In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus.
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.
Cevik
B
B
Department of Plant Protection, Faculty of Agriculture, Suleyman Demirel University, Isparta, Turkey. bcevik@ziraat.sdu.edu.tr
Lee
R F
RF
Niblett
C L
CL
D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002957:Citrus / Q000737:chemistry / Q000821:virology*; D017790:Closterovirus / Q000201:enzymology* / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry; D047468:Immunoprecipitation; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase