Abstract
Replication of non-segmented negative-strand RNA viruses requires the continuous supply of the nucleoprotein (N) in the form of a complex with the phosphoprotein (P). Here, we present the structural characterization of a soluble, heterodimeric complex between a variant of vesicular stomatitis virus N lacking its 21 N-terminal residues (NΔ21) and a peptide of 60 amino acids (P60) encompassing the molecular recognition element (MoRE) of P that binds RNA-free N (N0). The complex crystallized in a decameric circular form, which was solved at 3.0 Å resolution, reveals how the MoRE folds upon binding to N and competes with RNA binding and N polymerization. Small-angle X-ray scattering experiment and NMR spectroscopy on the soluble complex confirms the binding of the MoRE and indicates that its flanking regions remain flexible in the complex. The structure of this complex also suggests a mechanism for the initiation of viral RNA synthesis.
Highlights
Negative-sense RNA viruses include numerous major human pathogens such as influenza virus, rabies virus, measles virus and respiratory syncytial virus
After cleavage of the maltose binding protein (MBP) tag with the tobacco etch virus (TEV) protease, the resulting ND210-peptide of 60 amino acids (P60) complex was purified by Ni2+ chelate affinity chromatography followed by size-exclusion chromatography (SEC)
Because the N0-P complex is required for the replication of the virus [5,22], inhibition of its formation might represent an interesting target for blocking viral replication and could explain the recent observations that a homologous peptide (P60) from rabies virus P inhibits viral replication [23]
Summary
Negative-sense RNA viruses include numerous major human pathogens such as influenza virus, rabies virus, measles virus and respiratory syncytial virus. P binds to nascent RNA-free N, forming a N0-P complex (the superscript 0 denotes the absence of RNA) that prevents the polymerization of N and the non-specific encapsidation of host cell RNAs [4,5,6,7,8,9]. These processes are independent of each other [10] and, P has to fulfill two chaperone activities; blocking both RNA binding and self-assembly of N. The N0-binding region is localized in the Nterminal disordered region [7,8,9], and it has been demonstrated that in vesicular stomatitis virus (VSV), a prototypical rhabdovirus, this region of P contains transient helical elements and may constitute a short molecular recognition element (MoRE) that folds upon binding to its partner [13]
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