Inactivation of purified plant viruses and their nucleic acids by photosensitizing dyes

The reaction between tobacco mosaic virus (TMV ), alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), their ribonucleic acids (RNA), and three photosensitizing dyes was investigated. Optimal conditions for the inactivation of TMV by toluidine blue and acridine orange were: presence of light, pH 9, and 0.01 ionic strength. Inactivation of TMV by neutral red was optimal at pH 6, but this dye was less effective than the other two dyes. TMV-RNA was more sensitive to inactivation than was the whole virus.Inactivation of TMV by toludine blue at a low concentration followed a slow first-order reaction after an instantaneous decrease. At a high dye concentration, inactivation was first order over 98% of the range, followed by a period of decreased inactivation which also seemed to be first order.Inocula containing high concentrations of toluidine blue were less infectious than inocula from which dye had been removed. Thus dye at high concentrations seemed to affect not only the virus but also the host. TMV could be photosensitized when free toluidine blue was removed from the suspension prior to illumination, indicating that dye bound to the virus was responsible for inactivation.The capacity of TMV to bind acridine orange was reduced by suboptimal ionic strength of the suspension, but not by suboptimal pH; whereas infectivity was affected by both suboptimal pH and ionic strength. Binding of TMV to dyes was also indicated by a shift of maximal absorption of virus-treated dyes and dye-treated virus. No morphological changes of photosensitized TMV were visible under the electron microscope.AMV, AMV-RNA, CMV, and CMV-RNA were more rapidly inactivated than TMV or TMV-RNA. As with TMV, inactivation of these viruses was more gradual in the dark than in the light. CMV and CMV-RNA could also be inactivated by exposure to light without treatment with photoreactive dyes.