Influence of RNA Binding on the Conformational Dynamics of the Lassa Virus Nucleoprotein

Abstract

Lassa virus is an enveloped virus, with a bisegmented ambisense single-stranded RNA genome, encoding only four gene products. The most abundant protein is the nucleoprotein (NP) which encapsidates the viral genomic RNA. Multiple structures of the NP have been determined. The full-length NP was determined in the absence of RNA in a trimeric arrangement, which has been shown to be the dominant solution form of NP. A second structure of only the N-terminal domain of NP was determined in the presence of a short RNA strand, which revealed an RNA-binding groove. There were significant differences between the RNA bound and RNA-free forms in the NP N-terminal domain, highlighted by helix 6 adopting a distal (open) orientation, and helix 5 undergoing a partial loss of helicity, when RNA is bound.The primary focus of this investigation is to understand the mechanism by which the Lassa virus NP binds to RNA and how conformational changes in the NP are achieved. We have employed extensive equilibrium molecular dynamics (MD) simulations to investigate the influence of RNA on the global motions of the NP, and have identified that RNA-binding induces a closing motion. This observation is supported by free energy calculations using both umbrella sampling and metadynamics methods. The free energy profiles show the closed state is more favorable, both in the absence and presence of RNA, and that RNA-binding reduces the energy cost for opening. The observation that the RNA-bound form prefers a closed configuration appears in disagreement with the open-state crystal structure. However, we believe non-native crystal contacts are stabilizing the open form of NP, which is supported from energetic analysis of MD simulations of NP dimers present in the crystal unit cell.