Jacquenet Sandrine, Decimo Didier, Muriaux Delphine, Darlix Jean-Luc
Laboratoire de Médecine et Thérapeutique Moléculaire, INSERM CIC9501, 15 rue du Bois de la Champelle, 54500 Vandoeuvre-lès-Nancy, France.
Retrovirology. 2005 May 22;2:33. doi: 10.1186/1742-4690-2-33.
In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.
在HIV-1感染的细胞中，整合的前病毒转录产生全长9 kb的单一病毒RNA，其中大部分被剪接以产生单剪接的4 kb RNA和多剪接的2 kb RNA。这些剪接后的RNA是Env糖蛋白和病毒调节因子的信使。体外实验表明，细胞中的SR蛋白和hnRNP蛋白通过结合病毒RNA上的顺式调节元件来控制选择性剪接。为了更好地了解SR蛋白在体内对HIV-1基因组RNA剪接和病毒体产生的作用，我们使用了一种能高水平表达完整HIV-1以及ASF/SF2、SC35和9G8这三种SR蛋白之一的人类细胞系。结果表明，过表达SR蛋白会导致基因组RNA大量减少，并且每种SR蛋白都以特定方式改变了病毒9 kb RNA的剪接模式。事实上，ASF/SF2增加了Vpr RNA的水平，而SC35和9G8则使Tat RNA大量增加。正如预期的那样，过表达SR蛋白导致总Gag蛋白大量减少。然而，我们通过免疫共聚焦显微镜观察到，Gag蛋白在质膜和细胞内区室中积累，而大多数细胞中产生的Env蛋白则显著减少。由于SR蛋白对基因组RNA水平和HIV-1结构蛋白产生负面影响，产生的病毒体数量大大减少，但其仍保留部分感染性。总之，SR蛋白可以下调HIV-1复制的后期步骤。
