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Journal of Virology, July 2007, p. 7400-7409, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.02720-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein µ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation{triangledown}

Melina A. Agosto,1,2 Jason K. Middleton,3,4 Elaine C. Freimont,1 John Yin,3,4 and Max L. Nibert1,2*

Department of Microbiology and Molecular Genetics, Harvard Medical School,1 Ph.D. Training Program in Biological and Biomedical Sciences, Division of Medical Sciences, Harvard University, Boston, Massachusetts 02115,2 Department of Chemical and Biological Engineering,3 Graduate Training Program in Biotechnology, University of Wisconsin—Madison, Madison, Wisconsin 537064

Received 9 December 2006/ Accepted 4 May 2007

Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein µ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to µ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of µ1-based thermostabilization can occur by a general mechanism of µ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between µ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in µ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-4838. Fax: (617) 738-7664. E-mail: mnibert{at}hms.harvard.edu

{triangledown} Published ahead of print on 16 May 2007.

Journal of Virology, July 2007, p. 7400-7409, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.02720-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zhang, L., Agosto, M. A., Ivanovic, T., King, D. S., Nibert, M. L., Harrison, S. C. (2009). Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated {micro}1N Peptide. J. Virol. 83: 7004-7014 [Abstract] [Full Text]  
  • Agosto, M. A., Myers, K. S., Ivanovic, T., Nibert, M. L. (2008). A positive-feedback mechanism promotes reovirus particle conversion to the intermediate associated with membrane penetration. Proc. Natl. Acad. Sci. USA 105: 10571-10576 [Abstract] [Full Text]  


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