INVITRO REPLICATION OF MOUSE HEPATITIS-VIRUS STRAIN-A59
INVITRO REPLICATION OF MOUSE HEPATITIS-VIRUS STRAIN-A59
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DOI：
10.1128/jvi.61.6.1814-1820.1987
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发表时间：
1987-06-01
影响因子：
5.4
通讯作者：
MCGOWAN, JJ
中科院分区：
文献类型：
--
作者：
COMPTON, SR;ROGERS, DB;MCGOWAN, JJ
用溶血素制备细胞提取物，建立了小鼠肝炎病毒（MHV）A59株的体外复制系统。在MHV感染细胞的提取物中，放射性标记的UMP以线性速率掺入RNA中长达1小时，RNA与MHV特异性cDNA探针杂交，并在变性甲醛琼脂糖凝胶中迁移到与MHV基因组RNA相同的位置。[32 P]UMP掺入体外基因组大小的RNA与体内标记的MHV感染细胞中观察到的[3 H]尿苷掺入增加相关。当提取物与嘌呤霉素孵育时，[32 P]UMP掺入基因组大小的RNA被抑制。与加入免疫前血清相比，加入MHV-A59核衣壳蛋白N的抗血清可抑制基因组大小的RNA合成90%。相反，E1或E2糖蛋白的抗血清没有显着抑制RNA复制。体外合成的RNA在氯化铯梯度中条带化，作为核糖核蛋白复合物，具有从病毒体分离的MHV核衣壳的特征密度。这些实验表明，正在进行的蛋白质合成是MHV基因组RNA复制所必需的，并表明N蛋白在MHV复制中起着重要作用。
An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.