In vitro synthesis of biologically active beet necrotic yellow vein virus RNA

Abstract

Beet necrotic yellow vein virus (BNYVV) has a quadripartite plus-strand RNA genome in which the two smallest genome components, RNA 3 and 4, are not necessary for virus multiplication in leaves. Infectious transcripts of BNYVV RNA 3 and 4 have already been described ( V. Ziegler-Graff, S. Bouzoubaa, I. Jupin, H. Guilley, G. Jonard, and K. Richards (1988) J. Gen. Virol. 69, 2347–2357 ). In this paper we describe synthesis of a full-length RNA-1 transcript by bacteriophage T7 RNA polymerase-directed run-off transcription of cloned viral cDNA. A recombinant plasmid containing a full-length cDNA insert of RNA 2 could not be maintained in Escherichia coli. Therefore full-length transcript of RNA 2 was produced by transcription of cDNA ligation products without amplification in bacteria. When inoculated together to leaves of Chenopodium quinoa or Tetragonia expansa the RNA 1 and 2 transcripts were infectious; they also supported multiplication of the BNYVV RNA 3 and 4 transcripts, providing a totally synthetic inoculum of the virus. In one recombinant clone of RNA 2 a point mutation causing an arginine to serine substitution at position 119 of the viral coat protein was discovered. The mutation was detected because the resulting coat protein had altered electrophoretic mobility. RNA 2 transcripts containing this mutation were infectious but viral RNA was not encapsidated. The mutation also interfered with long distance movement of the virus in spinach, presumably as a consequence of the packaging deficiency.