Abstract
As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N0-P complex. We have previously demonstrated that residues 1-30 of P specifically bind to N0 Here, to isolate a stable N0-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N0-P-like complex, a deletion of the first 30 N-terminal residues of N (NΔ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N0-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) NΔ30 mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N0-P complexes. In both complexes, the P40-binding sites on N0 appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N0-P for the development of RSV antivirals.
Highlights
As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N
The crystal structures of the Mononegavirales N proteins complexed with RNA show that they all present a similar structural organization with two globular domains (NNTD and NCTD) that form the RNA groove, and N- and C-arms that are involved in N oligomerization [5]
In one case, the RNA groove access is blocked by the positioning of the N C-arm in the groove and the NCTD and NNTD domains are maintained in an open conformation
Summary
We have previously shown that a surrogate of the RSV N0-P complex can be obtained by co-expression of a K170A/R185A double N mutant with the N terminus of P in bacteria [17]. The data suggest a role of N residues Leu-258 and Val-284 in the interaction These results confirm the strong structural homology between RSV and hMPV N0-P complexes, with the P-binding domain on N0 overlapping both the NiϪ1 N-arm–. It is noteworthy that all N mutants were shown to be expressed in similar amounts in BSRT7 cells compared with WT N protein (Fig. 6C) These results show that conserved acidic and hydrophobic residues of the C-arm of N play a major role in the function of N within the polymerase complex. The N C-arm is directly involved in the inhibition of RNA binding To determine whether the decrease in the polymerase activity observed with the N C-arm Ala mutants was correlated to a defect of N0-P complex conformation, the same mutations were introduced in the plasmid expressing N⌬30 in E. coli, to generate 11 N variants. These data indicate that the residues Leu-368 and Leu370 are critical for stabilization of the N0-P complex, and could be directly involved in the interaction of the C-arm close to the N-RNA– binding groove, confirming our initial hypothesis
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