The four Brome mosaic virus (BMV) RNAs are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidates each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of post-translational 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. CryoEM 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 production. The degree of phosphorylation affected CP-RNA interaction to modulate interaction with the encapsidated RNA, the release of three of the BMV RNAs. CLIP-seq experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contained regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus with its three genomic RNAs encapsidated in separate virions. Through proteomic structural, and biochemical analyses this work shows that post-translational modifications, specifically phosphorylation, on the capsid protein regulates capsid-RNA interaction, the stability of the virions, and affected viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the regulation of chromatin packaging by nucleosomes.