Biochemical and biophysical analysis of distinctive human respiratory syncytial virus: phospoprotein, M2-1 antiterminator, and non-structural NS1 protein.

SEBASTIAN A. ESPERANTE, ESTEBAN PRETEL, GONZALO DE PRAT GAY

Granada

Congreso; 15th International Conference on Negative Strand Virus; 2013

Human respiratory syncytial virus (hRSV) is a worldwide distributed pathogen that infects most infants and elderly people. It shares several basic gene products with the paramyxoviridae family related to replication, attachment and fusion. With the aim of understanding fundamental, either comparative or distinctive, biochemical mechanisms, we tackled the characterization of: i) the phosphoprotein (P), the RNA polymerase cofactor; ii) the M2-1 transcription antiterminator, present only in pneumovirus, and iii) the non-structural protein NS1, a type I interferon antagonist, unique to RSVs. The P tetramer is highly stable with a modular unfolding coupled to dissociation. The M2-1 tetramer shares a strikingly similar stability (36.8 and 37.3 kcal/mol), but is highly dependent on pH. Both proteins interact with with a KD of 8 nM through a singular tetramer-tetramer interface. M2-1 bears an essential cys3-his1 zinc binding motif that can be found in Sendai and Ebola viruses and some eukaryotic transcription factors. We found that removal of the zinc atom leads to an Apo-M2-1 monomer, with secondary structure and stability identical to the tetramer. Dissociation is highly increased at pH 5.0 strongly suggesting that zinc removal, and therefore dissociation, is governed by the protonation of the histidine residue, indicative of an independent folding module with its non-specific RNA binding activity unaffected. Rather different solvent conditions cause irreversible self-oligomerization of NS1, leading to discrete stable and spherical species (NS1SOs). The convergence of these conditions, including a mild temperature change, suggest that N1SOs may accumulate in cells, where multiple conformational equilibria could be related to NS1 low binding specificity.