Fig 1.
Organization of the dsRNA in CHIKV spherules.
(A) Computational slice through a cryo-electron tomogram containing two CHIKV spherules at the plasma membrane. The right slice shows an overlay of the dsRNA tracing for one of the two spherules. Density is black. Scale bar, 25nm. (B) Average apparent persistence length of the dsRNA, individually measured in 12 spherules, with standard deviation indicated. The measurement was made using cosine correlation plots, one example of which is shown in S3C–S3G Fig. Unmasked subtomogram averages of five classes of dsRNA resulting from multireference alignment classification. For each class, the left panel shows a slice through the density. The right panel shows an isosurface representation where the density interpreted as dsRNA is shown in red, and adjacent densities in yellow. Scale bar, 10 nm. (H) Chart of the distribution of the genome percentage present in each average. The percentages of the dsRNA that was not present in our initial tracing, or traced but not resolved during the averaging process are also indicated.
Table 1.
Distribution of subtomograms in the five different classes shown in Fig 1.
The length of the dsRNA densities as measured in the averages is stated, as well as their contribution to the total length of the genome. The sum indicates the total percentage of the genome that could be resolved in the subtomogram average classes. This number (52%) is smaller than the fraction of the genome initially traced (80%) since only the consensus structure appears in the subtomogram average. Number of particles refers to the total number of subtomograms, from 22 spherules.
Fig 2.
Occupancy of the dsRNA within spherules.
(A) Volume fraction of the dsRNA, assuming a radius of 1.2 nm and using individually measured spherule volumes measured in tomograms. Each dot represents one spherule and the bar shows the mean. (B) Concentration of dsRNA in the spherules, expressed as molar concentration of polymerized NTPs and mg/ml dsRNA. Each dot represents one spherule and the bar shows the mean. (C) Average local density of dsRNA in 22 spherules as a function of the radial distance from the center of the spherule. The included spherules had radii in the range of 30–38 nm.
Fig 3.
Preferential orientation of the dsRNA in spherules.
(A) Schematic of the measurement of the angles between short segments of traced dsRNA and a reference vector anchored to the neck complex and pointing toward the spherule center. (B-D) Hypothetical relative frequency of angles, as defined in A, for the scenarios indicated in the spherule sketches. The dsRNA could pack with no preferred orientation to the neck complex vector (e.g., randomly) (B), with a preferred direction parallel (C), or perpendicular (D) to the neck complex vector. (E) Measurement of the relative frequency of angles in 22 spherules. (F) The traced dsRNA overlayed on a slice through the tomogram for one representative spherule included in the analysis. Scale bar, 25 nm.