Gosert Rainer, Kanjanahaluethai Amornrat, Egger Denise, Bienz Kurt, Baker Susan C
Institute for Medical Microbiology, University of Basel, Basel, Switzerland.
J Virol. 2002 Apr;76(8):3697-708. doi: 10.1128/jvi.76.8.3697-3708.2002.
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 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 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 MHV-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 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 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.
正链RNA病毒的复制复合体（RCs）与细胞膜密切相关。为了研究膜的改变并表征小鼠肝炎病毒（MHV）的RC，我们使用感染MHV的细胞进行了生化和超微结构研究。生化分级分离表明，所检测的MHV基因1多聚蛋白产物中的所有10种都与膜级分一起沉淀，这与RC与膜的结合一致。此外，MHV基因1产物p290、p210和p150以及p150裂解产物膜蛋白1（MP1，也称为p44）对Triton X-114提取具有抗性，表明它们是整合膜蛋白。超微结构分析揭示了MHV感染细胞细胞质中的双膜囊泡（DMV）。DMV既可以作为单独的实体存在，也可以作为小的囊泡簇存在。为了确定MHV蛋白和病毒RNA是否与DMV相关，我们进行了免疫细胞化学电子显微镜（IEM）。我们发现，使用针对源自MHV开放阅读框1a的蛋白的抗血清对DMV进行了标记。通过使用MHV特异性RNA探针的电子显微镜原位杂交（ISH），DMV对基因1和基因7序列都有高度标记。通过联合ISH和IEM，正链RNA和病毒蛋白定位于相同的DMV。最后，通过用5-溴尿苷5'-三磷酸标记来检测病毒RNA合成。发现新合成的病毒RNA与DMV相关。我们从这些数据得出结论，DMV携带MHV RNA复制复合体并且是MHV RNA合成的位点。
