Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3.
Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5' end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.
Pizarro
J L
JL
Unit of Virology, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Santiago.
Sandino
A M
AM
Pizarro
J M
JM
Fernández
J
J
Spencer
E
E
D004247:DNA / Q000378:metabolism; D003968:Diarrhea, Infantile / Q000382:microbiology*; D011756:Diphosphates / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007223:Infant; D008274:Magnesium / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D012401:Rotavirus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014758:Viral Core Proteins / Q000378:metabolism*
D011756:Diphosphates; D010710:Phosphates; D014758:Viral Core Proteins; D012313:RNA; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D004247:DNA; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D010450:Endopeptidases; D008274:Magnesium; D004229:Dithiothreitol