Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome-mediated mechanism that causes sitespecific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI- and MALDI-based FT-ICR, Orbitrap, and TOF mass spectroscopy. Top-down mass spectrometry of the products identified the backbone cleavage site as Cys46–Ser47 in the virus capsid protein, a location of viral genome–protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site-specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 Ca. Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome-induced protein cleavage without the addition of sensitizers.