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

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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.

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Acknowledgments

We are very grateful to Dr. Claudia Nome for helpful advice in immunoelectron microscopy, and Dr. Liliana Di Feo for guide in virus purification. We also thank Claudio Nikolaus who collaborated in UV chamber construction. This study was supported by the projects FONCyT 08-04416; CABBIO 00006 and Agencia Córdoba Ciencia SE. JDA is career member of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). ABSP hold a Doctoral Fellowship of CONICET and Ministerio de Ciencia y Tecnología de Córdoba, Argentina. AVL is professional member of CONICET.

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Authors and Affiliations

  1. Instituto de Fitopatología y Fisiología Vegetal (IFFIVE), Instituto Nacional de Tecnología Agropecuaria (INTA), Camino 60 cuadras Km 5 ½, X5020ICA, Córdoba, Argentina

    Fabiana A. Guzmán, Amalia B. Saavedra Pons, Graciela A. Truol, Andrés V. Luque & Luis R. Conci

  2. Intituto de Microbiología y Zoología Agrícola (IMIZA), Instituto Nacional de Tecnología Agropecuaria (INTA), Las Cabañas y De Los Reseros s/n. Ituzaingó, 1712, Castelar, Buenos Aires, Argentina

    Joel D. Arneodo

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  1. Fabiana A. Guzmán
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  3. Amalia B. Saavedra Pons
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  6. Luis R. Conci
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Correspondence to Luis R. Conci.

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Guzmán, F.A., Arneodo, J.D., Saavedra Pons, A.B. et al. Immunodetection and subcellular localization of Mal de Río Cuarto virus P9-1 protein in infected plant and insect host cells. Virus Genes 41, 111–117 (2010). https://doi.org/10.1007/s11262-010-0480-9

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