囊泡相关膜蛋白A调控牛病毒性腹泻病毒复制

Regulation of bovine viral diarrhea virus replication by vesicle-associated membrane protein A

摘要:

【背景】牛病毒性腹泻病毒(bovine viral diarrhea virus，BVDV)是致犊牛腹泻的重要病原之一，而目前BVDV与宿主因子互作机理研究较少，成为限制BVDV防控的重要原因。【目的】探明囊泡相关膜蛋白A (vesicle-associated membrane protein A，VAPA)对BVDV复制的影响。【方法】根据GenBank中VAPA基因，使用Benchling和CHOPCHOP等平台设计靶向VAPA的向导RNA (small guide RNA，sgRNA)，融合后克隆至慢病毒lentiCRISPR v2载体中，包装慢病毒后感染牛肾细胞(madin-darby bovine kidney，MDBK)，使用嘌呤霉素连续筛选5代，使用western blot检测VAPA蛋白敲除(knockout，KO)情况；BVDV感染VAPA KO细胞不同时间后，收集细胞提取总RNA，并将等质量的RNA反转录成cDNA，使用实时荧光定量PCR (real-time quantitative PCR，rt-qPCR)和免疫荧光分析(immunofluorescence assay，IFA)分别检测BVDV 5'-非翻译区(untranslated region，UTR) mRNA水平和双链RNA (double strand RNA，dsRNA)积累水平，于BVDV感染后不同时间观察致细胞病变(cytopathic effect，CPE)情况，并收集子代病毒液，使用Reed-Muench法计算子代病毒滴度变化。接种等量VAPA KO和乱码序列对照scramble细胞后不同时间进行活细胞计数，检测VAPA KO是否影响细胞活性。【结果】慢病毒感染后使用嘌呤霉素筛选，western blot检测VAPA蛋白明显降低，成功获得VAPA KO细胞；与对照scramble细胞相比，BVDV感染VAPA KO细胞后5'-UTR mRNA水平和dsRNA积累量均显著性降低，BVDV感染造成的CPE明显推迟并减轻，子代病毒滴度在12 h和36 h显著下降，在48 h极显著下降；与scramble细胞相比，VAPA KO细胞活性未受影响。【结论】VAPA KO后显著性抑制BVDV复制，为BVDV防控新技术的建立提供重要靶标。

Abstract:

[Background] Bovine viral diarrhea virus (BVDV) is among the important pathogens of calf diarrhea. However, there are few studies on the interaction mechanism between BVDV and host factors, which impedes the prevention and control of BVDV. [Objective] This study aims to explore the effect of vesicle-associated membrane protein A (VAPA) on BVDV replication. [Methods] According to the information of VAPA gene in GenBank, we designed the small guide RNA (sgRNA) targeting VAPA using Benchling and CHOPCHOP, cloned it to lentiCRISPR v2 vector, and then packed the lentivirus. Afterward, Madin-Darby bovine kidney (MDBK) cells were transfected with the lentivirus, followed by puromycin screening for 5 generations and detection of VAPA knockout (KO) by Western Blot. Total RNA was extracted from VAPA KO cells at different time after BVDV infection and then reverse-transcribed into cDNA. Real-time quantitative PCR (RT-qPCR) and immunofluorescence assay (IFA) were used to detect the 5'-untranslated region (UTR) mRNA level and the accumulation of double strand RNA (dsRNA) of BVDV, respectively. Cytopathic effect (CPE) was observed at different time after BVDV infection. Progeny virus titer was calculated with the Reed-Muench method. The same number of VAPA KO and scramble control cells were inoculated and then the living cells were counted at different time after virus infection to detect whether VAPA KO affected the cell viability. [Results] After lentivirus infection and puromycin screening, the results of Western Blot showed that VAPA protein was significantly decreased and thus VAPA KO cells were successfully established. The 5'-UTR mRNA level and dsRNA accumulation in VAPA KO cells infected with BVDV were significantly reduced compared with those of the scramble control cells. The CPE caused by BVDV infection was significantly delayed and attenuated and the titer of progeny virus decreased significantly at 12 h and 36 h and highly significantly at 48 h. The viability of VAPA KO cells was not affected compared with that of the scramble control cells. [Conclusion] VAPA KO can significantly inhibit BVDV replication. Therefore, this study provides an important target for the prevention and control of BVDV.