Structural basis for suppression of a host antiviral response by influenza A virus

Das, Kalyan; Chung, Li; Xiao, Rong; Radvansky, Brian M.; Aramini, James M.; Zhao, Li; Marklund, Jesper K.; Kuo, Rei Lin; Twu, Karen Y.; Arnold, Edward; Krug, Robert M.; Montelione, Gaetano T.

doi:10.1073/pnas.0805213105

Cited by 197 publications

(302 citation statements)

References 36 publications

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“…These findings are intriguing in light of evidence that the H1N1 human influenza virus protein NS1A targets CPSF30 to obstruct cellular mRNA processing (19)(20)(21), suggesting that the link between NS1A and cellular mRNA processing is RNA recognition by CPSF30.…”

Section: Significancementioning

confidence: 84%

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Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

Shimberg

Michalek

Oluyadi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys 3 His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys 3 His domains; 2Fe-2S clusters with a Cys 3 His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metaldependent cooperative binding mechanism.zinc | iron-sulfur | RNA | protein | binding

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Section: Significancementioning

confidence: 84%

“…There are also other AUrich RNA-binding proteins such as AUF-1 (p42), Hsp70, and HuR, which bind to their respective RNA partners in a cooperative manner (39)(40)(41). Moreover, a two-domain construct of CPSF30 has been crystallized bound to the NS1 influenza A virus protein, where it is present as a dimer (21).…”

Section: Cpsf30 Contains Ironmentioning

confidence: 99%

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

Shimberg

Michalek

Oluyadi

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, dimerization of the NS1 ED is not essential for efficient β-iSH2 binding (Fig. S2), and the NS1 ED has been suggested to adopt multiple different homotypic assemblies depending on its function (17,(20)(21)(22). The heterodimeric ED:β-iSH2 structure thus resembles a golf club-shaped complex ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

Hale

Kerry

Jackson

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

115

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Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-Å resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85β, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85β:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85β and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic α-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic α-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85β inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85β (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.C lass IA phosphoinositide 3-kinases (PI3Ks) are obligate heterodimeric enzymes consisting of a 110-kDa catalytic subunit (p110α, p110β, or p110δ) bound to a noncatalytic 85-kDa regulatory subunit (typically p85α or p85β) (1). Growth factor receptor-mediated activation of PI3K requires the relocalization of p85:p110 heterodimers to the plasma membrane, where disinhibition of p110 by p85 leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). PIP 3 is an intracellular lipid second messenger that recruits pleckstrin homology domain-containing effectors (including protein kinases such as Akt) to the membrane. Subsequent activation of these effectors stimulates a plethora of signaling cascades that regulate diverse biological processes, including cell survival, proliferation, and metabolism (2). Given that PI3K is among the most frequently mutated enzymes associated with human cancers (3, 4), there is considerable interest in trying to understand the structural basis for both normal and pathophysiological regulation of p110 by p85 (5-7). Such studies are likely to yield insights into the novel mechanisms by which PI3K can be aberrantly activated, and may provide the focus for designing selective inhibitors targeting specific diseases.During infection, the PI3K signaling pathway is activated by influenz...

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“…Recent models derived from the crystal structure of full-length NS1 indicate that, besides dimers, NS1 is also capable of forming larger homopolymeric structures, which might enhance NS1's anti-IFN function (79). However, dimeric NS1 might be the predominant form of NS1 involved in some of the protein-protein interactions established between NS1 and some cellular factors (80,81). Therefore, SUMOylation may exert a broad regulatory role on NS1 function simply by helping modulate the proportion of NS1 monomers, dimers, trimers, and polymers present in the cell.…”

Section: Discussionmentioning

confidence: 99%

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

Santos

Pal

Chacon

et al. 2013

J Virol

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Our pioneering studies on the interplay between the small ubiquitin-like modifier (SUMO) and influenza A virus identified the nonstructural protein NS1 as the first known SUMO target of influenza virus and one of the most abundantly SUMOylated influenza virus proteins. Here, we further characterize the role of SUMOylation for the A/Puerto Rico/8/1934 (PR8) NS1 protein, demonstrating that NS1 is SUMOylated not only by SUMO1 but also by SUMO2/3 and mapping the main SUMOylation sites in NS1 to residues K219 and K70. Furthermore, by using SUMOylatable and non-SUMOylatable forms of NS1 and an NS1-specific artificial SUMO ligase (ASL) that increases NS1 SUMOylation ϳ4-fold, we demonstrate that SUMOylation does not affect the stability or cellular localization of PR8 NS1. However, NS1's ability to be SUMOylated appears to affect virus multiplication, as indicated by the delayed growth of a virus expressing the non-SUMOylatable form of NS1 in the interferon (IFN)-competent MDCK cell line. Remarkably, while a non-SUMOylatable form of NS1 exhibited a substantially diminished ability to neutralize IFN production, increasing NS1 SUMOylation beyond its normal levels also exerted a negative effect on its IFN-blocking function. This observation indicates the existence of an optimal level of NS1 SUMOylation that allows NS1 to achieve maximal activity and suggests that the limited amount of SUMOylation normally observed for most SUMO targets may correspond to an optimal level that maximizes the contribution of SUMOylation to protein function. Finally, protein cross-linking data suggest that SUMOylation may affect NS1 function by regulating the abundance of NS1 dimers and trimers in the cell.

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Structural basis for suppression of a host antiviral response by influenza A virus

Cited by 197 publications

References 36 publications

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

Cleavage and polyadenylation specificity factor 30: An RNA-binding zinc-finger protein with an unexpected 2Fe–2S cluster

Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein

SUMOylation Affects the Interferon Blocking Activity of the Influenza A Nonstructural Protein NS1 without Affecting Its Stability or Cellular Localization

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