Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro

• Published in: Journal of general virology 1991-04, Vol.72 (4), p.769-777
• Main Authors: Mundry, Karl W, Watkins, Peter A. C, Ashfield, Thomas, Plaskitt, Kitty A, Eisele-Walter, Sylvia, Wilson, T. Michael A
• Format: Article
• Language: English
• Subjects: Base Sequence; Capsid - genetics; Capsid - isolation & purification; Codon; Endoribonucleases; Genes, Viral; Kinetics; Macromolecular Substances; Molecular Sequence Data; Oligonucleotide Probes; Protein Biosynthesis; Reticulocytes - metabolism; Ribonuclease H; Ribonuclease T1; Ribosomes - metabolism; RNA, Messenger - genetics; RNA, Viral - genetics; Tobacco Mosaic Virus - genetics; Triticum - metabolism
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/0022-1317-72-4-769

1 Biologisches Institut, Universität Stuttgart, Pfaffenwaldring 57, D-7000 Stuttgart 80, Germany and 2 Department of Virus Research and 3 Department of Cell Biology, John Innes Institute, AFRC Institute of Plant Science Research, Colney Lane, Norwich NR4 7UH, U.K. Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U- 32 P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2·5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker ( ) recovered remained constant throughout a 15 min period in SDS. Additional RNase T 1 -sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions ‘destabilized’ by ultracentrifugation at between pH 7·2 and 9·2 were quantified using L -[ 35 S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7·5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7·2 to 9·2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly. Present address: Center for Agricultural Molecular Biology, Cook College, Rutgers University, P.O. Box 231, New Brunswick, New Jersey 08903-0231, U.S.A. Received 12 June 1990; accepted 2 January 1991.