RNA control of HIV-1 particle size polydispersity

Abstract

HIV-1, an enveloped RNA virus, produces viral particles that are known to be much more heterogeneous in size than is typical of non-enveloped viruses. We present here a novel strategy to study HIV-1 Viral Like Particles (VLP) assembly by measuring the size distribution of these purified VLPs and subsequent viral cores thanks to Atomic Force Microscopy imaging and statistical analysis. This strategy allowed us to identify whether the presence of viral RNA acts as a modulator for VLPs and cores size heterogeneity in a large population of particles. These results are analyzed in the light of a recently proposed statistical physics model for the self-assembly process. In particular, our results reveal that the modulation of size distribution by the presence of viral RNA is qualitatively reproduced, suggesting therefore an entropic origin for the modulation of RNA uptake by the nascent VLP.

Figure 1. Biochemical characterization of VLPs and viral cores, and AFM imaging.

(a) Immunoblot of HIV-1 for mature and immature VLPs and cores. (b) Reverse transcription test on mature VLPs and cores in the presence or the absence of ψRNA in the VLPs or cores. (c) Typical images of mature VLPs. (d) Typical images of viral cores.

Figure 2. Automated image analysis.

(a) Example of the successive image analysis steps applied on a single image. (b) Cartoon of the principle of image analysis. (c) Various representation (color map, contour plots, 3D plots) of particles that were selected. Top VLP, down core. Interestingly, the contour map of the VLP shows a pronounced asymmetry close to its maximal height, reflecting the presence of an asymmetric object inside the VLP.

Figure 3. Statistical analysis of size distributions of VLPs and cores obtained through automated image analysis.

The number of particles is indicated by the value N. (a) and (c) 2D histograms of short and long diameters for respectively mature VLPs and cores. (b) Short diameter histogram obtained by projecting the 2D histograms for VLPs and cores. (d) and (f), long diameter histogram obtained projecting the 2D histograms for VLPs and cores. (e) Typical example of short and long diameters measured on a single particle.

Figure 4. Influence of the presence or absence of viral ψRNA on particle morphogenesis.

The number of particles is indicated by the value N. (a) and (c) 2D histogram of short and long diameters for respectively VLPs in the absence and in the presence of ψRNA. The size distribution is shifted toward smaller value, and its dispersion is reduced. (b) and (d) 2D histogram of short and long diameters for respectively cores in the absence and in the presence of ψRNA. The same shift in the distribution is observed, although with w weaker amplitude. (e) Box plot of equivalent diameters summarizing the previous results. The equivalent diameter is obtained by converting the 2D projected area of the particle into the diameter of a disk that would give the same area.

Figure 5. Model of entropic selection of viral genome at fixed particle size.

(a) Cartoon of the self-assembly. The model is specialized to bimodal products of self-assembly, in which the size of particle are equal and the RNA content are different. (b) Typical large RNA titration computed thanks to the model detailed in File S1. The value of parameters chosen for the calculation are found in the material and methods. Blue circles correspond to particles with large RNA, and green crosses correspond to particles lacking large RNA.(c) Phase diagram small RNA/large RNA. The boundary for which the concentration of both particles are equal is shown by blue filled circles. The line joining the circles is drawn to guide the eye.

Figure 6. Combined model of viral genome and particle size entropic selection.

The model is specialized to bimodal products of self-assembly, with different particle size and different RNA content. (a) Typical large RNA titration computed thanks to the model detailed in File S1. The value of parameters chosen for the calculation are found in the material and methods. In this case, the number of proteins in the “large particles” is twice the number of proteins in the small one. Red circles correspond to small particles with large RNA, and pink crosses correspond to large particles lacking large RNA. (b) Phase diagram small RNA/large RNA. The boundary for which the concentration of both particles are equal is shown by red filled circles. The line joining the circles is drawn to guide the eye. For comparison, the boundary at fixed particle sizes found in figure 5b is depicted by a blue dotted line.