The NS1 protein of influenza B virus binds 5’-triphosphorylated dsRNA to suppress RIG-I activation and the host antiviral response

Woltz R, Schweibenz B, Tsutakawa S, Zhao C, Ma L, Shurina B, Hura G, John R, Vorobiev S, Swapna G, Solotchi M, Tainer J, Krug R, Patel S, Montelione G, (2024) DOI

SASDT65 – Non-structural protein 1 (NS1B, C-terminal) plus double stranded RNA

Non-structural protein 1
RNA top strand
RNA bottom strand
MWexperimental 18 kDa
MWexpected 26 kDa
VPorod 23 nm3
log I(s) 2.50×10-3 2.50×10-4 2.50×10-5 2.50×10-6
Non-structural protein 1 RNA top strand RNA bottom strand small angle scattering data  s, nm-1
ln I(s)
Non-structural protein 1 RNA top strand RNA bottom strand Guinier plot ln 2.51×10-3 Rg: 2.8 nm 0 (2.8 nm)-2 s2
(sRg)2I(s)/I(0)
Non-structural protein 1 RNA top strand RNA bottom strand Kratky plot 1.104 0 √3 sRg
p(r)
Non-structural protein 1 RNA top strand RNA bottom strand pair distance distribution function Rg: 2.8 nm 0 Dmax: 10.3 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Non-structural protein 1 RNA top strand RNA bottom strand HADDOCK model
Samples of NS1B-Cterminal domain (CTD) were prepared at 250 μΜ (4 mg/mL), after 16-bp dsRNA was added to a 1:1 NS1B-CTD:16-bp dsRNA ratio in 2K buffer (40 mM ammonium acetate, pH 5.5, 450 mM NaCl, 5 mM CaCl2, 0.02% NaN3, 50 mM arginine). To minimize aggregation in the SAXS samples, samples were centrifuged at 8,500 x g for a minimum of 10 mins immediately prior to measurements. For this complex, SAXS data was collected on CHESS beamline G1 (MacCHESS: 161 Synchrotron drive, Ithaca NY – Cornell University) at 9.924 keV (0.1249 nm) with 7.7×1011 photons/s. The X-ray beam was collimated to 250 × 250 µm2 diameter and centered on a capillary sample cell with 1.5 mm path length and 25 μm thick quartz glass walls (Charles Supper Company, Natik, MA). The sample cell and full X-ray flight path, including beamstop, were kept in vacuo (< 1×10-3 Torr) to eliminate air scatter. Temperature was maintained at 4 °C. Images were collected on a dual Pilatus 100K-S detector system (Dectris, Baden, Switzerland). Sample-to-detector distance was calibrated using silver behenate powder (The Gem Dugout, State College, PA).

Sample detector distance = UNKNOWN

Non-structural protein 1 (NS1B)
Mol. type   Protein
Organism   Influenza B virus
Olig. state   Monomer
Mon. MW   15.9 kDa
 
UniProt   X2C382 (141-281)
Sequence   FASTA
 
RNA top strand (RNAtop)
Mol. type   RNA
Olig. state   Monomer
Mon. MW   5.1 kDa
Sequence   FASTA
 
RNA bottom strand (RNAbottom)
Mol. type   RNA
Olig. state   Monomer
Mon. MW   5.3 kDa
Sequence   FASTA