In vivo Uncoating of Tobacco Mosaic Virus after Infection of Tobacco Leaves

Abstract

In vivo uncoating of tobacco mosaic virus (TMV) was studied. As Shaw had reported, initiation of uncoating reaction takes place very efficiently. Coat protein is removed from the virus as a peptide which is precipitable with trichloroacetic acid. Short rod particles with partly exposed RNA are thus formed. Further uncoating to coat protein-free TMV-RNA (28S) seems to take place with very low efficiency which is comparable to that of formation of local lesions on the inoculated leaf. From the data on the intracellular distribution of these products of uncoating reaction, mechanisms and significance of these reactions are discussed.The first process after viral infection is the release of the viral aenome from its protein coat. Little information is available at present about the mechanism of this uncoating process in plant virus infection. With tobacco mosaic virus (TMV) labeled with 1+C in its protein moiety, Shaw [7] had shown that the removal of protein coat from TMV after infection of tobacco leaves begins almost immediately after infection and proceeds until about half the total virus protein had been removed at 5 hours. Results suggest that almost all TMV particles retained by the leaves are partially uncoated. In parallel with this findings, some naked parental RNA was detected after TMV infection by Reddi [6]. Furthermore he isolated RNase sensitive RNA from nucleus fraction of leaves obtained at 2 days after infection. As the nucleotide composition of this RNA resembles that of TMV-RNA, he claimed that it is TMV-RNA. If it is true, the size of this RNA should be 28S. Actually, there has been no report of isolation of a free 28S RNA from leaves shortly after infection.These works suggest that the initiation of uncoating reaction takes place very efficiently. However, it stops soon and the efficiency of completely uncoating to 28S TMV-RNA seems to be low. This may reflects the low efficiency of establishment of TMV infection [8]. Why does uncoating process stop soon after infection? In order to solve this problem, we investigated the details of uncoating process using 32P or 35S labeled TMV . In this paper, we report on the nature of the product of the uncoating reaction.

Polysomes Containing Infecting Viral Genome in Tobacco Leaves Infected with Tobacco Mosaic Virus

“…Xanthi) was used in this work. Methods of preparation and purification of 32P-or 35S-labelled TMV are reported previously [8].…”

“…Details of the method of density gradient analysis employed was described previously [5]. For incorporation of 14C-amino acids into the protein fraction , 1 gram of lamina slices were incubated in Hoagland medium [8] containing 14G amino acid (51/Ci/m1) for 10 min at 25 C under a fluorescent lamp. Slices were homogenized, extracts were obtained and analysed as described above.…”

“…This appears to be due to a release of RNase by detergents, since further addition of yeast RNA (method C) effectively prevented the breakdown of larger polysomes, as shown in curve C. Due to a diffusion of the yeast RNA from the top of the gradient, the ribosome peak is not clear in this case. Yeast RNA was one of the most potent inhibitors of RNase and inhibited a digestion of TMV-RNA by RNase in the extracts of tobacco leaves prepared by the method B [8]. Yeast RNA alone did not improve the yield of polysomes.…”

“…Figure 2b shows that there were, besides TMV (fraction 13), structures containing parental TMV-RNA which sediment slower than TMV (fraction 15 to top of the gradient). These are the partially uncoated products of TMV [11] which are short rod particles with partially exposed RNA [8].…”

“…However, some recent works have shown that almost all TMV particles retained by the leaves after inoculation are at least partially uncoated [4,8,10,11]. These reports stimulated us to study the fate of infecting TMV.…”