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Review
. 2021 Dec:51:74-79.
doi: 10.1016/j.coviro.2021.09.008. Epub 2021 Sep 30.

Cryo-electron microscopy of nodavirus RNA replication organelles illuminates positive-strand RNA virus genome replication

Nuruddin Unchwaniwala  1 , Hong Zhan  1 , Johan A den Boon  1 , Paul Ahlquist  1
Affiliations
Review

Cryo-electron microscopy of nodavirus RNA replication organelles illuminates positive-strand RNA virus genome replication

Nuruddin Unchwaniwala et al. Curr Opin Virol. 2021 Dec.

Abstract

The nodavirus flock house virus recently provided a well-characterized model for the first cryo-electron microscope tomography of membrane-bound, positive-strand RNA ((+)RNA) virus genome replication complexes (RCs). The resulting first views of RC organization and complementary biochemical results showed that the viral RNA replication vesicle is tightly packed with the dsRNA genomic RNA replication intermediate, and that (+)ssRNA replication products are released through the vesicle neck to the cytosol through a 12-fold symmetric ring or crown of multi-functional viral RNA replication proteins, which likely also contribute to viral RNA synthesis. Subsequent studies identified similar crown-like RNA replication protein complexes in alphavirus and coronavirus RCs, indicating related mechanisms across highly divergent (+)RNA viruses. As outlined in this review, these results have significant implications for viral function, evolution and control.

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Figures

Figure 1
Figure 1
Positive strand RNA virus genome maps. Schematic representation of the nodavirus, alphavirus, and coronavirus genomes. Their 4.4 kb, 11.7 kb and 30 kb genomes, respectively, are shown progressively scaled down for ease of presentation and alignment. A vertical dashed line presents the general separation of RNA replication protein (left) and other protein (right) coding regions, to emphasize that the essential RNA replication functions to the left of the line expend up to 70% or more of genome coding capacity. Colored highlights indicate regions of RNA replication protein functional and structural similarity. Proteolytic cleavage sites are indicated with triangles. Membrane-association domains are underlined in purple. Virion protein coding regions are indicated in gray.
Figure 2
Figure 2
Nodavirus RNA replication complexes. (a) 3D tomographic reconstruction of a mitochondrion in a chemically fixed, FHV-infected Drosophila S2 cell (from Ref. [19•]). The outer mitochondrial membrane (OMM) is outlined in blue, and FHV RC spherules in white. (b) Cryo-EM reconstruction of an FHV RC spherule on the OMM crowned at the aperture by a multimeric ring of protein A (modelled after Ref. [25••] with the higher resolution crown and membrane neck structure from Ref. [27••] included.) (c) Detailed views at 8.5 Å resolution of (top panel) an individual subunit revealing distinct apical and basal globular domains and a leg-like extension, and (lower panels) side and top views of the protein A 12-fold symmetric crown (based on Ref. [27••]).
Figure 3
Figure 3
Comparison of nodavirus RCs and crowns to those of alphaviruses and coronaviruses. Left column: the spherule RNA replication complexes of nodaviruses are crowned by 12-fold symmetric crown complex of protein A [27••]. Middle column: In the absence of other viral proteins, exogenously expressed alphavirus nsP1, which contains RNA capping domains similar to those of the nodavirus crown basal region, assembles into a 12-mer ring that is proposed to reside atop alphavirus spherules similarly to the basal ring of the nodavirus crown [29••,30••]. In active alphavirus RNA replication complexes, this nsP1 ring presumably represents the base of a larger complex that also contains additional alphavirus RNA replication proteins nsPs 2–4 (Figure 1). Right column: Coronaviruses replicate their RNA in double-membrane vesicles (DMVs) rather than invaginated spherules, but nevertheless also have crown-like protein apertures connecting the DMV interior to the cytosol [31••]. These coronavirus crowns contain viral protein nsp3 and additional undefined viral or cellular proteins. Emerging results suggest that coronavirus crowns also engage in dynamic interactions with other viral proteins and RNAs to mediate RNA synthesis and encapsidation.
Figure 4
Figure 4
Model for nodavirus genome replication complex formation and function. Nodavirus Protein A associates with the outer mitochondrial membrane (OMM), assembles into a 12-fold symmetric crown, and recruits a founding genomic (+)RNA template. Negative-strand RNA synthesis induces the formation of spherular invaginations, possibly by filling the growing vesicle with the dsRNA product of (−)RNA synthesis. Upon completion of (−)RNA synthesis, spherules reach full final size, and the replication complex switches to repetitive synthesis and capping of (+)RNA genome copies that are released into the cytosol. The question marks in the two rightmost panels indicate that certain issues regarding (+)RNA synthesis and release are under further study, including whether RNA polymerase activity for (+)RNA synthesis is provided by protein A in the crown or in another form.
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