Inhibition of virion-associated IPNV RNA polymerase, VP1, by radiolabeled nucleotide analogs.
Infectious pancreatic necrosis virus (IPNV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The structural protein VP1 has been postulated as the RNA-dependent RNA polymerase (RdRp), but its transcriptional activity has not been unequivocally identified from viral particles. Here, we assayed partially purified IPNV in an in vitro RNA synthesis system. To test the RdRp, dialdehyde-nucleotide analogs were used to covalently inhibit the polymerase-associated activity. Our results showed that dialdehyde-nucleotide analogs completely abrogated IPNV in vitro RNA synthesis. The protein involved in this process was identified as viral VP1, since: (a) after incubation of IPNV with [alpha-(32)P]2',3'-dialdehyde-UTP, labeled VP1 protein was identified and (b) VP1 was unable to bind [alpha-(32)P]GTP when particles were preincubated with 2',3'-dialdehyde-ATP. Thus, within viral particles, inhibition of the transcriptional activity is a result of the binding of 2',3'-dialdehyde-nucleotide analogs to the RdRp, VP1.
Villanueva
Rodrigo A
RA
Laboratorio de Virología, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, P.O. Box 40, Santiago 33, Chile. ravillan@UTMB.edu
Guacucano
Maritza
M
Pizarro
Jacqueline
J
Sandino
Ana María
AM
D036022:Capsid Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018061:Infectious pancreatic necrosis virus / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis; D014771:Virion / Q000201:enzymology*
D036022:Capsid Proteins; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases