scholarly journals A Recombinant Influenza A Virus Expressing anRNA-Binding-Defective NS1 Protein Induces High Levels of BetaInterferon and Is Attenuated inMice

2003 ◽  
Vol 77 (24) ◽  
pp. 13257-13266 ◽  
Author(s):  
Nicola R. Donelan ◽  
Christopher F. Basler ◽  
Adolfo García-Sastre
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Mdck Cells ◽  
Binding Activity ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Dsrna Binding

ABSTRACT Previously we found that the amino-terminal region of the NS1 protein of influenza A virus plays a key role in preventing the induction of beta interferon (IFN-β) in virus-infected cells. This region is characterized by its ability to bind to different RNA species, including double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order to investigate whether the NS1 RNA-binding activity is required for its IFN antagonist properties, we have generated a recombinant influenza A virus which expresses a mutant NS1 protein defective in dsRNA binding. For this purpose, we substituted alanines for two basic amino acids within NS1 (R38 and K41) that were previously found to be required for RNA binding. Cells infected with the resulting recombinant virus showed increased IFN-β production, demonstrating that these two amino acids play a critical role in the inhibition of IFN production by the NS1 protein during viral infection. In addition, this virus grew to lower titers than wild-type virus in MDCK cells, and it was attenuated in mice. Interestingly, passaging in MDCK cells resulted in the selection of a mutant virus containing a third mutation at amino acid residue 42 of the NS1 protein (S42G). This mutation did not result in a gain in dsRNA-binding activity by the NS1 protein, as measured by an in vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able to replicate in MDCK cells to titers close to those of wild-type virus. This mutant virus had intermediate virulence in mice, between those of the wild-type and parental NS1 R38AK41A viruses. These results suggest not only that the IFN antagonist properties of the NS1 protein depend on its ability to bind dsRNA but also that they can be modulated by amino acid residues not involved in RNA binding.

scholarly journals Functional Replacement of the Carboxy-Terminal Two-Thirds of the Influenza A Virus NS1 Protein with Short Heterologous Dimerization Domains

2002 ◽  
Vol 76 (24) ◽  
pp. 12951-12962 ◽  
Author(s):  
Xiuyan Wang ◽  
Christopher F. Basler ◽  
Bryan R. G. Williams ◽  
Robert H. Silverman ◽  
Peter Palese ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Influenza Viruses ◽  
Ns1 Protein ◽  
Wild Type ◽  
Dimerization Domain ◽  
Carboxy Terminal

ABSTRACT The NS1 protein of influenza A/WSN/33 virus is a 230-amino-acid-long protein which functions as an interferon alpha/beta (IFN-α/β) antagonist by preventing the synthesis of IFN during viral infection. In tissue culture, the IFN inhibitory function of the NS1 protein has been mapped to the RNA binding domain, the first 73 amino acids. Nevertheless, influenza viruses expressing carboxy-terminally truncated NS1 proteins are attenuated in mice. Dimerization of the NS1 protein has previously been shown to be essential for its RNA binding activity. We have explored the ability of heterologous dimerization domains to functionally substitute in vivo for the carboxy-terminal domains of the NS1 protein. Recombinant influenza viruses were generated that expressed truncated NS1 proteins of 126 amino acids, fused to 28 or 24 amino acids derived from the dimerization domains of either the Saccharomyces cerevisiae PUT3 or the Drosophila melanogaster Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that the attenuation was mainly due to an inability to inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure.

scholarly journals Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon

2000 ◽  
Vol 74 (24) ◽  
pp. 11566-11573 ◽  
Author(s):  
Xiuyan Wang ◽  
Ming Li ◽  
Hongyong Zheng ◽  
Thomas Muster ◽  
Peter Palese ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Wild Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Functional Link ◽  
Beta Interferon ◽  
Alpha Beta

ABSTRACT The alpha/beta interferon (IFN-α/β) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-α/β-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-α/β genes, including IFN-β. It is known that transactivation of the IFN-β promoter depends on NF-κB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-κB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-κB pathway during delNS1 virus infection prevented the transactivation of the IFN-β promoter, demonstrating a functional link between NF-κB activation and IFN-α/β synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-κB pathway and of IFN-β synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-κB pathway may thus play a key role in the pathogenesis of influenza A virus.

