Yukio Kiho

Unique nature of an attenuated strain of tobacco mosaic virus: autoregulation

An attenuated strain L11A of tobacco mosaic virus (TMV) multiplied like wild type strain L at an early stage of infection in tomato leaves. Four days after inoculation, however, multiplication of L11A was drastically reduced (autoregulation) compared with the constant multiplication of L. In mixed infections, L11A strongly inhibited the multiplication of homologous strain L. Experiments with cucumber mosaic virus (CMV) or tobacco plants revealed that the inhibitory mechanism of L11A is not host‐specific but virus‐specific, and the auto‐regulatory mechanism is effective only for TMV.

Disassembly of Tobacco Mosaic Virus by Membrane Lipid Isolated from Tobacco Leaves and Polyornithine

In vitro disassembly of tobacco mosaic virus (TMV) virions occurred in the presence of both polyornithine and a lipid fraction isolated from tobacco leaf membrane. The latter could be replaced by lecithine. Disassembly of 10 μg of TMV virions was attained in the presence of a 500‐mg leaf equivalent of membrane lipid and 20 μg of polyornithine in 1 ml of 0.01 M Tris‐HCl buffer, pH 7.4 at 30 C. Similarity and dissimilarity between the in vitro disassembly and the in vivo uncoating mechanisms are discussed.

Modification of Tobacco Mosaic Virus by Polyornithine and Lecithin

Combined action of polyornithine and lecithin modified tobacco mosaic virus (TMV) virions making them sensitive to ribonuclease (RNase), pronase or Triton X‐100. Sedimentational analysis and examination of the fluorescence spectrum revealed that the reaction product obtained after RNase treatment of modified TMV was a three‐component complex made of coat protein, polyornithine and lecithin. The minimum requirement for the modification was completely fulfilled by cetyltrimethylammonium bromide, suggesting that a positively charged nitrogen group and an alkyl group of moderate size, C10–18, are necessary components. These components react with the surface region of TMV which is considered to have an important role in connecting coat protein subunits in TMV virions.

Infectivity Suppressing and Virus‐Binding Activities of a Membrane Material Isolated from Tobacco Leaves

TMV binding substance (R) was isolated from a tobacco leaf membrane fraction and was purified by extraction with organic solvents and by column chromatography. Experimental results suggest that the binding of R with TMV results in inactivation of TMV. When tobacco leaves were inoculated with the R‐TMV complex, it was found that the formation of polysome containing infecting viral RNA was inhibited. Model experiments showed that the mode of R‐TMV adsorption to the membrane is different from that of TMV adsorption and that stripping of coat protein from TMV by SDS was inhibited by R. A possible explanation for the mechanism of this inhibition by R is that the R‐TMV complex follows a pathway which does not lead to establishment of infection. Although less efficient, R was still active when it was applied after virus inoculation. Due to its affinity to coat protein, R might also interfere with a later process of viral multiplication.

Sensitivity to Ultraviolet Light of Tobacco Mosaic Virus Modified by Cetyltrimethylammonium Bromide

Cetyltrimethylammonium bromide (CTAB) modified tobacco mosaic virus (TMV) virions so that the intrinsic fluorescence changed, viral infectivity decreased, sensitivity to RNase or UV irradiation increased, and coat protein subunits were released by the addition of Triton X‐100. The change in fluorescence emission at 320 nm shifted to 340 nm was observed at 100 μg of CTAB per ml. This represents a change in the tryptophan environment inside the virion. At a lower concentration of CTAB, intersubunit contact was weakened, resulting in the release of coat protein subunits and an increase in RNase sensitivity. The release of coat protein took place gradually and two relatively stable intermediates were observed. Increase in UV sensitivity was observed at a lower concentration of CTAB and formation of pyrimidine hydrate was involved in this inactivation. The nature of the minor structural change leading to UV inactivation is discussed.