Inderbitzin Anne, Kok Yik Lim, Jörimann Lisa, Kelley Audrey, Neumann Kathrin, Heinzer Daniel, Cathomen Toni, Metzner Karin J
Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.
Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
PeerJ. 2020 Nov 24;8:e10321. doi: 10.7717/peerj.10321. eCollection 2020.
The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene .
We investigated HIV-1 promoter activity after integration into specific sites in in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of , and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean/mCherry) and inactive (Cerulean/mCherry) HIV-1 promoters were characterised.
Upon targeted integration of the 5.3 kb vector LTatCL[M] into , the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M].
Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.
潜伏的HIV-1病毒库的持续存在是治愈HIV-1感染的主要障碍。HIV-1整合到细胞基因组中，并且在克隆扩增的潜伏HIV-1感染细胞中经常检测到一些靶向基因组位点，例如基因 。
我们研究了HIV-1载体整合到Jurkat T细胞中特定位点后的启动子活性。基于HIV-1的载体LTatCL[M]包含两种荧光团：（1）用于报告HIV-1启动子活性的天蓝蛋白；（2）由组成型启动子驱动并侧翼带有遗传绝缘子的mCherry。该载体通过CRISPR/Cas9技术以与 的相同和同向转录方向插入Jurkat T细胞 的内含子2和5中，并插入基因组安全港AAVS1中。对代表活跃（天蓝蛋白/mCherry）和不活跃（天蓝蛋白/mCherry）HIV-1启动子的单细胞克隆进行了表征。
将5.3 kb载体LTatCL[M]靶向整合到 后，HIV-1启动子逐渐沉默，这在162天的时间里通过天蓝蛋白表达的降低得以体现。沉默的HIV-1启动子可被肿瘤坏死因子-α和罗米地辛重新激活。这一观察结果与靶向内含子和转录方向无关。LTatCL[M]的单等位基因整合不会损害 mRNA和蛋白质表达。
基于HIV-1的载体LTatCL[M]的成功靶向整合允许对HIV-1启动子活性进行纵向分析。
