Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Dengue virus (DENV) and other mosquito-borne flaviviruses are highly dependent on the ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), for efficient infection. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. 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 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, implying 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.