scholarly journals Mutational Analysis of cis-Acting RNA Signals in Segment 7 of Influenza A Virus

2008 ◽  
Vol 82 (23) ◽  
pp. 11869-11879 ◽  
Author(s):  
Edward C. Hutchinson ◽  
Martin D. Curran ◽  
Eliot K. Read ◽  
Julia R. Gog ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Wild Type Virus ◽  
Wild Type ◽  
Genome Packaging ◽  
Virus Growth ◽  
Virus Particles ◽  
Synonymous Mutations ◽  
Coding Regions

ABSTRACT The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.

scholarly journals Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Carina F. Pereira ◽  
Eliot K. C. Read ◽  
Helen M. Wise ◽  
Maria J. Amorim ◽  
Paul Digard
Keyword(s):  
Influenza A Virus ◽  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Mutant Virus ◽  
Viral Gene ◽  
Ns1 Protein ◽  
Viral Mrnas

ABSTRACT Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.

scholarly journals Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins 1β and 18

2005 ◽  
Vol 86 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Jana Stasakova ◽  
Boris Ferko ◽  
Christian Kittel ◽  
Sabine Sereinig ◽  
Julia Romanova ◽  
...  
Keyword(s):  
Influenza A ◽  
Mutant Virus ◽  
Biologically Active ◽  
Wild Type Virus ◽  
Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Human Macrophages ◽  
Caspase 1 ◽  
Terminal Domains

Several NS1 mutant viruses of human influenza A/PR/8/34 (H1N1) virus were tested for their ability to induce pro-inflammatory cytokines in primary human macrophages. The findings revealed a pronounced difference in the virus-induced cytokine pattern, depending on the functionality of the NS1 protein-encoded domains. The PR8/NS1–125 mutant virus, which encodes the first 125 aa of the NS1 protein, thus lacking the C-terminal domains, induced significantly higher amounts of beta interferon, interleukin (IL) 6, tumour necrosis factor alpha and CCL3 (MIP-1α) when compared with the A/PR/8/34 wild-type virus. However, this mutant virus was as efficient as wild-type virus in the inhibition of IL1β and IL18 release from infected macrophages. Another group of viral mutants either lacking or possessing non-functional RNA-binding and dimerization domains induced 10–50 times more biologically active IL1β and five times more biologically active IL18 than the wild-type or PR8/NS1–125 viruses. The hallmark of infection with this group of mutant viruses was the induction of rapid apoptosis in infected macrophages, which correlated with the enhanced activity of caspase-1. These results indicated that the NS1 protein, through the function of its N-terminal domains, might control caspase-1 activation, thus repressing the maturation of pro-IL1β-, pro-IL18- and caspase-1-dependent apoptosis in infected primary human macrophages.

scholarly journals Immunogenicity and Protection Efficacy of Replication-Deficient Influenza A Viruses with Altered NS1 Genes

2004 ◽  
Vol 78 (23) ◽  
pp. 13037-13045 ◽  
Author(s):  
Boris Ferko ◽  
Jana Stasakova ◽  
Julia Romanova ◽  
Christian Kittel ◽  
Sabine Sereinig ◽  
...  
Keyword(s):  
Influenza A ◽  
Rna Binding ◽  
T Cell Response ◽  
Binding Domain ◽  
Wild Type Virus ◽  
Type Virus ◽  
Ns1 Protein ◽  
Wild Type ◽  
Influenza A Viruses ◽  
Rna Binding Domain

ABSTRACT We explored the immunogenic properties of influenza A viruses with altered NS1 genes (NS1 mutant viruses). NS1 mutant viruses expressing NS1 proteins with an impaired RNA-binding function or insertion of a longer foreign sequence did not replicate in murine lungs but still were capable of inducing a Th1-type immune response resulting in significant titers of virus-specific serum and mucosal immunoglobulin G2 (IgG2) and IgA, but with lower titers of IgG1. In contrast, replicating viruses elicited high titers of serum and mucosal IgG1 but less serum IgA. Replication-deficient NS1 mutant viruses induced a rapid local release of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-6. Moreover, these viruses also elicited markedly higher levels of IFN-α/β in serum than the wild-type virus. Comparable numbers of virus-specific primary CD8+ T cells were determined in all of the groups of immunized mice. The most rapid onset of the recall CD8+-T-cell response upon the wild-type virus challenge was detected in mice primed with NS1 mutant viruses eliciting high levels of cytokines. It is noteworthy that there was one NS1 mutant virus encoding NS1 protein with a deletion of 40 amino acids predominantly in the RNA-binding domain that induced the highest levels of IFN-α/β, IL-6 and IL-1β after infection. Mice that were immunized with this virus were completely protected from the challenge infection. These findings indicate that a targeted modification of the RNA-binding domain of the NS1 protein is a valuable technique to generate replication-deficient, but immunogenic influenza virus vaccines.

