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The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

The ability of the cyanobacterium Synechocystis PCC6803 to transport inorganic carbon in the form of bicarbonate rapidly decreased following a shift from bicarbonate-limited growth to either excess bicarbonate supply or to photoheterotrophic growth on glucose. Nonmetabolizable analogs of glucose did not exert this effect. The rate at which the bicarbonate uptake rate declined was too rapid to be accounted for by dilution of the activity by culture growth and suggested that posttranslational modification may be involved. Several proteins that were unphosphorylated during bicarbonate-limited growth became phosphorylated during the shifts to high CO(2) conditions and to photoheterotrophic growth. A similar alteration in the profile of phosphopolypeptides was observed following a shift into the dark. The changes in protein phosphorylation were not blocked by chloramphenicol or rifampicin.