RNA replication of mouse hepatitis virus takes place at double-membrane vesicles
RNA replication of mouse hepatitis virus takes place at double-membrane vesicles
复制标题
DOI：
10.1128/jvi.76.8.3697-3708.2002
复制
发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
Baker, SC
中科院分区：
文献类型：
--
作者：
Gosert, R;Kanjanahaluethai, A;Baker, SC
正链RNA病毒的复制复合物（RCs）与细胞膜紧密相关。为了研究膜的改变并鉴定小鼠肝炎病毒（MHV）的复制复合物，我们利用感染MHV的细胞进行了生化和超微结构研究。生化分级分离显示，所检测的10种MHV基因I多聚蛋白产物都与膜组分一起沉淀，这与复制复合物的膜结合特性相符。此外，MHV基因I产物p290、p210和p150以及p150裂解产物膜蛋白1（MP1，也称为p44）对用Triton X - 114提取具有抗性，表明它们是整合膜蛋白。超微结构分析显示在MHV感染细胞的细胞质中有双层膜囊泡（DMVs）。这些双层膜囊泡要么作为单独的个体存在，要么作为小的囊泡簇存在。为了确定MHV蛋白和病毒RNA是否与双层膜囊泡相关，我们进行了免疫细胞化学电子显微镜检查（IEM）。我们发现使用针对源自MHV开放阅读框1a的蛋白的抗血清可标记双层膜囊泡。通过使用MHV特异性RNA探针进行电子显微镜原位杂交（ISH），双层膜囊泡对基因I和基因7序列都有高度标记。通过原位杂交和免疫细胞化学电子显微镜检查相结合的方法，正链RNA和病毒蛋白定位在相同的双层膜囊泡中。最后，通过用5 - 溴尿苷5'-三磷酸标记检测到病毒RNA的合成。新合成的病毒RNA被发现与双层膜囊泡相关。我们从这些数据得出结论：双层膜囊泡携带MHV RNA复制复合物，并且是MHV RNA合成的位点。
The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene I polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene I products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MRV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame la of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene I and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.