人类免疫缺陷病毒-1 Rev蛋白调控卡波济肉瘤相关疱疹病毒的裂解性周期复制与潜伏感染

Effect of HIV-1 Rev protein on the lytic and latent cycle of KSHV

目的:研究人类免疫缺陷病毒-1(HIV-1)Rev蛋白的表达水平对原发性渗出性淋巴瘤(PEL)细胞BCBL-1中卡波济肉瘤相关疱疹病毒(KSHV)裂解性周期复制与潜伏的影响。方法:自表达质粒pCI-neo-Rev中扩增出Rev基因,插入到pHAGE-CMV-MCS-IZsGreen中构建成慢病毒载体pLenti-Rev,利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞。荧光显微镜观察293T细胞中绿色荧光蛋白(IZsGreen)表达;收集培养上清经0.45μm滤器过滤后即获得病毒悬液。梯度稀释法测定病毒滴度并感染细胞,RT-PCR和Western blot检测Rev在293T细胞中的表达。用慢病毒感染BCBL-1细胞,同时将表达质粒pCI-neo-Rev转染该细胞,采用Western blot分别检测Rev以及KSHV Rta和vIL-6蛋白的表达;Real-timePCR进一步从mRNA水平上定量验证Rev对KSHV复制的影响。结果:限制性内切酶酶切反应和基因测序证实成功构建了携带Rev基因的慢病毒表达载体,病毒滴度为2×107TU/ml。重组慢病毒感染BCBL-1细胞72h后,能够检测到Rev蛋白的表达。Western blot结果显示,低水平表达的Rev能够上调KSHV裂解期蛋白Rta、vIL-6的表达,而高水平表达的Rev则能够抑制Rta和vIL-6的表达。Real-time PCR进一步验证了该结果,表明高水平表达的Rev能够显著抑制BCBL-1中Rta的mRNA转录,96h尤为明显。结论:成功构建了含Rev基因的慢病毒表达载体,获得的病毒能够有效感染BCBL-1细胞,并在其中大量表达Rev蛋白。Rev蛋白的表达水平对KSHV的复制与潜伏可能起到复杂的调控作用。

Objective：To construct the recombinant lentivirns containing HIV-1 Rev gene and detect the effect of Rev protein expression on the lyric cycle replication and latent infection of KSHV. Methods：The fragment of Rev gene from expression vector pCI-neo-Rev was cloned into the lentivirus vector pHAGE-CMV-MCS-IZsGreen,then the recombinant plasmid pLenti-Rev,packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293T cells. Culture media were harvested and filtered through a 0.45 μm filter to obtain the recombinant lentivirus. The viral titer was checked by observing the expression of IZsGreen protein. After infected with the recombinant lentivirus,the mRNA transcription and protein expression of Rev in 293T and BCBL-1 cells were detected by reverse transcription-PCR and Western blot,respectively. Then,Rev-Flag, Rta and vIL-6 protein from BCBL-I infected with the recombinant lentivirus and transfected with pCI-neo-Rev were detected by Western blot. Further more, mRNA transcription of Rta in BCBL-1 transfected with pCI-neo-Rev was detected quantitatively by real-time PCR. Results：The recombinant lentivirus vector carrying Rev was constructed successfully with the viral titer of 2×10^7 TU/ml. BCBL-1 cells were efficiendy infected by the recombinant lentivirns and Rev protein was readily expressed in these ceils. The Western blot and real-time PCR results showed that Rta and vIL-6 expression were significantly upregulated by low Rev expression and vice versa. Conclusion：The lentivirus packaging system is available for BCBL-1, and Rev protein could express effectively in BCBL-1 infected with plenti-Rev. Forthermore, the Rev protein may regulate the lytic cycle replication and latent infection of KSHV in a complicated pattern.