反义寡聚核苷酸抗柯萨奇A24病毒感染的研究

The study about antisense oligonucleotides resistance to coxsackievirus A24 infection

目的针对柯萨奇病毒A组24型(coxsackievirus A24,CVA24)基因组特殊功能区设计系列反义寡聚核苷酸序列,通过实验观察特异性反义核酸对病毒感染细胞的抑制作用。同时进行抗病毒作用的量效评价。方法通过抑制空斑形成实验、病毒致细胞病变作用及免疫印迹等实验,了解反义核酸抑制及阻断CVA24病毒感染细胞的能力,及经反义核酸抑制后病毒结构蛋白在感染细胞中的表达状况,并对抗病毒效果较好的反义核酸进行量效关系测定。结果从本实验所设计的8条反义核酸中,筛选出5条能较好抑制CVA24的反义核酸序列,分别为SCA547、SCA551、SCA554、SCA558、SCA568。当病毒感染量为1 MOI时,5μmol/L的上述反义核酸的病毒活性抑制率均在50%以上。经过量效关系分析显示,0.3μmol/L的SCA568已经对细胞产生较强的保护作用,对病毒空斑形成抑制程度达80%;在2.5μmol/L的SCA568保护下,病毒空斑抑制率达到90%以上,是几条反义核酸中抗病毒效果最好的一条。SCA568对病毒早期结构蛋白的表达有较强的抑制作用。而抗病毒效果稍差的SCA723,随浓度的增加也表现出一定程度对病毒蛋白表达的抑制作用。结论本实验所筛选的5条反义核酸SCA547、SCA551、SCA554、SCA558、SCA568能较好地抑制CVA24活性,为进一步研究反义核酸治疗和预防CVA24感染、防止CVA24引起的流行性出血性结膜炎的暴发流行打下基础。

Objective To investigate inhibition of coxsackievirus A24（CVA24） gene expression by antisense ohgonucleotides（OND） complementary to the internal ribosome entry site （IRES） and translation initiation codon, and to observe the dose-response of the sequence specific inhibition of CVA24 plaque formation by antisense oligonucleotides. Methods Antiviral activities of these oligonucleotides were evaluated by plaque reduction assay, cytopathic effect, Western blot analysis, and the dose response analysis. The cells were treated with random oligonucleotides as an unspecified control. Results Eight phosphorothioate antisense oligonucleotides for CVA24 were synthesized. At a screening concentration of 5 μmol/L, 5 of the ONDs（SCA547, SCA551, SCA554, SCA558, SCA568）inhibited viral activity. Plaque formation was inhibited by over 50%. Analysis by dose response, the most effective site was located at the core sequence of the IRES（SCA568） near the terminus of the 5＇ UTR of the CVA24 RNA. The inhibition of SCA568 for CVA24 plaque formation were 80% and 90% at 0.3 μmol/L and 2.5 μmol/L of SCA568 concentration respectively. The effect of SCA568, SCA732 treatment on immediate early protein levels. VP1 of CVA24 was significantly reduced with 0. 125 μmol/L SCA568, and were undetectable with 2.5 μmol/L SCA568. Conclusion Antisense oligonucleotides against IRES and translation initiation codon are capable of specifically inhibiting the synthesis of viral protein and CVA24 replication. The selective inhibition of antisense oligonucleotide may lead to development of an effective antiviral agent for future clinical evaluation. Our result demonstrated a great potential of 5 antisenses to develope an effective treatment for hemorrhagic conjunctivitis as well as other diseases caused by CVA24 infection.