Fig 1.
Structure and function of natural and engineered viruses.
(a) Replication potential of natural infectious virus and defective interfering particles (DIPs). An infectious virus alone cannot replicate, but after it infects a permissive cell, viral progeny are produced. A DIP alone can enter a cell, but it cannot replicate. However, when an infectious virus and DIP infect the same cell, DIPs can replicate at the expense of infectious virus. Here figures highlight qualitative relationships between inputs and outputs. (b) Structure of natural and engineered virus genomes. (c) Production of particles, total and infectious (PFU), depends on level of natural (DIP) or engineered (DIP-GFP) input to co-infected cells. (d) Expression of virus or DIP reporter depends on level of natural (DIP) or engineered (DIP-GFP) inputs to co-infected cells. Values are normalized to a no-DIP control (PFU, RFP) or highest DIP input (GFP).
Fig 2.
Single-cell measures of reporter expression show trade-offs between infectious and defective virus.
(a) Experimental setup. Cells in all conditions were infected with a constant input of infectious virus (30 particles/cell) and varying amounts of DIP-GFP(0-to-84 particles/cell). Individual cells were imaged by time-lapse fluorescence microscopy, as detailed in Methods. For each tracked cell kinetic parameters were estimated for each RFP and GFP expression profile. (b) Example single cell kinetics. RFP and GFP kinetics are shown for two representative cells for DIP input levels 0 and 10. The average RFP and GFP expressions are also shown (dark red and green lines). (c) Anticorrelation between infectious and defective virus yields in single cells. Each gray point is an individual cell, and the green and red lines represent average defective and infectious virus yields respectively.
Fig 3.
Co-propagation of infectious and defective virus.
(a) Experimental setup. Cells were infected with a high MOI (30) of infectious virus and varying amounts of DIP-GFP(0-84). These infected cells were mixed with a large excess of uninfected cells, plated, and the spread progress of infectious and defective virus was observed via time-lapse microscopy. (b) Representative spreading infections. The first four columns contain merged images with red showing infectious virus expression, green showing defective virus expression, and yellow showing areas of both infectious and defective virus expression. The last two columns separate the red and green expression from the 25 hours post infection (hpi) images. The scale bar is 0.5 mm. (c) Infectious virus spread rate (μm/h) as a function of DIP-GFP input. Individual plaques shown as points, the average as the line. (d) The normalized intensity during the earliest detectable spread, near plaque centers, for infectious (red points and line) and defective (green points and line) virus versus DIP-GFP input.
Fig 4.
Spatial analysis of defective and infectious virus spread.
(a) Example image (DIP-GFP input = 10) of a plaque maximum intensity projection with concentric rings overlayed, where the center is the plaque origin and each ring has a width of 100 pixels (116 μm). (b) Example ring (r = 5) from the maximum intensity projection shown in (a). (c) The infectious virus (RFP) expression versus defective virus (GFP) expression for all pixels in (b). The color corresponds to point density with red being the highest and blue the lowest. The gray lines gate positive and negative populations. (d) The infectious virus (RFP) expression versus defective virus (GFP) expression for selected rings of the plaque; here, all pixel intensities that fall below both reporter thresholds have been excluded. For each ring, the contribution of each virus sub-population (e.g., infectious, defective or both) is shown as a percentage of the total population.
Fig 5.
Spatial distributions of cells infected with infectious, defective and both viruses.
The relative abundance of cells expressing reporter from infectious (RFP+), defective (GFP+), or both (RFP+, GFP+) viruses depends on distance from the plaque center (ring 1) as shown for representative plaques for DIP-GFP input levels (a) 0, (b) 1, and (c) 84 particles/cell. Plaques initiated with a DIP-GFP input of 10 particles/cell exhibited diverse patterns (d-g). Here, (d) shows the analysis for the plaque shown in Fig 4.