Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells

Abstract

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.

Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein

“…3), confirming the hypothesis that the viroplasm is the site for the replication and assembly of fijiviruses. Our results extend the limited information from previous electron microscopic studies that showed that P9-1 of two other fijiviruses (RBSDV and MRCV) exclusively localized in the matrix of viroplasms in virus-infected cells (9,12,19). With the evidence indicating that P9-1 alone was necessary for the formation of viroplasm-like structures in nonhost cells (Fig.…”

Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1

“…Being highly homologous to RBSDV P9-1 (64.5% identity) [34], Mal de Rio Cuarto virus (MRCV) P9-1, which was detected in viroplams in both infected plants and planthoppers [35], was found to be sufficient for the formation of viral inclusion body (VIB)-like structures when expressed in Spodoptera frugiperda Sf9 cells [36]. There might be distinct mechanisms involved in viroplasm formation in insect and plant hosts.…”

Characterization of homologous and heterologous interactions between viroplasm proteins P6 and P9-1 of the fijivirus southern rice black-streaked dwarf virus

“…P6 is also thought to self-interact and to form punctuate VLS in the cytoplasm and to recruit P9-1 [15]. It has ATPase activity and is associated with the formation of viroplasms [2,10]. The Mal de Rio Cuarto virus (MRCV) P9-1 can also self-interact and bind ssRNA.…”