Tools for Single-Cell Kinetic Analysis of Virus-Host Interactions

Abstract

Measures of cellular gene expression or behavior, when performed on individual cells, inevitably reveal a diversity of behaviors and outcomes that can correlate with normal or diseased states. For virus infections, the potential diversity of outcomes are pushed to an extreme, where measures of infection reflect features of the specific infecting virus particle, the individual host cell, as well as interactions between viral and cellular components. Single-cell measures, while revealing, still often rely on specialized fluid handling capabilities, employ end-point measures, and remain labor-intensive to perform. To address these limitations, we consider a new microwell-based device that uses simple pipette-based fluid handling to isolate individual cells. Our design allows different experimental conditions to be implemented in a single device, permitting easier and more standardized protocols. Further, we utilize a recently reported dual-color fluorescent reporter system that provides dynamic readouts of viral and cellular gene expression during single-cell infections by vesicular stomatitis virus. In addition, we develop and show how free, open-source software can enable streamlined data management and batch image analysis. Here we validate the integration of the device and software using the reporter system to demonstrate unique single-cell dynamic measures of cellular responses to viral infection.

Fig 1. Comparison of general approaches that enable fluorescence-based cytometry.

The categories and capabilities of each are roughly assessed. Capability Key: ● = High, $\mathrm{●}$ = Medium, $\mathrm{●}$ = Low, (blank) = not significant.

Fig 2. Infection with the recombinant VSV strains leads to the production of RFP as a reporter of viral protein production.

(a) Although VSV-rWT does stimulate the early stages of immune recognition, its matrix (M) protein suppresses export of host transcripts, thereby suppressing the subsequent innate immune response and resulting in a GFP⁻RFP⁺cell. (b) The well characterized mutant of M protein, M51R [34], abolishes this function and readily stimulates the innate immune response [38]. Thus, an infection with VSV-M51R allows production of type-I interferons and activation of interferon stimulated genes, including the IFIT2 reporter, resulting in a GFP⁺RFP⁺cell.

Fig 3. Traditional microwell array (MA) design.

(a) Typical device dimensions. (b) A lid can be secured to the top of the PDMS MA to either allow or block cross-talk or communication between the microwells [46].

Fig 4. New microwell array (MA) device design and assembly for single-cell studies.

(a) Regions of microwells are isolated from one another using grooves or ‘moats’ allowing multiple conditions to be tested on a single device using surface tension to maintain the droplets. Each isolated region is referred to as a bull’s-eye given its appearance. (inset, d, f) Twelve bull’s-eyes in a 2×6 array fit on a standard glass slide. The device is trimmed to include 2×5 and only the outer 8 can be imaged through the clamp device. (b-c) The array of bull’s-eyes containing ∼ 2500 microwells each are placed on a microscope slide. Cells in suspension within each droplet settle into microwells randomly for easy loading and are visualized using nuclear staining. Cells are identified and counted using image analysis. Only wells containing a single cell are considered. The image in (b) is a stitched phase contrast image, (c) is the same image with fluorescent nuclei visible, and the magnified image indicates the size and shape of individual microwells. (d-g) Device assembly. (d) The slide of bull’s-eyes is placed in the base of the microscope insert. (e) The droplets are removed and a glass slide is placed over the bull’s-eyes. (f) The pressure distributor of the microscope stage insert is placed over the glass slide lid. (g) The top plate is fastened with a bracket using two hex screws. The device is sealed and ready for time-lapse imaging.

Fig 5. Data acquisition and analysis.

a) Images are acquired in multiple colors and locations over time. The resultant list of files is imported into JEX. JEX performs analysis on the imported data, and stores the outputs into the same database. All function parameters and data from intermediate steps can be recorded in the JEX database. b) The database of information is stored in a simply named and transparent folder structure for perusal and use outside of JEX, but is also easily accessed via the JEX user interface. c) The workflow for baseline microscopy experiments generally consists of: 1) an initial background and illumination correction, 2) stitching the image array for each color and timepoint within each well, 3) identification of cells based on Hoechst staining, 4) quantification of cell location and fluorescence intensity for each color, and 5) plotting of results. The workflow for MA-based experiments includes additional functions for identifying microwells, counting the number of cells within each microwell, and quantifying whole-microwell and single-cell fluorescence. See S1 Text for details of all steps.

Fig 6. Movie frames of FC and microscopy PDC plots at 6, 12, and 18 hpi.

The green and red fluorescence of each cell in VSV-rWT and VSV-M51R infections are shown in each scatter plot. All timepoints for the microscopy data are taken from the same sample whereas the FC data at each timepoint represents data from separate samples obtained in parallel. Furthermore, the microscope sample was sacrificed at 18 hpi and used for the FC 18 hpi timepoint. Thus, the 18 hpi timepoint is the same sample for both methods and is directly comparable. The summary plots on the right-hand side show gated population percentages over time, illustrating the similarity in sensitivity of the two methods but highlighting the improved temporal resolution of the microscopy approach. The gray cross-hair in each plot represents the overall population mean.

Fig 7. Microwell-array-based cytometry plots (VSV-M51R & VSV-rWT) show agreement with results from baseline cytometry experiments from Fig 6.

Fig 8. Sample VSV-rWT viral and innate immune reporter protein expression kinetics from six individual cells.

Kinetic parameters have been extracted from the trajectories of both the viral (red) and host (green) trajectories and displayed in the top left corner of each figure. Maximum yields from individual cells vary greatly (a-b), host reporter expression can be greater or lesser than viral gene expression given similar starting conditions (c-d), and cells can lyse (d) or remain intact during imaging. Single-cell analysis can detect and quantify cells displaying rare behavior, such as those that appear to have a basal level of innate immune activation all the time (e) and cells infected with wt-VSV that express ZS-Green despite encoding a functional matrix gene.