Structural studies of mammalian reovirus by electron cryomicroscopy

Xing Zhang, Purdue University

Abstract

Mammalian reovirus (MRV) is a double-stranded RNA virus of the Reoviridae family. MRV uses viral polymerases λ3 inside the viral cores to synthesize viral mRNAs. To locate the polymerases, the atomic model of λ3 was fitted into a reconstructed density map of MRV at 7.6-Å resolution. The polymerase was found attached to the interior surface of the λ1 shell, with its molecular center ∼28-Å from the icosahedral five-fold axis. A small portion of λ3 overlaps the five-fold axis, which is consistent with the finding that only one polymerase binds at an five-fold axis. Polymerase contacts the λ1 shell with its mRNA exit channel facing the shell. These results suggest that, during transcription, the viral mRNA synthesized by λ3 may pass directly through a peripentonal channel of the λ1 shell, and finally into the central cavity of the λ2 pentamer, where a cap structure is added to the mRNA. Several new features of the membrane penetration protein μ1, that were disordered in the crystal structure of the μ13σ3 3 heterohexamer, were revealed by electron cryomicroscopy (cryoEM). These new features include a loop-like structure formed by residues 2 to 9 that suggests the N-terminal myristoyl group can enter a hydrophobic pocket within the same μ1 trimer, a U-shaped structure formed by residues 72–96 that may mediate the interactions between adjacent μ1 trimers, and spoke-like structures formed by the C-terminal 33 residues of μ1. These new features may be related to virus assembly, stability, transcription regulation and cell entry. Assembly of MRV shells was studied by analyzing association energies between viral proteins to explore possible assembly intermediates of the inner and outer shells. As a result, the primary assembly intermediates of the inner shell were predicted to be dimers and tetramers of the λ1 protein. Assembly of the outer shell appears to be initiated by the binding of Q-type of μ13σ33 heterohexamers to the λ2 pentamers of cores. This proposal was supported by a cryoEM reconstruction of a partially assembled intermediate particle of reovirus. The assembly order of the R-, S-, and T-types of heterohexamers was also predicted as S→R→T.

Degree

Ph.D.

Advisors

Baker, Purdue University.

Subject Area

Microbiology

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