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 Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

2007 ◽  
Vol 81 (11) ◽  
pp. 5995-6006 ◽  
Author(s):  
Krister Melén ◽  
Leena Kinnunen ◽  
Riku Fagerlund ◽  
Niina Ikonen ◽  
Karen Y. Twu ◽  
...  
Keyword(s):  
Host Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Ns1 Protein ◽  
Nonstructural Protein 1 ◽  
Importin Α ◽  
Localization Signal ◽  
Ns1a Protein

ABSTRACT Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

scholarly journals The NS1 Protein of Influenza A Virus Blocks RIG-I-Mediated Activation of the Noncanonical NF-κB Pathway and p52/RelB-Dependent Gene Expression in Lung Epithelial Cells

2012 ◽  
Vol 86 (18) ◽  
pp. 10211-10217 ◽  
Author(s):  
Andrea Rückle ◽  
Emanuel Haasbach ◽  
Ilkka Julkunen ◽  
Oliver Planz ◽  
Christina Ehrhardt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Influenza A Virus ◽  
Influenza A ◽  
Target Gene ◽  
Lung Epithelial Cells ◽  
Ns1 Protein ◽  
Antiviral Immune Response ◽  
Lung Epithelial ◽  
Infected Cells

Influenza A virus (IAV) infection of epithelial cells activates NF-κB transcription factors via the canonical NF-κB signaling pathway, which modulates both the antiviral immune response and viral replication. Since almost nothing is known so far about a function of noncanonical NF-κB signaling after IAV infection, we tested infected cells for activation of p52 and RelB. We show that the viral NS1 protein strongly inhibits RIG-I-mediated noncanonical NF-κB activation and expression of the noncanonical target gene CCL19.

scholarly journals The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein

2012 ◽  
Vol 93 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Nicole C. Robb ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Viral Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Mrna Splicing ◽  
Ns1 Protein ◽  
Binding Ability ◽  
Alternatively Spliced ◽  
Transcription And Replication

The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA3, and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA.

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 Polymerase Is an Integral Component of the CPSF30-NS1A Protein Complex in Infected Cells

2008 ◽  
Vol 83 (4) ◽  
pp. 1611-1616 ◽  
Author(s):  
Rei-Lin Kuo ◽  
Robert M. Krug
Keyword(s):  
Influenza A Virus ◽  
Protein Complex ◽  
Influenza A ◽  
Critical Role ◽  
Viral Polymerase ◽  
Polymerase Complex ◽  
H5n1 Influenza ◽  
Integral Component ◽  
Infected Cells ◽  
Ns1a Protein

ABSTRACT The NS1A protein of influenza A virus binds the cellular CPSF30 protein, thereby inhibiting the 3′-end processing of all cellular pre-mRNAs, including beta interferon pre-mRNA. X-ray crystallography identified the CPSF30-binding pocket on the influenza virus A/Udorn/72 (Ud) NS1A protein and the critical role of two hydrophobic NS1A amino acids outside the pocket, F103 and M106, in stabilizing the CPSF30-NS1A complex. Although the NS1A protein of the 1997 H5N1 influenza A/Hong Kong/483/97 (HK97) virus contains L (not F) at position 103 and I (not M) at position 106, it binds CPSF30 in vivo to a significant extent because cognate (HK97) internal proteins stabilize the CPSF30-NS1A complex in infected cells. Here we show that the cognate HK97 polymerase complex, containing the viral polymerase proteins (PB1, PB2, and PA) and the nucleocapsid protein (NP), is responsible for this stabilization. The noncognate Ud polymerase complex cannot carry out this stabilization, but it can stabilize CPSF30 binding to a mutated (F103L M106I) cognate Ud NS1A protein. These results suggested that the viral polymerase complex is an integral component of the CPSF30-NS1A protein complex in infected cells even when the cognate NS1A protein contains F103 and M106, and we show that this is indeed the case. Finally, we show that cognate PA protein and NP, but not cognate PB1 and PB2 proteins, are required for stabilizing the CPSF30-NS1A complex, indicating that the NS1A protein interacts primarily with its cognate PA protein and NP in a complex that includes the cellular CPSF30 protein.

Antiviral activity of double‐stranded RNA‐binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase

2018 ◽  
Vol 32 (8) ◽  
pp. 4380-4393 ◽  
Author(s):  
Chi‐Ping Chan ◽  
Chun‐Kit Yuen ◽  
Pak‐Hin Hinson Cheung ◽  
Sin‐Yee Fung ◽  
Pak‐Yin Lui ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Viral Rna ◽  
Double Stranded Rna
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