下调LANA对卡波氏肉瘤相关疱疹病毒复制的影响

Impact of Down-regulating LANA on KSHV Replication

本文旨在探讨下调潜伏相关核抗原(Latency-associated nuclear antigen,LANA)对卡波氏肉瘤相关疱疹病毒(Kaposi’s sarcoma associated herpesvirus,KSHV)复制的影响。以四环素(Doxycycline,DOX)处理KSHV阳性的iSLK.219细胞,在不同时间点收集细胞,采用实时荧光定量PCR(real time quantitative PCR,qPCR)检测裂解复制期LANA mRNA表达水平。采用脂质体法转染si-LANA至iSLK.219细胞中下调LANA表达水平,以转染非特异siRNA为对照,24h后加入DOX激活病毒裂解复制,在裂解复制激活后不同时间点收集细胞和培养液上清。采用台盼蓝染色法检测细胞活力,倒置荧光显微镜观察红色荧光蛋白(Red fluorescence protein,RFP)表达评价KSHV裂解复制水平;qPCR检测细胞内和上清液中KSHV基因组的拷贝数以判断病毒复制水平。诱导KSHV裂解复制后24h和48h LANA表达分别为潜伏期的2.2和2.6倍。敲低LANA后iSLK.219细胞裂解复制水平在24h、48h和72h比未敲低组分别下降了8.18、3.25和3.98倍。敲低LANA细胞内病毒基因组拷贝数没有明显增加,而未敲低组在细胞内病毒基因组拷贝数在72h增加到2.1倍。相较于未敲低组,上清液中的病毒拷贝数在所有时间点均显著减少。对细胞活力的分析显示敲低LANA降低了细胞活力。敲低LANA抑制KSHV裂解复制,LANA可能通过维持细胞存活和增加细胞内病毒基因组拷贝数促进KSHV病毒的裂解复制。

To investigate the effect of knockdown of latent associated nuclear antigen(LANA)on Kaposi’s sarcoma-associated Herpes virus(KSHV)replication.Firstly,KSHV latent infected iSLK.219 cells were treated with doxycycline(DOX),and cells were collected at different timepoints.Real-time quantitative PCR(qPCR)was used to measure expression levels of LANA mRNA and thereby determine the dynamic changes in LANA expression over time during lytic reactivation.Next,si-LANA was transfected into iSLK.219 cells by the liposomal method,non-specific siRNA was transfected as a control,and 24 h after transfection DOX was added to activate lytic replication.Cells and supernatants were harvested at different timepoints after DOX treatment.Cell viability was determined by Trypan Blue staining,expression levels of red fluorescent protein(RFP)were measured under an inverted fluorescence microscope,and the lytic replication level of KSHV was evaluated according RFP brightness;the number of copies of the KSHV genome in cells and supernatants was determined by qPCR.Expression of LANA at 24 h and 48 h after induction of KSHV lytic replication was 2.2and 2.6 times higher than in the latent period,respectively.The lytic replication level of iSLK.219 cells after LANA knockdown decreased by 8.18,3.25,and 3.98 times compared with the si-control group at 24 h,48 h,and 72 h,respectively.There was no significant increase in intracellular virus genome copies after LANA knockdown,while virus genome copies in the si-control group were increased 2.1 time after 72h.The number of virus copies in the supernatants of the si-LANA group was decreased significantly at all timepoints compared with the si-control group.Analysis of cell viability showed that LANA knockdown reduced cell viability.LANA knockdown inhibits the lytic replication of KSHV,and LANA may promote the lytic replication of KSHV by enhancing cell viability and increasing the copy number of the intracellular viral genome.