AN INTERNAL RIBOSOMAL ENTRY MECHANISM PROMOTES TRANSLATION OF MURINE LEUKEMIA-VIRUS GAG POLYPROTEIN PRECURSORS
AN INTERNAL RIBOSOMAL ENTRY MECHANISM PROMOTES TRANSLATION OF MURINE LEUKEMIA-VIRUS GAG POLYPROTEIN PRECURSORS
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DOI：
10.1128/jvi.69.4.2214-2222.1995
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发表时间：
1995-04-01
影响因子：
5.4
通讯作者：
DARLIX, JL
中科院分区：
文献类型：
--
作者：
BERLIOZ, C;DARLIX, JL
基因组逆转录病毒RNA是用于翻译gag和pol基因的信使，gag和pol基因分别编码病毒体核心的主要结构蛋白和酶的前体。长的5'非翻译区（前导序列）由独立的结构良好的结构域形成，所述结构域参与病毒生命周期的关键步骤，例如前病毒DNA合成的起始、基因组RNA二聚化和包装以及gag翻译的起始。这些功能特征以及cap和gag起始密码子之间存在稳定的二级结构表明，gag的翻译起始可能通过不同于典型核糖体扫描过程的机制进行。有趣的是，小鼠白血病病毒还编码糖基化的gag前体，称为glyco-gag，其起始于CUG密码子上游，并且与AUG（gag）在相同的开放阅读框中。我们研究了Friend鼠白血病病毒（F-MLV）gag和glyco-gag前体在兔网织红细胞裂解物系统和鼠细胞中的翻译起始。通过gag和glyco-gag起始密码子的定点突变，我们发现gag和glyco-gag合成的起始不利用经典的核糖体扫描。当脊髓灰质炎病毒蛋白酶2A在鼠细胞中共表达时，MLV-lacZ RNA的表达未被修饰，表明MLV gag前体的翻译起始是帽非依赖性机制。此外，将F-MLV前导序列插入双顺反子neo-MLV-lacZ mRNA中的两个基因之间，并在体外和体内检测其促进表达的能力，获得的结果表明，内部核糖体进入机制促进F-MLV gag前体的翻译，这一发现使我们构建了一个nem双顺反子逆转录病毒载体，其中F-MLV前导序列可以促进重组基因组RNA的包装和3'基因的表达。
The genomic retroviral RNA is the messenger for the translation of the gag and pol genes encoding the precursors to the major structural proteins and enzymes, respectively, of the virion core. The long 5' untranslated region, the leader, is formed of independent well-structured domains involved in key steps of the viral life cycle such as the initiation of proviral DNA synthesis, genomic RNA dimerization and packaging, and the initiation of gag translation. These functional features and the presence of stable secondary structures between the cap and the gag initiation codon suggested that translation initiation of gag might proceed through a mechanism different from the canonical ribosome scanning process, Interestingly enough, murine leukemia viruses code also for a glycosylated gag precursor, named glyco-gag, initiated at a CUG codon upstream and in the same open reading frame as the AUG(gag). We have investigated the translation initiation of gag and glyco-gag precursors of Friend murine leukemia virus (F-MLV) in the rabbit reticulocyte lysate system and in murine cells, Through site-directed mutagenesis of gag and glyco-gag initiation codons, we show that initiation of gag and glyco-gag synthesis does not utilize the classical ribosome scanning, When poliovirus protease 2A is coexpressed in murine cells, expression of MLV-lacZ RNA is not modified, indicating that translation initiation of MLV gag precursors is a cap-independent mechanism, In addition, the F-MLV leader was inserted between two genes in a dicistronic neo-MLV-lacZ mRNA, and its ability to promote expression was examined in vitro and in vivo, Results obtained demonstrate that an internal ribosome entry mechanism promotes translation of F-MLV gag precursors, This finding led us to construct a nem dicistronic retroviral vector in which the F-MLV leader can promote both packaging of recombinant genomic RNA and expression of the 3' gene.