Journal of Virology, October 1998, p. 8205-8213, Vol. 72, No. 10
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
1 during Proteolytic Disassembly of Virions Is
Determined by a Sequence Polymorphism in the
1 Neck
Departments of Pediatrics1 and Microbiology and Immunology3 and Elizabeth B. Lamb Center for Pediatric Research,2 Vanderbilt University School of Medicine, Nashville, Tennessee 37232, and Institute for Molecular Virology, University of Wisconsin, Madison, Wisconsin 537064
Received 6 March 1998/Accepted 18 June 1998
A requisite step in reovirus infection of the murine intestine is
proteolysis of outer-capsid proteins to yield infectious subvirion
particles (ISVPs). When converted to ISVPs by intestinal proteases,
virions of reovirus strain type 3 Dearing (T3D) lose 90% of their
original infectivity due to cleavage of viral attachment protein
1.
In an analysis of eight field isolate strains of type 3 reovirus, we
identified one additional strain, type 3 clone 31 (T3C31), that loses
infectivity and undergoes
1 cleavage upon conversion of virions to
ISVPs. We examined the
1 deduced amino acid sequences of T3D and the
eight field isolate strains for a correlation between sequence
variability and
1 cleavage. The
1 proteins of T3D and T3C31
contain a threonine at amino acid position 249, whereas an isoleucine
occurs at this position in the
1 proteins of the remaining strains.
Thr249 occupies the d position of a heptad
repeat motif predicted to stabilize
1 oligomers through
-helical
coiled-coil interactions. This region of sequence comprises a portion
of the fibrous tail domain of
1 known as the neck. Substitution of
Thr249 with isoleucine or leucine resulted in resistance to
cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid
position 249 is an independent determinant of T3D
1 cleavage susceptibility and that an intact heptad repeat is required to confer
cleavage resistance. We performed amino-terminal sequence analysis on
the
1 cleavage product released during trypsin treatment of T3D
virions to generate ISVPs and found that trypsin cleaves
1 after
Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of
these results to reovirus infection in vivo was assessed by treating
virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of
1 cleavage susceptibility generated by using purified protease was reproduced in
assays using the intestinal wash. These results provide a mechanistic explanation for
1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of
reovirus pathogenesis.
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