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Journal of Virology, January 2006, p. 671-681, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.671-681.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Reovirus Variants Selected for Resistance to Ammonium Chloride Have Mutations in Viral Outer-Capsid Protein {sigma}3

Kimberly M. Clark,1,2,{dagger} J. Denise Wetzel,2,3,{dagger} Yingqi Gu,4 Daniel H. Ebert,2,5,{ddagger} Stephanie A. McAbee,2,3,§ Emily K. Stoneman,2,3,¶ Geoffrey S. Baer,2,5,|| Yuwei Zhu,3 Gregory J. Wilson,2,3 B. V. V. Prasad,4 and Terence S. Dermody2,3,5*

Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37241; Departments of,1 Pediatrics,3 Microbiology and Immunology,5 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,2 Department of Biochemistry, Baylor College of Medicine, Houston, Texas 770304

Received 18 July 2005/ Accepted 19 October 2005

Mammalian reoviruses are internalized into cells by receptor-mediated endocytosis. Within the endocytic compartment, the viral outer capsid undergoes acid-dependent proteolysis resulting in removal of the {sigma}3 protein and proteolytic cleavage of the µ1/µ1C protein. Ammonium chloride (AC) is a weak base that blocks disassembly of reovirus virions by inhibiting acidification of intracellular vacuoles. To identify domains in reovirus proteins that influence pH-sensitive steps in viral disassembly, we adapted strain type 3 Dearing (T3D) to growth in murine L929 cells treated with AC. In comparison to wild-type (wt) T3D, AC-adapted (ACA-D) variant viruses exhibited increased yields in AC-treated cells. AC resistance of reassortant viruses generated from a cross of wt type 1 Lang and ACA-D variant ACA-D1 segregated with the {sigma}3-encoding S4 gene. The deduced {sigma}3 amino acid sequences of six independently derived ACA-D variants contain one or two mutations each, affecting a total of six residues. Four of these mutations, I180T, A246G, I347S, and Y354H, cluster in the virion-distal lobe of {sigma}3. Linkage of these mutations to AC resistance was confirmed in experiments using reovirus disassembly intermediates recoated with wt or mutant {sigma}3 proteins. In comparison to wt virions, ACA-D viruses displayed enhanced susceptibility to proteolysis by endocytic protease cathepsin L. Image reconstructions of cryoelectron micrographs of three ACA-D viruses that each contain a single mutation in the virion-distal lobe of {sigma}3 demonstrated native capsid protein organization and minimal alterations in {sigma}3 structure. These results suggest that mutations in {sigma}3 that confer resistance to inhibitors of vacuolar acidification identify a specific domain that regulates proteolytic disassembly.

* Corresponding author. Mailing address: Lamb Center for Pediatric Research, D7235 MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723. E-mail: terry.dermody{at}vanderbilt.edu.

{dagger} K.M.C. and J.D.W. contributed equally to this study.

{ddagger} Present address: Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114.

§ Present address: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232.

Present address: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109.

|| Present address: Department of Orthopaedic Surgery, University of Virginia School of Medicine, Charlottesville, VA 22908.

Journal of Virology, January 2006, p. 671-681, Vol. 80, No. 2
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.2.671-681.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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