Coxsackievirus expression of the murine secretory protein interleukin-4 induces increased synthesis of immunoglobulin G1 in mice
Coxsackievirus expression of the murine secretory protein interleukin-4 induces increased synthesis of immunoglobulin G1 in mice
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DOI：
10.1128/jvi.74.17.7952-7962.2000
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发表时间：
2000-09-01
影响因子：
5.4
通讯作者：
Kolbeck, P
中科院分区：
文献类型：
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作者：
Chapman, NM;Kim, KS;Kolbeck, P
我们将编码鼠白细胞介素-4（mIL-4）的序列（包括分泌信号）克隆到柯萨奇病毒B3的人工减毒株CVB 3/0的基因组中，位于衣壳蛋白1D和病毒蛋白酶2Apro的连接处。两株嵌合CVB 3的构建，在一种情况下，相同的序列编码2Apro切割位点（CVB 3/0-mIL 4/47）的插入编码序列的两侧和？在另一种情况下，不相同的序列在核苷酸水平上变化而不改变氨基酸序列（CVB 3-PL 2-mIL-4/46），HeLa转染产生的子代病毒以与亲本CVB 3/0毒株相似的速率复制，尽管表达mIL-4的毒株的产量比亲本病毒的产量低约10倍。免疫印迹分析的病毒蛋白质分离的HeLa细胞接种的嵌合病毒的任一菌株表明，嵌合病毒合成的衣壳蛋白1D在约两倍的水平比亲本病毒。用酶联免疫吸附试验（ELISA）检测两株嵌合病毒接种的HeLa细胞中mIL-4蛋白的表达。用任一嵌合病毒接种的HeLa细胞的裂解物诱导需要mIL-4的鼠MC-9细胞系的增殖，证明了CVB 3表达的mIL-4的生物活性。对来自CVB 3/0-mIL 4/47在HeLa细胞中连续传代的病毒RNA的逆转录（RT）-PCR分析证明了在第四代时发生的mIL-4编码序列的缺失，而对CVB 3-PL 2-mIL-4/46 RNA的类似分析证明在HeLa细胞中通过10代在病毒群体中检测到mIL-4编码序列。通过ELISA测定的mIL-4蛋白水平与通过传代病毒基因组的RT-PCR分析测定的稳定性和损失数据一致。对CVB 3-PL 2-mIL 4/46在小鼠体内复制期间插入物稳定性的研究表明，在感染后14天，在胰腺、心脏和肝脏中存在病毒mIL-4插入物。对CVB 3-PL 2-mIL 4/46和亲本CVB 3/0株的小鼠抗体应答的比较表明，在接种CVB 3-PL 2-mIL 4/46的小鼠中，CVB 3结合血清免疫球蛋白GI水平增加。
We cloned the sequence encoding murine interleukin-4 (mIL-4), including the secretory signal, into the genome of CVB3/0, an artificially attenuated strain of coxsackievirus B3, at the junction of the capsid protein 1D and the viral protease 2Apro. Two strains of chimeric CVB3 were constructed using, in one case, identical sequences to encode 2Apro cleavage sites (CVB3/0-mIL4/47) on either side of the inserted coding sequence and? in the other case, nonidentical sequences that varied at the nucleotide level without changing the amino acid sequences (CVB3-PL2-mIL4/46), Transfection of HeLa tells yielded progeny viruses that replicated with rates similar to that of the parental CVB3/0 strain, although yields of mIL-4-expressing strains were approximately 10-fold lower than those of the parental virus. Western blot analysis of viral proteins isolated from HeLa cells inoculated with either strain of chimeric virus demonstrated that the chimeric viruses synthesized capsid protein 1D at approximately twofold-higher levels than the parental virus. mIL-4 protein was detected by enzyme-linked immunosorbent assay (ELISA) in HeLa cells inoculated with either strain of chimeric virus. Lysates of HeLa cells inoculated with either chimeric virus induced the proliferation of the mIL-4-requiring murine MC-9 cell line, demonstrating biological activity of the CVB3-expressed mIL-4, Reverse transcription (RT)-PCR analysis of viral RNA derived from sequential passaging of CVB3/0-mIL4/47 in HeLa cells demonstrated deletion of the mIL-4 coding sequence occurring by the fourth passage, while similar analysis of CVB3-PL2-mIL4/46 RNA demonstrated detection of the mIL-4 coding sequence in the virus population through 10 generations in HeLa cells. mIL-4 protein levels determined by ELISA were consistent with the stability and loss data determined by RT-PCR analysis of the passaged viral genomes. Studies of insert stability of CVB3-PL2-mIL4/46 during replication in mice showed the presence of the viral mIL-4 insert in pancreas, heart, and liver at 14 days postinfection, Comparison of the murine antibody responses to CVB3-PL2-mIL4/46 and the parental CVB3/0 strain demonstrated an increased level of CVB3-binding serum immunoglobulin GI in mice inoculated with CVB3-PL2-mIL4/46.