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).

我们采用核糖体谱（ribosome profiling）技术，对RPLP1/2敲低细胞中的病毒与细胞翻译过程进行评估。结果发现，核糖体在包膜蛋白N端编码序列中发生暂停，该序列紧邻两个相邻跨膜结构域（transmembrane domains, TMDs）的编码序列下游。RPLP1/2的功能是增强该位点的核糖体延伸效率，并提升病毒蛋白的稳定性，这可能通过改善登革病毒（Dengue virus, DENV）蛋白的共翻译折叠实现。

我们还分析了RPLP1/2敲低对细胞翻译的影响。结果显示，RPLP1/2仅对少量细胞mRNA的核糖体密度产生中度影响。然而，对所有细胞mRNA上的核糖体位置进行荟萃分析后发现，在RPLP1/2敲低细胞中，起始密码子下游的核糖体积累略有增加，表明RPLP1/2可提升延伸效率。值得注意的是，我们发现编码多个跨膜结构域的mRNA上的核糖体密度，受RPLP1/2敲低的影响不成比例，这提示RPLP1/2在跨膜蛋白生物发生中发挥作用。综上，本研究揭示了RPLP1/2在登革病毒及细胞翻译过程中的功能机制。

整体实验设计：将A549细胞以每10cm培养皿1.5×10⁶个的密度铺板。其中3个培养皿转染NSC小干扰RNA（small interfering RNA, siRNA），另外3个培养皿分别转染siP1_1、siP2_1或siP2_4 siRNA，转染步骤详见后续转染章节。转染48小时后，按照此前报道的方法，以感染复数（multiplicity of infection, MOI）为10的DENV-2（NGC毒株）在10ml总体系中感染细胞，每15分钟轻晃培养板一次，共持续1小时，随后继续培养1.5小时（总感染时长2.5小时）。随后无需使用环己酰亚胺（cycloheximide, CHX）预处理，直接将细胞置于液氮中快速冻存，使用含CHX的低温裂解缓冲液在冰上裂解细胞。RIBOseq（核糖体测序）策略参考Ingolia及其团队的方法，并进行了少许修改，具体如下：核酸酶消化后，将样品进行多糖体梯度离心，收集核糖体组分。参照Reid及其团队的方法，使用Trizol LS（Thermo Fisher Scientific）提取组分中的核酸，随后按照制造商方案使用Ribo-Zero gold rRNA Removal Kit（Illumina，加利福尼亚州圣地亚哥）去除rRNA。接头连接与文库构建过程采用NEBNext Small RNA Library Prep Set（Illumina）完成。