Toward a new world monkey model of HIV-1 infection

描述(由申请人提供)：逆转录病毒在其自然宿主物种以外的物种中的复制可以在病毒生命周期的多个步骤中被阻止。近年来，在描述这些限制背后的机制方面取得了重大进展。在这项建议中，我们试图利用和扩展这些观察结果来产生一种在猫头鹰猴子PBMC(OmPBMC)中有效复制的HIV-1，这是开发新的HIV-1感染动物模型的第一步。目前尚不存在HIV-1感染的动物模型。这样的模型将在HIV-1治疗的评估、疫苗研究和HIV-1发病机制的研究中有用。在初步数据中，我们发现，两种新世界猴子，猫头鹰猴子(Aotus Trivirgatus)和普通绒猴(CallirixJacchus)的入境限制可以很容易地克服，但松鼠猴(Saimiri Scureus)的入境限制不是很容易克服。我们还观察到，绒猴没有进入后障碍，正如之前已经证明的那样，猫头鹰猴子的进入后障碍可以通过病毒Gag基因点突变来克服。我们发现稳定表达OWL猴子CD4和CXCR4的OWL猴子细胞系OMK允许强大的病毒复制，表明这些细胞中不存在额外的限制。然而，在OWL猴PBMC中组装的HIV-1病毒粒子随后感染受体表达的OMK细胞的能力大大减弱，并且这些PBMC比OMK细胞表达更高水平的APOBEC3G。我们发现NL4-3vif部分下调猫头猴APOBEC3G，但不能下调绒猴APOBEC3G。这些数据表明，APOBEC3蛋白是在猫头鹰猴PBMC中复制HIV-1的关键但可克服的障碍。因此，本研究的重点是了解和克服APOBECs在OWL-猕猴细胞中的部分限制，并进一步使HIV-1在OWL-猕猴PBMC中高效复制。适应的病毒将在其env和nef基因上进行进一步修改，以增强面对猫头鹰猴子免疫反应时的病毒复制。修改后的病毒将进一步传代并用于连续挑战新英格兰灵长类研究中心饲养的猫头鹰猴子，这项工作将在那里进行。

DESCRIPTION (provided by applicant): Replication of retroviruses in species other than their natural host species can be blocked at multiple steps in the viral lifecycle. In recent years, significant progress has been made in describing mechanisms underlying these restrictions. In this proposal we seek to use and extend these observations to generate an HIV-1 that efficiently replicates in owl monkey PBMC (omPBMC), a first step toward developing a new animal model of HIV-1 infection. No animal model of HIV-1 infection currently exists. Such a model would be useful in the evaluation of HIV-1 therapuetics, in vaccine studies, and in studies of HIV-1 pathogenesis. In preliminary data we show that the entry restrictions of two new world monkeys, the owl monkey (Aotus trivirgatus) and the common marmoset (Callithrixjacchus), but not those of the squirrel monkey (Saimiri sciureus), can be readily overcome. We also observe that marmosets exhibit no post-entry barrier, and that, as has been previously demonstrated, the post-entry barrier in owl monkeys can be overcome through a point mutation in the viral gag gene. We show that the owl monkey cell line, OMK, stably expressing owl monkey CD4 and CXCR4 permits robust viral replication, indicating that no additional restrictions are present in these cells. However, HIV-1 virions assembled in owl monkey PBMC are greatly attenuated in their ability to subsequently infect receptor-expressing OMK cells, and these PBMC express substantially higher levels of APOBEC3G than OMK cells. We show that NL4-3 vif partially down-regulates owl monkey APOBEC3G but is unable to down-regulate marmoset APOBEC3G. These data suggest that APOBEC3 proteins are a critical but surmountable impediment to replication of HIV-1 in owl monkey PBMC. Accordingly, the focus of this proposal is to understand and overcome the partial APOBECS-mediated restriction in owl-monkey cells, and to further adapt HIV-1 for efficient replication in owl-monkey PBMC. Adapted viruses will be further modified in their env and nef genes to enhance viral replication in the face of the owl monkey immune response. Modified virus will be further passaged and used to serially challenge owl monkeys housed at the New England Primate Research Center, where this work will be performed.