scholarly journals The RNA Binding Domain of Influenza A Virus NS1 Protein Affects Secretion of Tumor Necrosis Factor Alpha, Interleukin-6, and Interferon in Primary Murine Tracheal Epithelial Cells

2007 ◽  
Vol 81 (17) ◽  
pp. 9469-9480 ◽  
Author(s):  
Celeste M. Newby ◽  
Leah Sabin ◽  
Andrew Pekosz
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Binding Domain ◽  
Wild Type Virus ◽  
Ns1 Protein ◽  
Necrosis Factor Alpha ◽  
Tracheal Epithelial ◽  
Rna Binding Domain

ABSTRACT Primary differentiated respiratory epithelial cell cultures closely model the in vivo environment and allow for studies of innate immune responses generated specifically by epithelial cells, the primary cell type infected by human influenza A virus strains. We used primary murine tracheal epithelial cell (mTEC) cultures to investigate antiviral and cytokine responses to influenza A virus infection, focusing on the contribution of the RNA binding domain of the NS1 protein. rWSN NS1 R38A replication is attenuated in mTEC cultures; however, viral antigen is detected predominantly in ciliated cells, similar to wild-type virus. NS1 and NS1 R38A proteins display a primarily cytoplasmic localization in infected mTEC cultures. Increased production of tumor necrosis factor alpha, interleukin-6, and beta interferon is observed during rWSN NS1 R38A infection, and cytokines are secreted in a directional manner. Cytokine pretreatment of mTEC cultures and Vero cells suggest that rWSN NS1 R38A is more sensitive to the presence of antiviral/inflammatory cytokines than wild-type virus. Our results demonstrate that the RNA binding domain is a critical regulator of both cytokine production and cytokine sensitivity during influenza A virus infection of primary tracheal epithelial cells.

scholarly journals A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus

2019 ◽  
Vol 48 (1) ◽  
pp. 304-315 ◽  
Author(s):  
Guifang Chen ◽  
Li-Chung Ma ◽  
Shanshan Wang ◽  
Ryan L Woltz ◽  
Emily M Grasso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Binding Activity ◽  
Restriction Factor ◽  
Amino Acid Residues ◽  
Ns1 Protein ◽  
Double Stranded Rna ◽  
Host Restriction ◽  
Host Restriction Factor ◽  
Dsrna Binding

Abstract Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

scholarly journals Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K

2007 ◽  
Vol 88 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Yeun-Kyung Shin ◽  
Qiang Liu ◽  
Suresh K. Tikoo ◽  
Lorne A. Babiuk ◽  
Yan Zhou
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mutant Virus ◽  
Akt Pathway ◽  
Binding Motif ◽  
Ns1 Protein ◽  
Phosphatidylinositol 3 Kinase ◽  
Binding Motifs ◽  
Sh2 Domains ◽  
Phosphatidylinositol 3

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism is not clear. Here, it is reported that influenza A virus NS1 protein is responsible for PI3K/Akt pathway activation. It was demonstrated that the NS1 protein interacts with the p85 regulatory subunit of PI3K via direct binding to the SH3 and C-terminal SH2 domains of p85. Consensus binding motifs for SH3 and SH2 domains were found in influenza A virus NS1, namely an SH2-binding motif (YXXXM) at aa 89, SH3-binding motif 1 (PXXP) around aa 164 and SH3-binding motif 2 around aa 212. Mutant virus encoding NS1 protein with mutations in the SH-binding motifs failed to interact with SH domains of p85 and did not activate the PI3K/Akt pathway. The mutant virus is attenuated in Madin–Darby canine kidney cells. Our study has established a novel function of NS1: by interacting with p85 via the SH-binding motifs, NS1 can activate the PI3K/Akt pathway.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

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