Bartels Hanni, Luban Jeremy
Department of Microbiology and Molecular Medicine, University of Geneva, Geneva 1205, Switzerland.
Retrovirology. 2014 Sep 12;11:73. doi: 10.1186/s12977-014-0073-0.
All retroviruses synthesize essential proteins via alternatively spliced mRNAs. Retrovirus genera, though, exploit different mechanisms to coordinate the synthesis of proteins from alternatively spliced mRNAs. The best studied of these retroviral, post-transcriptional effectors are the trans-acting Rev protein of lentiviruses and the cis-acting constitutive transport element (CTE) of the betaretrovirus Mason-Pfizer monkey virus (MPMV). How members of the gammaretrovirus genus translate protein from unspliced RNA has not been elucidated.
The mechanism by which two gammaretroviruses, XMRV and MLV, synthesize the Gag polyprotein (Pr65Gag) from full-length, unspliced mRNA was investigated here. The yield of Pr65Gag from a gag-only expression plasmid was found to be at least 30-fold less than that from an otherwise isogenic gag-pol expression plasmid. A frameshift mutation disrupting the pol open reading frame within the gag-pol expression plasmid did not decrease Pr65Gag production and 398 silent nucleotide changes engineered into gag rendered Pr65Gag synthesis pol-independent. These results are consistent with pol-encoded RNA acting in cis to promote Pr65Gag translation. Two independently-acting pol fragments were identified by screening 17 pol deletion mutations. To determine the mechanism by which pol promoted Pr65Gag synthesis, gag RNA in total and cytoplasmic fractions was quantitated by northern blot and by RT-PCR. The pol sequences caused, maximally, three-fold increase in total or cytoplasmic gag mRNA. Instead, pol sequences increased gag mRNA association with polyribosomes ~100-fold, a magnitude sufficient to explain the increase in Pr65Gag translation efficiency. The MPMV CTE, an NXF1-binding element, substituted for pol in promoting Pr65Gag synthesis. A pol RNA stem-loop resembling the CTE promoted Pr65Gag synthesis. Over-expression of NXF1 and NXT, host factors that bind to the MPMV CTE, synergized with pol to promote gammaretroviral gag RNA loading onto polysomes and to increase Pr65Gag synthesis. Conversely, Gag polyprotein synthesis was decreased by NXF1 knockdown. Finally, overexpression of SRp20, a shuttling protein that binds to NXF1 and promotes NXF1 binding to RNA, also increased gag RNA loading onto polysomes and increased Pr65Gag synthesis.
These experiments demonstrate that gammaretroviral pol sequences act in cis to recruit NXF1 and SRp20 to promote polysome loading of gag RNA and, thereby license the synthesis of Pr65Gag from unspliced mRNA.
所有逆转录病毒都通过可变剪接的mRNA合成必需蛋白质。然而，逆转录病毒属利用不同机制来协调可变剪接mRNA的蛋白质合成。这些逆转录病毒转录后效应器中研究得最透彻的是慢病毒的反式作用Rev蛋白和β逆转录病毒梅森- Pfizer猴病毒（MPMV）的顺式作用组成型转运元件（CTE）。γ逆转录病毒属成员如何从未剪接的RNA翻译蛋白质尚未阐明。
本文研究了两种γ逆转录病毒XMRV和MLV从全长未剪接mRNA合成Gag多蛋白（Pr65Gag）的机制。发现仅含gag的表达质粒产生的Pr65Gag产量比等基因的gag-pol表达质粒低至少30倍。破坏gag-pol表达质粒中pol开放阅读框的移码突变并未降低Pr65Gag的产生，并且引入gag中的398个沉默核苷酸变化使Pr65Gag合成不依赖pol。这些结果与pol编码的RNA顺式作用促进Pr65Gag翻译一致。通过筛选17个pol缺失突变鉴定出两个独立作用的pol片段。为了确定pol促进Pr65Gag合成的机制，通过Northern印迹和RT-PCR对总RNA和细胞质RNA中的gag RNA进行定量。pol序列使总gag mRNA或细胞质gag mRNA最多增加三倍。相反，pol序列使gag mRNA与多核糖体的结合增加约100倍，这一增加幅度足以解释Pr65Gag翻译效率的提高。MPMV CTE（一种NXF1结合元件）在促进Pr65Gag合成方面可替代pol。一个类似于CTE的pol RNA茎环促进Pr65Gag合成。与MPMV CTE结合的宿主因子NXF1和NXT的过表达与pol协同作用，促进γ逆转录病毒gag RNA加载到多核糖体上并增加Pr65Gag合成。相反，NXF1敲低会降低Gag多蛋白的合成。最后，与NXF1结合并促进NXF1与RNA结合的穿梭蛋白SRp20的过表达也增加了gag RNA加载到多核糖体上并增加了Pr65Gag合成。
这些实验表明，γ逆转录病毒pol序列顺式作用招募NXF1和SRp20，以促进gag RNA加载到多核糖体上，从而许可从未剪接的mRNA合成Pr65Gag。
