Hwang Y K, Brinton M A
Department of Biology, Georgia State University, Atlanta 30302, USA.
J Virol. 1998 May;72(5):4341-51. doi: 10.1128/JVI.72.5.4341-4351.1998.
The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.
动脉炎病毒猴出血热病毒（SHFV）负链RNA的3'非编码区（NCR）[3'（-）NCR RNA]长度为209个核苷酸（nt）。由于这个被命名为3'（-）209的3'区域是全长正链RNA起始的位点，并且是5'前导序列合成的模板，该序列存在于全长和亚基因组mRNA上，所以它可能包含RNA合成的顺式作用信号，并与细胞和病毒蛋白相互作用形成复制复合体。凝胶迁移率变动分析表明，MA104 S100细胞质提取物中的细胞蛋白与SHFV 3'（-）209 RNA形成了两种复合体，竞争凝胶迁移率变动分析的结果表明这些相互作用是特异性的。在紫外线诱导的交联分析中检测到了分子量分别为103、86、55和36 kDa的四种蛋白，其中三种蛋白（103、55和36 kDa）也通过蛋白质印迹分析检测到。未感染和SHFV感染的提取物获得了相同的凝胶迁移率变动和紫外线诱导的交联模式，表明检测到的这四种蛋白是细胞蛋白而非病毒蛋白。四种细胞蛋白的结合位点被定位到SHFV负链RNA 3'端117至184 nt之间的区域（68 nt序列）。预计这个68 nt序列会形成两个茎环，即SL4和SL5。在竞争凝胶迁移率变动分析中，另一种动脉炎病毒乳酸脱氢酶升高病毒C（LDV-C）的3'（-）NCR RNA与SHFV 3'（-）209 RNA竞争。紫外线诱导的交联分析表明，四种与SHFV 3'（-）209 RNA结合的分子量相同的MA104细胞蛋白也与LDV-C 3'（-）NCR RNA和马动脉炎病毒3'（-）NCR RNA结合。然而，这些病毒RNA中的每一种也与一种额外的MA104蛋白结合。MA104细胞蛋白的结合位点在LDV-C 3'（-）NCR和SHFV 3'（-）209 RNA中位于相似位置。这些数据表明，一组细胞蛋白的结合位点在所有动脉炎病毒RNA中是保守的，并且这些细胞蛋白可能被用作病毒复制复合体的组成部分。
