Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.
Part 1: RNAi in 384 Well Plates
Part 2: Infecting with Vaccinia Virus
Part 3: Staining Plates
Part 4: Imaging and Image Analysis
Part 5: Representative Images and Interpretation of Infection Rates
Figure 1. Uninfected wells do not show any staining for vaccinia virus proteins, while a representative infected well contains cells staining positive for vaccinia-expressed beta-galactosidase (βgal) protein, as measured by immunofluorescence microscopy. Virus=green, nuclei = blue.
Figure 2. Automated image analysis software quantifies infection in each image using parameters set by the user. In a representative image, the total number of cell nuclei and the number of cells expressing viral antigen are counted based on size and the intensity of staining above the local background staining.
Figure 3. Knockdown of thread (dIAP) serves as a positive control for robust RNAi depletion of target genes. Knockdown of this anti-apoptotic factor leads to dramatic cell death. Nuclei=blue.
Figure 4. Knockdown of luciferase serves as a negative control for the effect of double-stranded RNA treatment on infection. Depletion of luciferase has no effect on infection relative to cells left untreated. Virus=green, nuclei = blue.
Figure 5. Knockdown of βgal protein serves as a positive control for a reduction in infection, since the assay uses βgal protein levels as a read out of infection. Depletion of βgal results in a decrease in the percentage of infected cells. Virus=green, nuclei = blue.
Figure 6. Knockdown of cellular factor Rab5 results in a decrease in the percentage of infected cells. Since Rab5 is known to participate in endocytosis, this factor likely contributes to vaccinia virus entry. This represents an example outlier from the screen. Virus=green, nuclei = blue.
Genome-wide RNAi screening in Drosophila provides a robust and efficient method for examining the cellular component of viral infection. A number of mammalian viruses infect Drosophila cells, which can be used to identify components of the host intrinsic immune response, and host protein factors required for viral replication that may represent novel therapeutic targets. Before performing a screen, the assay must be carefully optimized for cell type, cell number, and infection level, and must be well controlled, including both positive and negative viral and cellular targets11. It is important to ensure sufficient separation between positive and negative controls prior to screening to maximize the dynamic range of the assay. Once the screen has been performed, candidates can be divided into functional categories to determine which host mechanisms contribute to infection. For mammalian viruses such as vaccinia, the contribution of mammalian homologs by RNAi should be determined as part of secondary analysis. Taken together, these robust screening methods will allow us to gain insight into complex mechanisms by which viruses interact with their host cells.
The authors have nothing to disclose.
This work was supported by grants from NIAID (R01AI074951, U54AI057168) and the Penn Genome Frontiers Institute to SC; from NIH T32HG000046 to TSM; from NIH T32GM07229 and T32AI007324 to LRS.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Cell culture | ||||
Schneider’s media | GIBCO | 11720 | ||
GlutaMAX 100X | GIBCO | 35050 | ||
Penicillin/streptomycin (pen/strep) | GIBCO | 15140 | ||
Fetal Bovine Serum (FBS) | SIGMA | F6178 | ||
Screening | ||||
Genome-wide dsRNA library | Ambion | |||
384 well plates, black with clear bottom | Corning | 3712 | ||
Aluminum seals | Corning | 6569 | ||
Clear seals | Denville | B1212-4 | ||
Well Mate | Matrix | 201-10001 | ||
24 pin manifold | Drummond | 3-000-101 | ||
Hoechst 33342 | Sigma | B2261 | ||
Fluorescently-labeled secondary antibodies | Invitrogen | |||
ImageXpressMicro automated microscope | Molecular Devices | |||
MetaXpress image analysis software | Molecular Devices |