Social evolution of innate immunity evasion in a virus

Abstract

Antiviral immunity has been studied extensively from the perspective of virus-cell interactions, yet the role of virus-virus interactions remains poorly addressed. Here, we demonstrate that viral escape from interferon (IFN)-based innate immunity is a social process in which IFN-stimulating viruses determine the fitness of neighbouring viruses. We propose a general and simple social evolution framework to analyse how natural selection acts on IFN shutdown and validate it in cell cultures and mice infected with vesicular stomatitis virus. Furthermore, we find that IFN shutdown is costly because it reduces short-term viral progeny production, thus fulfilling the definition of an altruistic trait. Hence, in well-mixed populations, the IFN-blocking wild-type virus is susceptible to invasion by IFN-stimulating variants and spatial structure consequently determines whether IFN shutdown can evolve. Our findings reveal that fundamental social evolution rules govern viral innate immunity evasion and virulence and suggest possible antiviral interventions.

Fig. 1. Social evolution model for innate immunity evasion.

Top left: partition of individual fitness according to social neighborhood. One virus blocks IFN (W) and another that does not (D). The W virus in a W neighborhood is used as reference and has log fitness (f) equal to zero. IFN-mediated paracrine signaling has an indirect fitness effect b that applies to W and D. The direct effect of blocking IFN on the actor, independent of neighborhood effects, is denoted c, and can a priori be positive or negative. Because fitness is defined logarithmically, independent effects are additive and hence the fitness of D in a D neighborhood is c – b. Top right: fitness of each variant, which depends on spatial structure through r_W and r_D. Bottom: three possible scenarios (W-infected cells in red, D-infected cells in green, region of immunized cells in blue). Bottom left: no spatial structure, both viruses share the same neighborhood. Bottom middle: maximal spatial structure. Analysis of these two cases allows obtaining b and c. Bottom right: intermediate situation. If f_W and f_D are measured and b and c are known, r_W, r_D, and r can be inferred.

Fig. 2. Interaction between VSV WT and Δ51 variants.

a. Maximal titers of Δ51 (green) and WT (red) in mono-infected cultures at 45 hpi. b. Total titers at 45 hpi in cultures infected with Δ51 and WT at different input ratios (MOI = 0.001 FFU/cell). The black dashed line shows the least-squares linear regression. The red dashed line shows the expected total titer assuming no interaction between the two variants. This was obtained based on the titers reached by pure WT and Δ51 infections as follows: T(p) = pT_Δ51 + (1 − p) T_WT, where T is titer, p is the fraction of Δ51 at inoculation, and T_Δ51 and T_WT are the titers reached by pure Δ51 and WT infections, respectively. c. WT titer at 45 hpi in MEFs primed for 1 h with a conditioned medium obtained from a previous Δ51 infection (MOI = 0.001 FFU/cell). d. Time-dependence of anti-VSV IFN effects. MEFs were treated with a 1/5 dilution of conditioned medium at the indicated times. All treatments reduced titer significantly (one sample t-tests against 1.0: P = 3.5 × 10^–6, P = 2.9 × 10^–6, and P = 0.024 for t = – 1 hpi, 0 hpi, and 3 hpi, respectively) except the 6 hpi treatment (P = 0.290). In a-d error bars indicate the SEM of n = 3 independent measures, and in b-d the three individual data points are also shown. e. Range of action of innate immune signaling from single Δ51-infected cells. In the picture, a single cell infected with Δ51 (GFP-positive, apoptotic, shown with arrow) generates a region of cells resistant to the WT virus (lack of red fluorescence). The approximate size of the immunized region was determined by visual inspection. Histogram: distribution of the number of immunized cells obtained after analyzing 30 images (mean: 54.0 ± 9.6 cells).

Fig. 3. Real-time fluorescence microscopy of VSV WT and Δ51 in MEFs.

