A central question of biological research is how macromolecules interact to form the large, dynamic structures that carry out many essential processes of the living world, and developments such as the tools of structural biology are now making this question accessible. Reovirus is one such large macromolecular machine, transcribing its own mRNA from its ten genome segments within the viral capsid. How the virus accomplishes the twin feats of packing the genome segments and transcribing them in the capsid, as well as how the transcriptase enzyme complex is arranged and functions, are longstanding questions for both virology and studies of macromolecular assemblies in general. A set of biochemical approaches is proposed here to address several fundamental issues in reovirus genome arrangement and transcription. First, it has long been hypothesized that each copy of the reovirus transcription machinery is dedicated to synthesizing mRNA from a particular genome segment, but evidence directly supporting this assumption has never been found. A method based on RT-PCR of mRNA from single virus particles will be developed to address this question. Second, the architecture of the transcription machinery remains elusive, as it has been largely intractable to biophysical analysis thus far. The arrangement of this complex will be investigated by protein-protein cross-linking and mass spectrometry. ? ? ?
