Chan Chi N, Trinité Benjamin, Lee Caroline S, Mahajan Saurabh, Anand Akanksha, Wodarz Dominik, Sabbaj Steffanie, Bansal Anju, Goepfert Paul A, Levy David N
Department of Basic Science, New York University College of Dentistry, New York, NY, 10010, USA.
Department of Ecology and Evolutionary Biology, University of California, Irvine, School of Biological, Sciences, Irvine, CA, 92697, USA.
Retrovirology. 2016 Jan 5;13:1. doi: 10.1186/s12977-015-0234-9.
HIV-1 integration is prone to a high rate of failure, resulting in the accumulation of unintegrated viral genomes (uDNA) in vivo and in vitro. uDNA can be transcriptionally active, and circularized uDNA genomes are biochemically stable in non-proliferating cells. Resting, non-proliferating CD4 T cells are prime targets of HIV-1 infection and latently infected resting CD4 T cells are the major barrier to HIV cure. Our prior studies demonstrated that uDNA generates infectious virions when T cell activation follows rather than precedes infection.
Here, we characterize in primary resting CD4 T cells the dynamics of integrated and unintegrated virus expression, genome persistence and sensitivity to latency reversing agents. Unintegrated HIV-1 was abundant in directly infected resting CD4 T cells. Maximal gene expression from uDNA was delayed compared with integrated HIV-1 and was less toxic, resulting in uDNA enrichment over time relative to integrated proviruses. Inhibiting integration with raltegravir shunted the generation of durable latency from integrated to unintegrated genomes. Latent uDNA was activated to de novo virus production by latency reversing agents that also activated latent integrated proviruses, including PKC activators, histone deacetylase inhibitors and P-TEFb agonists. However, uDNA responses displayed a wider dynamic range, indicating differential regulation of expression relative to integrated proviruses. Similar to what has recently been demonstrated for latent integrated proviruses, one or two applications of latency reversing agents failed to activate all latent unintegrated genomes. Unlike integrated proviruses, uDNA gene expression did not down modulate expression of HLA Class I on resting CD4 T cells. uDNA did, however, efficiently prime infected cells for killing by HIV-1-specific cytotoxic T cells.
These studies demonstrate that contributions by unintegrated genomes to HIV-1 gene expression, virus production, latency and immune responses are inherent properties of the direct infection of resting CD4 T cells. Experimental models of HIV-1 latency employing directly infected resting CD4 T cells should calibrate the contribution of unintegrated HIV-1.
HIV-1整合容易出现高失败率，导致体内和体外未整合病毒基因组（uDNA）的积累。uDNA具有转录活性，并且环化的uDNA基因组在非增殖细胞中具有生化稳定性。静息、非增殖的CD4 T细胞是HIV-1感染的主要靶标，而潜伏感染的静息CD4 T细胞是治愈HIV的主要障碍。我们先前的研究表明，当T细胞激活发生在感染之后而非之前时，uDNA会产生有感染性的病毒粒子。
在此，我们在原代静息CD4 T细胞中表征了整合和未整合病毒表达的动力学、基因组持久性以及对潜伏逆转剂的敏感性。未整合的HIV-1在直接感染的静息CD4 T细胞中大量存在。与整合的HIV-1相比，uDNA的最大基因表达延迟，且毒性较小，导致随着时间的推移，相对于整合的前病毒，uDNA富集。用raltegravir抑制整合将持久潜伏的产生从整合基因组转移到未整合基因组。潜伏的uDNA被潜伏逆转剂激活产生新的病毒，这些逆转剂也能激活潜伏的整合前病毒，包括蛋白激酶C激活剂、组蛋白脱乙酰酶抑制剂和P-TEFb激动剂。然而，uDNA反应表现出更宽的动态范围，表明相对于整合前病毒，其表达受到不同的调控。与最近对潜伏整合前病毒的研究结果相似，一两次应用潜伏逆转剂未能激活所有潜伏的未整合基因组。与整合前病毒不同，uDNA基因表达不会下调静息CD4 T细胞上HLA I类分子的表达。然而，uDNA确实能有效地使感染细胞对HIV-1特异性细胞毒性T细胞的杀伤作用产生预敏感性。
这些研究表明，未整合基因组对HIV-1基因表达、病毒产生、潜伏和免疫反应的贡献是静息CD4 T细胞直接感染的固有特性。采用直接感染静息CD4 T细胞的HIV-1潜伏实验模型应校准未整合HIV-1的贡献。