Pure WT-mCherry, pure Δ51-GFP, and mixed WT-mCherry/Δ51-GFP infections were carried out in the same w12 multi-well dish, which also included mixed WT-mCherry/WT-GFP controls (Supplementary Fig. 2). Left: representative images of three time points. Right: average area occupied by GFP and mCherry signals (n = 2 replicate wells for pure WT and Δ51 infections, n = 4 replicates for mixed infections). Notice that these graphs were obtained by image analysis of entire culture wells, not just the representative images shown on the left panels. SEM values correspond to the technical error among wells of the same experimental block. Two additional experimental blocks were performed with similar results. For the trypsin treatment (performed at 8 and 24 hpi), fewer data points were analyzed because cell detachment prevented imaging at each time point. This treatment was performed in a separate w12 well, which included its own controls (Supplementary Fig. 2). The progression of the infection is shown in Supplementary Videos 1-3, and whole-well images are shown in Supplementary Fig. 1.

Fig. 4. Fitness cost of IFN shutdown.

a. Spread of pure VSV WT-GFP and pure VSV Δ51-GFP infections. Left: representative images of three time points. Right: average area occupied by the GFP signal (n = 2 replicate wells). Notice that these graphs were obtained by image analysis of entire culture wells, not just the representative images shown on the left panels. Infections were carried out in the same multi-well dish (experimental block), and image acquisition/analysis was performed identically for all wells. Similar results were obtained in another experimental block. b. Competition assays between VSV WT-mCherry and Δ51-GFP. Three 48 hpi transfers were performed in undisturbed cells (top) and in cells subjected to trypsin treatment at 8 and 24 hpi (bottom). The Δ51 fraction (GFP/total fluorescent area) after each transfer is shown. Each of the n = 4 lines represents one replicate of the competition assay.

Fig. 5. Metapopulation structure selects for IFN shutdown.

MEFs in a 96-well format were inoculated with a limiting dilution of an approximately 1:1 mix of the WT and a NmAb-resistant Δ51 virus. Titers produced by each variant in each well were determined by the plaque assay. Left: Box plots of the WT and Δ51 titers in wells showing only one variant (pure; n = 20 for WT and n = 35 for Δ51) or a mixture of the two variants (mixed; n = 16). The lower and upper limits of the box indicate 25^th and 75^th percentiles, and the middle line shows the median. Whiskers show the 10^th and 90^th percentiles, and outlying points are plotted individually. Middle: titers produced in each individual well. Right: overall WT and Δ51 yield in the metapopulation (sum of all wells).

Fig. 6. Fluorescence microscopy of VSV-infected mouse brains.

a. Brain full sagittal section (except cerebellum) of a mouse succumbing to an infection by VSV WT-mCherry (nuclei stained with DAPI). Scale bar: 1 mm. b, c. Atlases showing a schematic representation of the infection pattern observed in two additional, parallel sections. Given that animals were inoculated intranasally, the observed pattern is consistent with primary infection of the OB glomerular layer (GL) originating from olfactory axons and spreading along the RMS. Isolated infected areas were also found in the olfactory tubercle. The infection may have progressed from the RMS towards lateral ventricles, producing infected areas adjacent to ventricular walls (lateral septal nucleus, striatum adjacent to the anterior region of the lateral ventricle, and hippocampus adjacent to the posterior ventricle). Hence, the ventricular system probably acted as a route for disseminating the infection towards the thalamus and hypothalamus. The thalamus appears as another major infection site, from which the virus may have reached the spinal cord, producing paralysis. Examination of the brains from two additional animals inoculated with WT-mCherry showed similar infection patterns. The OB of a pure WT infection and OB/RMS regions of a mixed infection are shown in Supplementary Fig. 5. d-f. Individual infected regions in the OB glomerular layer of n = 3 mice infected with a 1:1 mix of WT-mCherry and Δ51-GFP, one at 3 dpi (d) and two at 4 dpi (e, f). Scale bars: 50 µm. In these three animals, no signs of infection were found in the rest of the brain. Quantitation of the area occupied by Δ51-GFP and WT-mCherry is shown on Supplementary Table 1.