Analysis of the TRIM5α-mediated restriction of retrotransposons

LINE-1反转录转座子是人类基因组中唯一自主活跃的可移动遗传元件。大约17%的人类基因组起源于LINE，它仍然拥有大约100个潜在的活跃拷贝。尽管LINE-1元件“跳跃”到新的位置很少见，但新的LINE-1插入已被证明会导致严重的遗传疾病，如血友病、肌肉营养不良或癌症。因此，对转座元件的有效控制对于维持基因组的完整性是必不可少的。对于控制外源性病毒感染，细胞限制因素是关键。其中之一，TRIM5α，已被证明以物种特异性的方式阻止各种逆转录病毒，最有可能的是通过与逆转录病毒衣壳结构的直接相互作用。虽然恒河猴而不是人的TRIM5α有效地抑制了HIV-1的感染，但我们发现恒河猴和人的TRIM5α对LINE-1元件的逆转录转位的限制程度相似。这甚至更令人惊讶，因为细胞内复制的Line-1元件不会形成衣壳结构，而衣壳结构是TRIM5α介导的阻断逆转录病毒感染的经典靶点。因此，内源性LINE-1元件的抑制代表了重要的HIV限制因子TRIM5α的一种新功能。在该提案的AIM1中，我们将确定人类TRIM5a是如何阻止内源性LINE-1元件的，以及这些元件生命周期的哪些步骤被阻止。TRIM5α对内源性遗传元件的阻断也可能解释了TRIM5α在进化过程中强烈的正向选择，这已被证明早于灵长类慢病毒的出现，如艾滋病毒-1。在人类TRIM5α中可以发现几个单核苷酸多态，但它们只有在阻止HIV的能力方面略有不同。因此，在这项提议的第二个目标中，我们将询问不同的TRIM5α等位基因是否对人类LINE-1显示出不同的活性。与人类基因组相比，小鼠基因组包含数量更多的仍然活跃的内源性逆转录病毒和逆转录元件。有趣的是，TRIM5α的遗传位点在小鼠中也有很强的扩展，并包含七种不同的TRIM5α同源基因，TRIM12a-c和TRIM30a-d。由于到目前为止还没有发现驱动这种扩增的病毒，我们将在研究的第三个目标中询问小鼠体内丰富的内源性逆转录病毒和逆转录元件是否可能与小鼠TRIM5基因座的扩增有关，并确定TRIM5蛋白对不同内源性逆转录病毒和逆转录元件复制的影响。总之，这项应用将确定人类TRIM5α如何阻止内源性LINE-1逆转录元件复制的机制。此外，该项目还将阐明人类TRIM5α的不同等位基因和小鼠不同的TRIM5同源基因在多大程度上阻止逆转录元件，从而有助于小鼠和人类的基因组稳定性。

LINE-1 retrotransposons are the only autonomously active mobile genetic elements within the human genome. About 17 % of the human genome is of LINE origin and it still harbors about 100 potentially active copies. Although "jumping" of LINE-1 elements into new locations is rare, new LINE-1 insertions have been shown to cause severe genetic disorders, like hemophilia, muscular dystrophy, or cancer. The efficient control of transposable elements is therefore essential to maintain genome integrity. For the control of exogenous viral infections, cellular restriction factors are key. One of them, TRIM5α, has been shown to block various retroviruses in a species-specific manner, most likely by direct interaction with the retroviral capsid structure. While rhesus but not human TRIM5 alpha efficiently inhibits HIV-1 infection, we found that both rhesus and human TRIM5α restrict the retrotransposition of LINE-1 elements to a similar degree. This is even more surprising since intracellular replicating LINE-1 elements do not form a capsid structure, which is the classical target of the TRIM5 alpha-mediated block to retroviral infection. Thus, the inhibition of endogenous LINE-1 elements represents a novel function of the important HIV restriction factor TRIM5 alpha. Within Aim1 of this proposal, we will determine how human TRIM5a blocks endogenous LINE-1 elements and which steps of the life cycle of these elements are blocked. The block to endogenous genetic elements by TRIM5 alpha might also explain the strong positive selection of TRIM5α during evolution, which has been shown to predate the appearance of primate lentiviruses, like HIV-1. Several single nucleotide polymorphisms can be found in human TRIM5 alpha, but the only differ marginally in their ability to block HIV. Thus, within the second aim of this proposal we will ask whether the various TRIM5α alleles show a differential activity against human LINE-1. The murine genome contains a much larger number of still active endogenous retroviruses and retroelements compared to the human genome. Interestingly, the genetic locus of TRIM5 alpha also strongly expanded in mice and harbors seven different TRIM5 alpha orthologues, TRIM12a-c and TRIM30a-d. Since no virus driving this expansion has been identified so far, we will ask within the third aim of the study whether the abundance of endogenous retroviruses and retroelements in mice might be responsible for the expansion of the murine TRIM5 locus and determine the effect of the TRIM5 proteins on the replication of different endogenous retroviruses and retroelements. Together, this application will identify the mechanism how human TRIM5α blocks the replication of endogenous LINE-1 retroelements. Also, this project will clarify to what extent different alleles of human TRIM5 alpha and the different murine TRIM5 orthologues block retroelements and therefore contribute to genome stability in mice and men.