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The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs.

机译:1型人类免疫缺陷病毒的Rev蛋白促进多体体结合和gag / pol和vpu / env mRNA的翻译。

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摘要

Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.
机译:从gag-Rev响应元件(RRE)表达质粒产生的gag mRNA的Rev依赖性表达的生化检查显示,细胞质gag mRNA的水平与产生的Gag蛋白之间存在很大差异。在没有Rev的情况下产生的大量mRNA定位在细胞质中,而产生的Gag蛋白水平却非常低。在Rev的存在下,mRNA的水平增加了4到16倍,而Gag蛋白的产量增加了800倍。这些发现表明,Rev除了促进细胞核到细胞质的转运外,还增加了细胞质病毒mRNA的利用。 Poly(A)的选择和细胞质gag mRNA的体外翻译证实,在不存在Rev的情况下产生的mRNA具有功能。若要分析缺少Rev的翻译缺陷,我们检查了细胞质gag mRNA与核糖体的关联。在无Rev的情况下产生的gag mRNA从多核糖体中排除,而在有Rev的情况下产生的gag mRNA与多核糖体和产生的Gag蛋白相关。这些观察结果表明,Rev的存在是将gag mRNA有效负载到多核糖体上所必需的。该作用需要在mRNA上存在RRE。从减正转前病毒克隆产生的mRNA的分析证实,Rev的存在可促进gag / pol和vpu / env mRNA的多体装载。还通过原位杂交检查gag mRNA的定位。该分析表明,在存在Rev的情况下,大多数gag mRNA在细胞质中被发现,而在没有Rev的情况下,大多数gag mRNA在细胞核中和在细胞核周围的区域中被发现。这些结果表明,在不存在和存在Rev的情况下,gag mRNA的大部分保留在不同的细胞质区室中。这些发现表明,沿着稳定,正确定位,含RRE的mRNA的高效翻译。

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