Quantitative RSV Gene Expression Analysis

FISH-PLA was performed as previously described [33 (link)]. Briefly, at the indicated timepoints, specified cells in a 96 well glass bottom plate were fixed with 1% paraformaldehyde for 10 minutes at RT. Cells were then permeabilized with 0.2% Triton X-100 for 5 minutes at RT. Cells were blocked for endogenous biotin with an endogenous biotin blocking kit (Thermo Fisher). MTRIPs were made as described above targeting RSV genome RNA, RSV NS1 mRNA, or polyadenylated transcripts. Controls included no primary antibodies, NeutrAvidin without a V5 epitope tag, NeutrAvidin bound to a scrambled PNA oligonucleotide, NeutrAvidin bound to biotin without an oligonucleotide, no FISH, and mock infected cells. MTRIPs were then incubated with cells overnight at 37°C in a hybridization buffer of 2x SSC with 0.5% tRNA, 0.5% ssDNA and 0.2% BSA in 1x PBS. RSV genome FISH, poly(A) FISH, and associated controls were at 5 nM, while NS1 mRNA FISH and associated controls were at 10 nM. PLA was then performed as described above between the V5 tag and RSV L. Cells were positively selected for successful infection by presence of viral protein immunofluorescence. Biological replicates were used, where n = 30 cells counted.