Studies on the Formation of Tobacco Mosaic Virus Ribonucleic Acid, Viii. Fate of Parental Viral Rna in the Host Cell

Abstract

and Summary.-Fate of parental viral RNA in the host cell was determined with the use of P32-labeled tobacco mosaic virus (TMIV)-RNA as inoculum. There was a wide variation in the retention of TMV-RNA that was rubbed on the tobacco leaf surface. On the average, about 0.73 jig of viral RNA was retained by a tobacco leaf. About 63 per cent of the retained RNA was degraded in the leaf cells. On the average, about 0.27 ,ug of TMIV-RNA was conserved in a tobacco leaf.The conserved parental TMV-RNA was remarkably stable and was located in the nuclear fraction of leaf homogenate. It could be isolated in an undegraded form and was RNase-sensitive, presumably single-stranded. About 2 per cent of it was associated with progeny TMV.RNA in TMV exists deeply embedded in the protein matrix as a single chain following the pitch of the protein helix and is insensitive to the action of RNase. After the viral particle enters the host cell, the prerequisite for the initiation of infection is the release of viral RNA from its protein envelope. With P32-labeled TMV as inoculum, direct evidence for the uncoating of viral RNA in the host cell was obtained: in the extracts of tobacco leaf inoculated with P32-labeled TMV, significant amounts of RNase-sensitive radioactive RNA were discernible within three hours after inoculation; this material, having the nucleotide composition of TMV-RNA, increased with time and was associated with the nuclear fraction of leaf extract.' However, the information concerning the fate of uncoated viral RNA (parental viral RNA) in the host cell is meager, even though it is well established that it has the essential information to direct all the processes which ultimately lead to the synthesis of viral particles. The present studies were aimed at elucidating the fate of parental viral RNA in the host cell.Experimental Procedures.-(1) Plants: Nicotiana tabacum var. Turkish was used in all the experiments described in this paper. Nicotiana glutinosa was used for TMV-RNA infectivity assays.(2) Preparation of P32-labeled TM11V-RNA: Tobacco plants 8 to 10 weeks old were transferred from a greenhouse to a room in which the temperature was maintained at 25°. Plants of same size and general appearance were selected. Bottom leaves were removed and discarded. The remaining leaves were dusted with carborundum and rubbed with TMV inoculum prepared as previously described,2 after which they were rinsed with water. The infected plants were immediately removed from the pots, and their roots were washed with tap water. Each plant was placed in a bottle that contained 40 ml of Hoagland's nutrient solution to which was added 1 me P,32. The phosphate content of Hoagland's nutrient solution was reduced to one tenth of that prescribed to ensure the maximum uptake of p,32 by the plants. The rest of the culture conditions were exactly the same as previously described.3 After 6 days, the plants were harvested, and TMV was prepared according to the procedure previously described' with the following modifi-604