GSE133111
Ribosome profiling of A549 cells depleted of RPLP1 and RPLP2 and infected with DENV.
We used ribosome profiling to evaluate viral and cellular translation in RPLP1/2-depleted cells. This revealed that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). RPLP1/2 function to enhance ribosome elongation at this position and increase viral protein stability, possibly by improving co-translational folding of DENV proteins. We also analyzed the effects of RPLP1/2 depletion on cellular translation. We find that RPLP1/2 moderately affects ribosome density for a small subset of cellular mRNAs. However, meta-analysis of ribosome positions on all cellular mRNAs revealed slightly increased accumulation of ribosomes downstream of start codons in RPLP1/2-depleted cells, suggesting that RPLP1/2 enhance elongation efficiency. Importantly, we found that ribosome density on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, suggesting a role for RPLP1/2 in transmembrane protein biogenesis. Together, our findings reveal insights into the function of RPLP1/2 in DENV and cellular translation. Overall design: A549 cells were plated at 1.5 x 106 cells per 10 cm dish. Three 10cm dishes were transfected with NSC siRNA whereas three other dishes were transfected with either siP1_1, siP2_1 or siP2_4 siRNAs as described in the transfections section. 48 h later cells were infected as described previously with DENV-2 (NGC strain) at MOI of 10 in a total volume of 10 ml, rocked every 15 minutes for 1 h and the infection was allowed to proceed for more 1.5 h (2.5 h total time). Cells were then flash frozen in liquid nitrogen without cycloheximide pretreatment and cold lysis buffer containing CHX was used to lyse the cells on ice. The RIBOseq strategy was adapted from Ingolia and colleagues with a few modifications described next. After nuclease digestion, samples were run in a polysome gradient and the ribosome fractions were collected. As described by Reid and colleagues, fractions were extracted using Trizol LS (Thermo Fisher Scientific), and rRNAs were removed using the Ribo-Zero gold rRNA removal kit (Illumina, San Diego CA) according to the manufacturer's protocol. For adapter ligation and library building we used NEBNext Small RNA Library Prep Set (Illumina).