobrido ◽  
David J. Topham
Keyword(s):  
Immune Responses ◽  
Influenza A ◽  
Innate Immune ◽  
Host Protein ◽  
Ns1 Protein ◽  
Innate Immune Responses ◽  
Virus Surveillance ◽  
Dsrna Binding ◽  
The Subject ◽  
Host Protein Synthesis

ABSTRACT Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

scholarly journals ISG15 conjugation system targets the viral NS1 protein in influenza A virus–infected cells

2010 ◽  
Vol 107 (5) ◽  
pp. 2253-2258 ◽  
Author(s):  
Chen Zhao ◽  
Tien-Ying Hsiang ◽  
Rei-Lin Kuo ◽  
Robert M. Krug
Keyword(s):  
Antiviral Activity ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Rna Binding ◽  
Acceptor Site ◽  
Ns1 Protein ◽  
Conjugation System ◽  
Infected Cells ◽  
Ns1a Protein

ISG15 is an IFN-α/β–induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-α/β. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-α, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-β. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a “loss of function” mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.

scholarly journals Nucleolar Relocalization of RBM14 by Influenza A Virus NS1 Protein

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Grant Beyleveld ◽  
Daniel J. Chin ◽  
Elena Moreno Del Olmo ◽  
Jade Carter ◽  
Isabel Najera ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Host Factor ◽  
Host Factors ◽  
Host Protein ◽  
Ns1 Protein ◽  
Network Analyses ◽  
Syncytial Virus ◽  
Virus Biology

ABSTRACTViruses utilize a number of host factors in order to carry out their replication cycles. Influenza A virus (IAV) and human respiratory syncytial virus (RSV) both infect the tissues of the respiratory tract, and as such we hypothesize that they might require similar host factors. Several published genome-wide screens have identified putative IAV host factors; however, there is significant discordance between their hits. In order to build on this work, we integrated a variety of “OMICS” data sources using two complementary network analyses, yielding 51 genes enriched for both IAV and RSV replication. We designed a targeted small interfering RNA (siRNA)-based assay to screen these genes against IAV under robust conditions and identified 13 genes supported by two IAV subtypes in both primary and transformed human lung cells. One of these hits, RNA binding motif 14 (RBM14), was validated as a required host factor and furthermore was shown to relocalize to the nucleolus upon IAV infection but not with other viruses. Additionally, the IAV NS1 protein is both necessary and sufficient for RBM14 relocalization, and relocalization also requires the double-stranded RNA (dsRNA) binding capacity of NS1. This work reports the discovery of a new host requirement for IAV replication and exposes a novel example of interplay between IAV NS1 and the host protein, RBM14.IMPORTANCEInfluenza A virus (IAV) and respiratory syncytial virus (RSV) present major global disease burdens. There are high economic costs associated with morbidity as well as significant mortality rates, especially in developing countries, in children, and in the elderly. There are currently limited therapeutic options for these viruses, which underscores the need for novel research into virus biology that may lead to the discovery of new therapeutic approaches. This work extends existing research into host factors involved in virus replication and explores the interaction between IAV and one such host factor, RBM14. Further study to fully characterize this interaction may elucidate novel mechanisms used by the virus during its replication cycle and open new avenues for understanding virus biology.

scholarly journals RNase L limits host and viral protein synthesis via inhibition of mRNA export

2021 ◽  
Author(s):  
James M. Burke ◽  
Alison R. Gilchrist ◽  
Sara L. Sawyer ◽  
Roy Parker
Keyword(s):  
Protein Synthesis ◽  
Influenza A Virus ◽  
Viral Protein ◽  
Influenza A ◽  
Cytokine Production ◽  
Mrna Export ◽  
Virus Protein ◽  
Rnase L ◽  
Viral Mrna ◽  
Viral Protein Synthesis

AbstractRNase L is widely thought to limit viral protein synthesis by cleaving host rRNA and viral mRNA, resulting in translation arrest and viral mRNA degradation. Herein, we show that the mRNAs of dengue virus and influenza A virus largely escape RNase L-mediated mRNA decay, and this permits viral protein production. However, activation of RNase L arrests nuclear mRNA export, which strongly inhibits influenza A virus protein synthesis and reduces cytokine production. Importantly, the heterogeneous and temporal nature of the mRNA export block in individual cells permits sufficient production of antiviral cytokines from transcriptionally induced host mRNAs. This defines RNase L-mediated arrest of mRNA export as a key antiviral shutoff and cytokine regulatory pathway.One Sentence SummaryRNase L-mediated shutoff of nuclear mRNA export limits viral protein synthesis and regulates antiviral cytokine production.

Sign in / Sign up

Export Citation Format

Share Document