Relief of Preintegration Inhibition and Characterization of Additional Blocks for HIV Replication in Primary Mouse T Cells

Development of a small animal model to study HIV replication and pathogenesis has been hampered by the failure of the virus to replicate in non-primate cells. Most studies aimed at achieving replication in murine cells have been limited to fibroblast cell lines, but generating an appropriate model requires overcoming blocks to viral replication in primary T cells. We have studied HIV-1 replication in CD4+ T cells from human CD4/ CCR5/Cyclin T1 transgenic mice. Expression of hCD4 and hCCR5 in mouse CD4+ T cells enabled efficient entry of R5 strain HIV-1. In mouse T cells, HIV-1 underwent reverse transcription and nuclear import as efficiently as in human T cells. In contrast, chromosomal integration of HIV-1 proviral DNA was inefficient in activated mouse T cells. This process was greatly enhanced by providing a secondary T cell receptor (TCR) signal after HIV-1 infection, especially between 12 to 24 h post infection. This effect was specific for primary mouse T cells. The pathways involved in HIV replication appear to be PKCθ−, CARMA1-, and WASp-independent. Treatment with Cyclosporin A (CsA) further relieved the pre-integration block. However, transcription of HIV-1 RNA was still reduced in mouse CD4+ T cells despite expression of the hCyclin T1 transgene. Additional post-transcriptional defects were observed at the levels of Gag expression, Gag processing, Gag release and virus infectivity. Together, these post-integration defects resulted in a dramatically reduced yield of infectious virus (300–500 fold) after a single cycle of HIV-1 replication. This study implies the existence of host factors, in addition to those already identified, that are critical for HIV-1 replication in mouse cells. This study also highlights the differences between primary T cells and cell lines regarding pre-integration steps in the HIV-1 replication cycle.

用于研究人类免疫缺陷病毒（HIV）复制与致病机制的小型动物模型开发，长期受制于该病毒无法在非人灵长类细胞中完成复制这一瓶颈。此前多数旨在实现HIV在小鼠细胞中复制的研究，仅局限于成纤维细胞系；而构建合格的动物模型，需攻克病毒在原代T细胞内的复制阻滞问题。本研究针对转人CD4/CC趋化因子受体5（CCR5）/细胞周期蛋白T1（Cyclin T1）基因的小鼠的CD4+ T细胞，开展了HIV-1复制相关研究。小鼠CD4+ T细胞中表达的人源CD4（hCD4）与CCR5（hCCR5），可介导R5嗜性HIV-1毒株高效入侵宿主细胞。在小鼠T细胞内，HIV-1的逆转录与核输入过程，其效率与人类T细胞中相当。与之相对，活化的小鼠T细胞内，HIV-1前病毒DNA的染色体整合效率低下。在HIV-1感染后施加T细胞受体（TCR）共刺激信号，可显著提升整合效率，尤其在感染后12至24小时内施加该信号时效果最为突出。该促进效应仅在原代小鼠T细胞中观测得到。参与HIV复制的相关通路，似乎不依赖蛋白激酶Cθ（PKCθ）、CARMA1及Wiskott-Aldrich综合征蛋白（WASp）。使用环孢菌素A（Cyclosporin A, CsA）处理，可进一步缓解整合前阻滞效应。尽管小鼠细胞表达了转人的细胞周期蛋白T1（hCyclin T1），但小鼠CD4+ T细胞内HIV-1 RNA的转录水平仍显著降低。此外，在转录后层面还存在多项缺陷：包括Gag蛋白表达、Gag蛋白加工、病毒颗粒释放及病毒感染性等环节。上述整合后的多重缺陷共同作用，使得HIV-1单轮复制后，感染性病毒颗粒的产量骤降300至500倍。本研究提示，除已被报道的宿主因子外，还存在其他对HIV-1在小鼠细胞内复制至关重要的宿主调控因子。本研究同时强调，在HIV-1复制周期的整合前步骤中，原代T细胞与细胞系之间存在显著差异。