The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion produc- T he timing of viral genome release into the infected host cell is critically important to the outcome of infection, as it initiates the race between viral processes and the cellular immune responses against the virus. Even small changes in the timing of viral genome release can result in decreased fitness of the virus (1, 2). The regulation of the release of the viral RNAs is poorly understood, especially for icosahedral viruses.Brome mosaic virus (BMV) is a plant-infecting RNA virus that has served as a model system to study the regulation of RNA virus infection (3). A particularly intriguing feature of BMV is that its tripartite positive-strand RNA genome is encapsidated in three distinct virions. All three virions are required for successful infection. Upon entry into cells, RNA1 and RNA2 direct the translation of the viral replication-associated proteins, while RNA3 encodes the movement protein required for cell-to-cell spread and the coat protein (CP). The CP is translated from subgenomic RNA4. In a typical infection, RNA4 is coencapsidated with RNA3 in a 1:1 ratio (4), although the host species can influence the encapsidation of the viral RNAs (5).The BMV CP has multiple regulatory activities during infection (6, 7). Each BMV capsid contains 180 subunits of the CP arranged in a Tϭ3 symmetry. A CP subunit has structural features similar to those of a histone protein. Both have an intrinsically disordered N-terminal arm rich in positively charged residues followed by sequences that fold into a globular domain (7). The N-terminal arm contributes to the differential encapsidation of BMV RNA, has the ability to translocate from the internal cavity to the outside of the virion, and is preferentially cleaved during proteolysis (8,9). Virions formed by CPs that lack the first 8 residues were found to encapsidate RNA2 well but were labile for the encapsidation of RNA1 (8).The BMV virions can be separated by their density into two populations, one enriched for virions containing RNA1, called B1 virions, and the other enriched for virions that encapsidate RNA2 and RNA3/4, called B2.3/4 virions (9). The B1 virions release RNA1 more rapidly than the B2.3/4 v...