Potential Mechanisms of Photodynamic Inactivation of Virus by Methylene Blue I. RNA-Protein Crosslinks and Other Oxidative Lesions in Qþ Bacteriophage

J. Edward Schneider, Jr.*, Tahereh Tabatabaie, Lindsay Maidt, Renee Horning Smith, Xuan Nguyen, Quentin Pye and Robert A. Floyd

*To whom correspondence should be addressed at: Oklahoma Medical Research Foundation, Free Radical Biology and Aging Research Program, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA. Fax: 405-271-1795; e-mail: Ed-Schneider@OMRF.OUHSC.EDU

ABSTRACT

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Qþ were exposed to methylene blue plus light (MB+L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8- dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Qþ phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Qþ genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Qþ RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8- oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB+L, crosslink formation between Qþ RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA- protein complexes (including full-length Qþ RNA) was observed at the lowest concentration of MB tested (0.5 mM), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Qþ inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Qþ following MB+L exposure.