Functional redundancy revealed by the deletion of the mimivirus GMC-oxidoreductase genes

Abstract

The mimivirus 1.2 Mb genome was shown to be organized into a nucleocapsid-like genomic fiber encased in the nucleoid compartment inside the icosahedral capsid. The genomic fiber protein shell is composed of a mixture of two GMC-oxidoreductase paralogs, one of them being the main component of the glycosylated layer of fibrils at the surface of the virion. In this study, we determined the effect of the deletion of each of the corresponding genes on the genomic fiber and the layer of surface fibrils. First, we deleted the GMC-oxidoreductase, the most abundant in the genomic fiber, and determined its structure and composition in the mutant. As expected, it was composed of the second GMC-oxidoreductase and contained 5- and 6-start helices similar to the wild-type fiber. This result led us to propose a model explaining their coexistence. Then we deleted the GMC-oxidoreductase, the most abundant in the layer of fibrils, to analyze its protein composition in the mutant. Second, we showed that the fitness of single mutants and the double mutant were not decreased compared with the wild-type viruses under laboratory conditions. Third, we determined that deleting the GMC-oxidoreductase genes did not impact the glycosylation or the glycan composition of the layer of surface fibrils, despite modifying their protein composition. Because the glycosylation machinery and glycan composition of members of different clades are different, we expanded the analysis of the protein composition of the layer of fibrils to members of the B and C clades and showed that it was different among the three clades and even among isolates within the same clade. Taken together, the results obtained on two distinct central processes (genome packaging and virion coating) illustrate an unexpected functional redundancy in members of the family Mimiviridae, suggesting this may be the major evolutionary force behind their giant genomes.

Keywords: MS-based proteomics; Mimivirus; cryo-EM; genomic fiber; giant virus; glycosylation; helical reconstruction; layer of fibrils.

Figure 1.

Mimivirus mutants’ generation and phenotypic characterization. (A) Schematic representation of the vector and strategy utilized for deletion of qu_143 (KO_qu143) and qu_946 (KO_qu946) in mimivirus reunion strain; goi: gene of interest. Selection cassette was introduced by homologous recombination and recombinant viruses were generated, as shown in (B). Primer annealing sites are also shown and the sequence of the primers is included in Table S1. (B) Graphic depicting the strategy for the selection of recombinant viruses. Viral infection was performed 1-h post-transfection; Ntc: Nourseothricin; P, passage. (C) Growth competition assays revealed no significant defects in the lytic cycle of deletion strains. The competition was also performed in the presence of Nourseothricin, which allows the outcompetition of the recombinant strains due to the expression of a Nourseothricin selection cassette. Measurements were performed by qPCR of an endogenous locus (present in wt and recombinant strains) and the Nourseothricin selection cassette (only present in recombinant viruses).

Figure 2.

Micrograph of negative stained genomic fiber of (A) wild-type mimivirus, (B) KO_qu143 mutant, (C) KO_qu946 mutant and (D) 2KO double mutant. Scale bars 100 nm.

Figure 3.

Clustering of the 2D classes obtained with qu_143 genomic fiber. Automatic sorting of the 2D classes using the fiber width W1 and pairwise correlations of the 2D classes resulted in two main clusters (5-start Cl1 in cyan; 6-start Cl2 in orange) and a smaller cluster (Cl0 in dark blue). Each cross corresponds to a 2D class and its associated W1. Representative 2D classes are displayed with their respective W1.

Figure 4.

Workflow of the 5- and 6-start helices reconstruction processes. Segment extraction was performed with a box size of 400 pixels (pix) binned (box size 100 pix, 4.3436 Å/pix). The distance between consecutive boxes was equal to the axial rise calculated by indexation of the power spectrum. After clustering, 2D classes were selected for Cl1 and Cl2 and 3D classification was carried out using the selected segments, helical symmetry parameters from the power spectrum indexation and a 300 Å or 330 Å featureless cylinder as 3D reference for Cl1 and Cl2, respectively. 3D refinement of the two boxed 3D classes was achieved using one low pass filtered 3D class as reference on the unbinned segments. A first 3D refinement was performed with solvent flattening followed by CTF refinement and polishing of the selected segments. A last 3D refinement was achieved with solvent flattening. The EM maps colored by local resolution from 5 Å (blue) to 3 Å (red) with Euler angle distribution of the particles used for the 3D reconstruction are presented.

Figure 5.

Compositional and NMR analysis of the fibrils of mimivirus reunion strain wt and mutants. (A) GC-MS chromatogram profiles of the sugars composing the fibrils of wt (a), KO_qu946 (b), KO_qu143 (c) and 2KO (d). (B) Comparison of the ¹H NMR spectra of mimivirus reunion strain wt and related mutants with that of mimivirus prototype strain. The anomeric signals related to poly_1 (C and D units) are in black, while those of poly_2 are in red. (C) Structures of mimivirus polysaccharides as reported in Notaro et al. (2021).

Figure 6.

Model explaining the transition from a 6- to a 5-start helix. (A) Flat models of the transition from a 6- to a 5-start involving a decrease of the helix diameter by ∼3 nm. The small cluster could thus correspond to a 25-nm diameter 4-start helix. (B) Model of the different helices. A longitudinal section of the GMC-oxidoreductase shell is represented around the central channel and each helix was positioned to produce a certain continuity of the DNA strands.

Figure 7.

Fluorescent virion (A) images and (B) quantification in infected cells overexpressing GFP (Vc2) or in cells infected by the mutant Nqu143-GFP. Scale bar 5 µm. (CTVF: corrected total virion fluorescence).