The genome formula of a multipartite virus is regulated both at the individual segment and the segment group levels.

Differential accumulation of the distinct genome segments is a common feature of viruses with segmented genomes. The reproducible and specific pattern of genome segment accumulation within the host is referred to as the "genome formula". There is speculation and some experimental support for a functional role of the genome formula by modulating gene expression through copy number variations. However, the mechanisms of genome formula regulation have not yet been identified. In this study, we investigated whether the genome formula of the octopartite nanovirus faba bean necrotic stunt virus (FBNSV) is regulated by processes acting at the individual segment vs. viral population levels. We used a leaf infiltration system to show that the two most accumulated genome segments of the FBNSV possess a greater intrinsic accumulation capacity in Vicia faba tissues than the other segments. Nevertheless, processes acting at the individual segment level are insufficient to generate the genome formula, suggesting the involvement of additional mechanisms acting at the supra-segment level. Indeed, the absence of segments with important functions during systemic infection strongly modifies the relative frequency of the others, indicating that the genome formula is a property of the segment group. Together, these results demonstrate that the FBNSV genome formula is shaped by a complex process acting at both the individual segment and the segment group levels

Comparison of DNA-R accumulation depending on the segment with which it was infiltrated.

The accumulation of DNA-R when infiltrated with each of the seven other segments (C+R, M+R, N+R, S+R, U1+R, U2+R or U4+R) or alone with the same (R) or doubled (R+R) OD of infiltrated bacteria was determined by qPCR. For each box, the horizontal central bar represents the median and the edges of the rectangle the first and third quartiles. The vertical outer bars delineate the minimum and maximum values of the distribution, excluding outliers. The dots represent outliers. Letters above the boxes indicate segments with which DNA-R accumulation is statistically significant (Kruskal-Wallis tests and Bonferroni correction for multiple tests; S8 Table). (TIF)</p

Infection rates and phenotypes of FBNSV complete and incomplete infections in <i>V</i>. <i>faba</i>.

Infection rates and symptom severity were determined three (FBNSVcomplete, FBNSVN-, FBNSVU4-, FBNSVC- and FBNSVC-,U4-) or four (FBNSVU2-) weeks after inoculation. The presence of the segments was controlled by qPCR. (DOCX)</p

Statistical analysis of the comparison of DNA-R accumulation depending on the segment with which it is infiltrated.

Statistical analyses were performed through Kruskal-Wallis tests using RStudio (package “agricolae”). The p-value indicating a statistically significant difference after Bonferroni correction (p≤0.05) is in red. (DOCX)</p

Statistical analysis of the comparison of segment accumulation in infiltrations in pairs of segments across experimental replicates.

We provide the output of a full model, ratio = replicate * segment. Statistical analyses were performed through Scheirer Ray Hare tests using RStudio (package “rcompanion”). The p-values indicating statistically significant differences after Bonferroni correction (p≤0.05) are in red. (DOCX)</p

S1 Fig -

Symptoms of V. faba infected with FBNSVcomplete (A and C) or FBNSVU2- (B and D) four weeks after agro-inoculation. (TIF)</p

Comparison of segment accumulation when infiltrated in pairs with DNA-R across experimental replicates.

The relative accumulation ratio of each segment with respect to DNA-R obtained for the two experimental replicates are represented in dark and light blue respectively. For each box, the horizontal central bar represents the median and the edges of the rectangle the first and third quartiles. The vertical outer bars delineate the minimum and maximum values of the distribution, excluding outliers. The dots represent outliers. No statistically significant differences were found between the two experimental replicates (Scheirer Ray Hare test, p≤0.05; S9 Table). (TIF)</p

Statistical analysis of the comparison of the segment relative frequency between complete and incomplete infections.

For each type of incomplete infection, we first provide the output of a full model, frequency = segment * modality where modality corresponds to the incomplete vs complete infection treatments. After the full tests we provide the output of per segment comparisons across modalities to identify segments whose relative frequency statistically significantly differed between incomplete and complete infections. These analyses were performed through Scheirer Ray Hare tests and Dunn tests using RStudio (packages “rcompanion” and “FSA”). The p-values indicating statistically significant differences after Bonferroni correction (p≤0.05) are in red. (DOCX)</p

Sequences of the primers used to quantify segment accumulation by qPCR.

Sequences of the primers used to quantify segment accumulation by qPCR.</p

Comparison of the FBNSV genome formula in complete and incomplete infections without C and/or U4.

FBNSV genome formula in incomplete infections FBNSVC-, U4- (blue) compared to FBNSVcomplete (A; grey) or FBNSVC- (B; red). Genome segments accumulation in symptomatic V. faba plants was estimated by qPCR and the relative frequency of the segments was determined. To allow meaningful comparisons, the relative frequency of each segment was calculated without considering the accumulation of segments C and U4 in FBNSVcomplete and without considering U4 in FBNSVC-. Standard deviations are represented by grey triangles (complete infections) or red crosses (incomplete infections). Asterisks associated to segment names indicate when the differences in frequencies between complete and incomplete infections are statistically significant (Scheirer Ray Hare (p≤0.05) and post-hoc Dunn tests, Bonferroni correction). (TIF)</p