Abstract
Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids usually prevents their full-length structural characterisation at high resolution. Here, we describe purification of full-length recombinant metastable helical nucleocapsid of Hantaan virus (Hantaviridae family, Bunyavirales order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerisation via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the nucleocapsid.
Highlights
The Bunyavirales order is one of the largest groups of segmented negative-strand RNA viruses that include many pathogenic strains (Sun et al, 2018)
Expression of recombinant full-length HTNV-NP in insect cells led to formation of recombinant NCs that have a diameter consistent with native NCs (Figure 1—figure supplement 1)
A NP74-429 construct obtained by trypsin limited proteolysis still forms a rigid helix, which means that the N-ter1-73 is not necessary for helix stabilisation (Figure 2—figure supplement 1A,B)
Summary
The Bunyavirales order is one of the largest groups of segmented negative-strand RNA viruses (sNSV) that include many pathogenic strains (Sun et al, 2018). NC flexibility appears as a hallmark in NSVs as NCs from non-segmented NSV such as Ebola or measles virus require C-terminal NP truncations in order to obtain rigid NCs and high-resolution 3D structures (Gutsche et al, 2015; Sugita et al, 2018; Wan et al, 2017). In this context, HTNVNCs are intriguing as they were shown to be able to form rather rigid helices of 10 nm diameter within viruses (Battisti et al, 2011; Huiskonen et al, 2010) and during cell infection (Goldsmith et al, 1995). We aimed at obtaining their high-resolution 3D structure, identifying the determinants of NP polymerisation and visualising RNA organisation
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