Institute of Virology, University of Zurich, Zurich, Switzerland.
Department of Microbiology, University of Medicine and Pharmacy of Tîrgu Mureș, Târgu Mureș, Romania.
Sci Rep. 2019 Feb 4;9(1):1260. doi: 10.1038/s41598-018-37989-3.
In order to evaluate the influence of CDK5 inhibitory peptide (CIP) on Human alphaherpesvirus 1 (HSV-1) replication, we constructed two recombinant adeno-associated-virus 2 (rAAV2) vectors encoding CIP fused with cyan-fluorescent-protein (CFP), with or without nuclear localization signal. A third vector encoding non-fused CIP and CFP was also constructed. HeLa and HEK 293T cells were infected with the rAAV-CIP vectors at multiplicity of infection (MOI) of 5000, in the absence or presence of a recombinant HSV-1 that encodes a yellow-fluorescent-protein (rHSV48Y; MOI = 1). Cells co-infected with rHSV48Y and rAAV vectors that did not express the CIP gene (rAAV-CFP-Neo) served as controls. At 24 h after infection, the effect of CIP on rHSV48Y replication was assessed by PCR, qRT-PCR, Western-blot, flow-cytometry, epifluorescence and confocal microscopy. We show that in cultures co-infected with rAAV-CFP-Neo, 27% of the CFP-positive cells present rHSV48Y replication compartments. By contrast, in cultures co-infected with CIP-encoding rAAV2 vectors and rHSV48Y only 6-20% of the cells positive for CIP showed rHSV48Y replication compartments, depending on the CIP variant. Flow-cytometry showed that less than 40% of the rHSV48Y/rAAV-CIP, and more than 75% of rHSV48Y/rAAV-CFP-Neo co-infected cells were positive for both transgene products. The microscopy and flow-cytometry data support the hypothesis that CIP is inhibiting HSV-1 replication.
为了评估 CDK5 抑制肽 (CIP) 对人类单纯疱疹病毒 1 (HSV-1) 复制的影响，我们构建了两个重组腺相关病毒 2 (rAAV2) 载体，分别编码与青色荧光蛋白 (CFP) 融合的 CIP，或不具有核定位信号。还构建了第三个编码未融合 CIP 和 CFP 的载体。在不存在或存在编码黄色荧光蛋白 (rHSV48Y；MOI = 1) 的重组 HSV-1 的情况下，将 rAAV-CIP 载体以 5000 的感染复数 (MOI) 感染 HeLa 和 HEK 293T 细胞。作为对照，用未表达 CIP 基因的 rAAV-CFP-Neo 共感染 rHSV48Y 和 rAAV 载体的细胞。在感染后 24 小时，通过 PCR、qRT-PCR、Western-blot、流式细胞术、荧光显微镜和共聚焦显微镜评估 CIP 对 rHSV48Y 复制的影响。我们发现，在共感染 rAAV-CFP-Neo 的培养物中，27%的 CFP 阳性细胞存在 rHSV48Y 复制室。相比之下，在共感染 CIP 编码 rAAV2 载体和 rHSV48Y 的培养物中，只有 6-20%的 CIP 阳性细胞显示 rHSV48Y 复制室，具体取决于 CIP 变体。流式细胞术显示，rHSV48Y/rAAV-CIP 的不到 40%，以及 rHSV48Y/rAAV-CFP-Neo 的超过 75%共感染细胞对两种转基因产物均呈阳性。显微镜和流式细胞术数据支持 CIP 抑制 HSV-1 复制的假设。
