Lemay Julie, Maidou-Peindara Priscilla, Bader Thomas, Ennifar Eric, Rain Jean-Christophe, Benarous Richard, Liu Lang Xia
Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France.
Retrovirology. 2008 Jun 10;5:47. doi: 10.1186/1742-4690-5-47.
Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex - the reverse transcription complex (RTC) - consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein - the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT.
人类免疫缺陷病毒1型（HIV-1）遗传物质的逆转录是该病毒复制周期中的关键步骤。这一由逆转录酶（RT）催化的过程在生化水平上已得到充分表征。然而，在受感染的细胞中，逆转录发生在一种多蛋白复合物——逆转录复合物（RTC）中，该复合物由与病毒蛋白（包括RT）相关的病毒基因组RNA以及可能尚未明确的细胞蛋白组成。关于与RTC相互作用的细胞蛋白，尤其是与逆转录酶相互作用的细胞蛋白，人们了解甚少。我们在此报告，HIV-1逆转录受一种核质穿梭蛋白——RNA结合蛋白HuR水平的影响。在酵母双杂交筛选中观察到RT与HuR之间存在直接的蛋白质-蛋白质相互作用，并通过均相时间分辨荧光（HTRF）在体外得到证实。我们将与HuR相互作用的结构域定位到RT的核糖核酸酶H结构域，将与RT结合的结构域定位到HuR的C末端，该末端与HuR的第三个RRM RNA结合结构域部分重叠。用特异性小干扰RNA（siRNA）沉默HuR会极大地损害逆转录的早期和晚期步骤，显著抑制HIV-1感染。此外，通过诱变和免疫沉淀研究，我们无法检测到HuR与病毒RNA的结合。这些结果表明，HuR可能通过一种涉及与HIV-1 RT蛋白质-蛋白质相互作用的未知机制参与并调节HIV-1的逆转录反应。
