Borgland S L, Bowen G P, Wong N C, Libermann T A, Muruve D A
Department of Medicine, University of Calgary, Calgary, Alberta, Canada.
J Virol. 2000 May;74(9):3941-7. doi: 10.1128/jvi.74.9.3941-3947.2000.
The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaBalpha resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB.
腺病毒载体用于基因治疗一直受到明确的细胞免疫和体液免疫反应的限制。我们之前已经表明，腺病毒载体在体内能快速诱导C-X-C趋化因子即干扰素诱导蛋白10（IP-10）的表达。各种第一代5型腺病毒载体，包括腺病毒CMVβ-半乳糖苷酶载体和补骨脂素紫外线灭活腺病毒，在感染小鼠肾上皮细胞（REC）后3小时就同样能诱导IP-10 mRNA的表达。使用小鼠IP-10 5'侧翼区缺失突变体进行的荧光素酶报告实验表明，腺病毒CMVβ-半乳糖苷酶载体对IP-10启动子的转录激活依赖于转录起始位点上游-161至-96碱基对区域。在电泳迁移率变动分析中，腺病毒CMVβ-半乳糖苷酶载体、腺病毒CMV-绿色荧光蛋白载体、FG140以及转录缺陷型腺病毒能诱导蛋白与位于IP-10启动子-113位含有核因子κB（NF-κB）共有序列的寡核苷酸结合。超迁移分析证实，在感染各种复制缺陷型腺病毒的REC细胞中，NF-κB p65而非p50或cRel的结合活性增加。腺病毒CMVβ-半乳糖苷酶载体与表达IκBα的腺病毒载体共感染REC细胞，导致在6小时和16小时时腺病毒CMVβ-半乳糖苷酶载体诱导的IP-10表达受到抑制，这进一步强化了腺病毒诱导的IP-10激活依赖于NF-κB的结论。IP-10的诱导似乎是直接的，因为用腺病毒载体感染未能诱导强效IP-10刺激物即干扰素γ和肿瘤坏死因子α的表达。总之，这些发现表明腺病毒载体通过衣壳依赖性激活NF-κB直接诱导IP-10的表达。
