Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication

RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.

RNA病毒（RNA virus）拥有5′和3′非翻译区（untranslated regions, UTRs），其中蕴含RNA合成所需的特异性信号。冠状病毒基因组在5′端带有帽子结构，其3′UTR由300至500个核苷酸（nucleotides, nt）以及一段poly(A)尾（poly(A) tail）组成。为加深对冠状病毒复制机制的理解，我们针对两类II群病毒——牛冠状病毒（bovine coronavirus, BCV）与小鼠肝炎冠状病毒（mouse hepatitis coronavirus, MHV），开展了宿主因子参与该过程的相关研究。我们利用凝胶迁移率变动分析（gel mobility shift assays），鉴定出了可与BCV 3′UTR（287 nt加poly(A)尾）特异性结合的宿主蛋白。针对MHV 3′UTR（301 nt加poly(A)尾）的竞争实验结果显示，两种病毒间的蛋白-核酸相互作用具有保守性。紫外交联实验（UV cross-linking experiments）中，我们检测到分子量分别为99、95和73 kDa的蛋白；同时在40~50 kDa及30 kDa范围内也发现了标记信号较弱的蛋白。73 kDa蛋白的结合反应依赖于poly(A)尾。通过对紫外交联产物进行免疫沉淀（immunoprecipitation），我们确认该73 kDa蛋白即为细胞质poly(A)结合蛋白（cytoplasmic poly(A)-binding protein, PABP）。我们借助缺陷基因组（defective genomes）BCV Drep、MHV MIDI-C及其多个突变体的复制实验，探究了poly(A)尾的重要性。将带有长度仅为5或10个A残基的截短poly(A)尾的缺陷基因组转染（transfection）至辅助病毒感染的细胞后，可检测到其复制。缺失poly(A)尾的BCV Drep RNA无法完成复制；而MHV MIDI-C RNA在缺失poly(A)尾后，需经过数次病毒传代才能检测到复制现象。所有突变体均表现出复制动力学延迟的特征。在复制实验中，可检测到这些突变体的RNA出现了poly(A)尾的延伸或从头添加，这与突变体RNA的出现呈正相关。带有截短poly(A)尾的RNA在体外与PABP的结合能力较弱，这提示其与该蛋白的相互作用减弱可能会影响RNA复制。本研究数据充分表明，poly(A)尾是冠状病毒复制过程中一类重要的顺式作用信号（cis-acting signal）。