Guzmán Fabiana A, Arneodo Joel D, Pons Amalia B Saavedra, Truol Graciela A, Luque Andrés V, Conci Luis R
Instituto de Fitopatología y Fisiología Vegetal (IFFIVE), Instituto Nacional de Tecnología Agropecuaria (INTA), Camino 60 cuadras Km 5 (1/2), X5020ICA, Córdoba, Argentina.
Virus Genes. 2010 Aug;41(1):111-7. doi: 10.1007/s11262-010-0480-9. Epub 2010 Apr 24.
Mal de Río Cuarto virus (MRCV), a member of the genus Fijivirus, family Reoviridae, has a genome consisting of 10 dsRNA segments. The segment 9 (S9) possesses two non-overlapping open reading frames (ORF-1 and ORF-2) encoding two putative proteins, MRCV P9-1 and MRCV P9-2, both of unknown function. The MRCV S9 ORF-1 was RT-PCR amplified, expressed in pET-15b vector, and the recombinant protein produced was used to raise an antiserum in rabbit. Western blot with the specific MRCV P9-1 antiserum detected a protein of about 39 kDa molecular weight present in crude protein extracts from infected plants and insects. However, no reaction was observed when this antiserum was tested against purified virus. In contrast, only virus particles were detected by a MRCV-coat antiserum used as a validation control. These results suggest that MRCV S9 ORF-1 encodes a non-structural protein of MRCV. Immunoelectron microscopy assays confirmed these results, and localized the MRCV P9-1 protein exclusively in electron-dense granular viroplasms within the cytoplasm of infected plants and insects cells. As viroplasms are believed to be the replication sites of reoviruses, the intracellular location of MRCV P9-1 protein suggests that it might be involved in the assembly process of MRCV particles.
里奥夸尔托病毒（MRCV）是呼肠孤病毒科斐济病毒属的成员，其基因组由10个双链RNA片段组成。第9片段（S9）具有两个不重叠的开放阅读框（ORF-1和ORF-2），编码两种假定的蛋白质，即MRCV P9-1和MRCV P9-2，二者功能均未知。对MRCV S9 ORF-1进行RT-PCR扩增，在pET-15b载体中表达，并利用产生的重组蛋白在兔体内制备抗血清。用特异性的MRCV P9-1抗血清进行蛋白质免疫印迹检测，在来自受感染植物和昆虫的粗蛋白提取物中检测到一种分子量约为39 kDa的蛋白质。然而，用该抗血清检测纯化病毒时未观察到反应。相比之下，用作验证对照的MRCV衣壳抗血清仅检测到病毒颗粒。这些结果表明，MRCV S9 ORF-1编码MRCV的一种非结构蛋白。免疫电子显微镜检测证实了这些结果，并将MRCV P9-1蛋白专门定位在受感染植物和昆虫细胞胞质内的电子致密颗粒状病毒质中。由于病毒质被认为是呼肠孤病毒的复制位点，MRCV P9-1蛋白在细胞内的定位表明它可能参与了MRCV颗粒的组装过程。
