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In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus

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Summary

Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.

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Authors and Affiliations

  1. Department of Plant Protection, Faculty of Agriculture, Suleyman Demirel University, Isparta, Turkey

    B. Çevik

  2. USD-ARS National Clonal Germplasm Repository for Citrus and Dates, CA, USA

    R. F. Lee

  3. Department of Plant Pathology, University of Florida, FL, USA

    C. L. Niblett

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  1. B. Çevik
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  2. R. F. Lee
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  3. C. L. Niblett
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Correspondence to B. Çevik.

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Correspondence: Bayram Çevik, Department of Plant Protection, Faculty of Agriculture, Suleyman Demirel University, Isparta 32260, Turkey

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Çevik, B., Lee, R. & Niblett, C. In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus. Arch Virol 153, 315–321 (2008). https://doi.org/10.1007/s00705-007-1060-8

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