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

Arch Virol. 2008;153(2):315-21. doi: 10.1007/s00705-007-1060-8. Epub 2008 Jan 14.


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.

MeSH terms

  • Blotting, Western
  • Cell Fractionation
  • Cell Membrane / chemistry
  • Citrus / chemistry
  • Citrus / virology*
  • Closterovirus / enzymology*
  • Closterovirus / genetics
  • Cytoplasm / chemistry
  • Immunoprecipitation
  • RNA-Dependent RNA Polymerase / biosynthesis*
  • RNA-Dependent RNA Polymerase / genetics
  • Time Factors
  • Viral Proteins / biosynthesis*
  • Viral Proteins / genetics


  • Viral Proteins
  • RNA-Dependent RNA Polymerase