Cleavage susceptibility of reovirus attachment protein sigma1 during proteolytic disassembly of virions is determined by a sequence polymorphism in the sigma1 neck

Abstract

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 sigma1. 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 sigma1 cleavage upon conversion of virions to ISVPs. We examined the sigma1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and sigma1 cleavage. The sigma1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the sigma1 proteins of the remaining strains. Thr249 occupies the d position of a heptad repeat motif predicted to stabilize sigma1 oligomers through alpha-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of sigma1 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 sigma1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the sigma1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves sigma1 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 sigma1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for sigma1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

FIG. 1

Changes in viral infectivity during generation of ISVPs by using chymotrypsin. Purified virions of T3D, T3C9, T3C18, T3C31, T3C43, T3C44, T3C45, T3C84, and T3C93 at a concentration of 2 × 10¹² particles per ml were treated with chymotrypsin at 37°C for 180 min. Infectious titers of virion preparations before and after treatment were determined by plaque assay using L cells. Changes in viral infectivity are expressed as the ratio of log₁₀ viral titer at 180 and 0 min of chymotrypsin treatment. Shown are the means and standard deviations of three independent experiments.

FIG. 2

Time course of change in T3C31 infectivity during generation of ISVPs by using chymotrypsin. Purified virions of strain T3C31 at a concentration of 2 × 10¹² particles per ml were treated with chymotrypsin at 37°C. At the times indicated, reactions were terminated, and infectious titers of virion preparations were determined by plaque assay using L cells. Changes in viral infectivity are expressed as the ratio of log₁₀ viral titer relative to 0 min of chymotrypsin treatment. Shown are the means and standard deviations of three independent experiments.

FIG. 3

Electrophoretic analysis of viral structural proteins of type 3 reovirus strains following treatment with chymotrypsin to generate ISVPs. Purified ³⁵S-labeled virions of T3D, T3C9, T3C18, T3C31, T3C43, T3C44, T3C45, T3C84, and T3C93 at a concentration of 2 × 10¹² particles per ml were treated with chymotrypsin (CHT) at 37°C for 60 min. Equal numbers of treated and untreated viral particles (2 × 10¹¹) were dissociated in sample buffer and loaded into wells of an SDS–10% polyacrylamide gel. After electrophoresis, gels were prepared for fluorography and exposed to film. Viral proteins are labeled.

FIG. 4

Changes in viral HA capacity during generation of ISVPs by using chymotrypsin. Purified virions of T3D, T3C9, T3C18, T3C31, T3C45, and T3C93 at a concentration of 2 × 10¹² particles per ml were treated with chymotrypsin at 37°C for 180 min. HA activity of virion preparations was determined by endpoint titration using human type O erythrocytes and serial dilutions of virus. Changes in HA activity are expressed as the ratio of log₂ HA titer at 180 and 0 min of chymotrypsin treatment. Shown are the means and standard deviations of three independent experiments.

FIG. 5

Identification of residues important for infectivity loss and ς1 cleavage of reovirus strains T3D and T3C31. (A) Model of ς1 structure. Predicted ς1 secondary structure (47) and morphologic domains of ς1 [T(i), T(ii), T(iii), T(iv), and H] described previously (24) are shown and scaled proportionally to the domains identified in electron microscopic images of ς1 isolated from virions (24). Amino acid positions are scaled according to their predicted relationships to individual ς1 morphologic domains (47). (B) Alignment of ς1 amino acid sequences. Deduced ς1 amino acid sequences of strains T3D and T3C31 were aligned with those of T3C9, T3C18, T3C43, T3C44, T3C45, T3C84, and T3C93 (18) and examined for correlation of sequence variability with viral infectivity changes and ς1 cleavage susceptibility during the generation of ISVPs by using chymotrypsin. The ς1 proteins of T3D and T3C31 contain a threonine residue at position 249, whereas all other ς1 proteins contain an isoleucine at that position. Shown is an alignment of amino acid residues 239 through 252, which are proposed to form α-helical coiled coil comprising a portion of the ς1 neck (47). In the alignment of ς1 sequences, residues in boxes are found in the a or d position of a heptad repeat motif characteristic of α-helical coiled coils (42). The mean (±standard deviation) ratio of log₁₀ viral titer at 180 and 0 min of chymotrypsin treatment is shown for each strain.

FIG. 6

Cleavage susceptibility of expressed T3D ς1 protein altered by site-directed mutagenesis. Wild-type (wt) and mutant ³⁵S-labeled ς1 proteins of T3D were expressed in Sf21 insect cells by using baculovirus vectors and purified by using anti-ς1 MAb G5. The threonine residue at amino acid position 249 of T3D ς1 was substituted with isoleucine (T249I), leucine (T249L), or asparagine (T249N). (A) MAb G5-conjugated Sepharose containing expressed ς1 protein was treated with various concentrations of chymotrypsin (CHT) at 10°C for 180 min. Treatment mixtures were heated at 100°C in sample buffer and subjected to electrophoresis in an SDS–10% polyacrylamide gel. Digestion products were visualized by autoradiography. Positions of molecular weight standards (in kilodaltons) are shown. Bands corresponding to full-length ς1 are indicated. , 0 to 67.5 μg of chymotrypsin per ml. (B) MAb G5-conjugated Sepharose containing expressed ς1 protein was treated with various concentrations of trypsin (TRY) at 4°C for 60 min. Treatment mixtures were processed as described for panel A. , 0 to 18 μg of trypsin per ml.

FIG. 7

Cleavage susceptibility of expressed T3C9 and T3C84 ς1 proteins. ³⁵S-labeled ς1 proteins of T3C9 and T3C84 were expressed in Sf21 insect cells by using baculovirus vectors and purified by using anti-ς1 MAb G5. MAb G5-conjugated Sepharose containing expressed ς1 protein was treated with various concentrations of either chymotrypsin (CHT) at 10°C for 180 min or trypsin (TRY) at 4°C for 60 min. Treatment mixtures were heated at 100°C in sample buffer and subjected to electrophoresis in an SDS–10% polyacrylamide gel. Digestion products were visualized by autoradiography. Positions of molecular weight standards (in kilodaltons) are shown. Bands corresponding to full-length ς1 are indicated. , 0 to 67.5 μg of chymotrypsin per ml or 0 to 18 μg of trypsin per ml.

FIG. 8

Amino-terminal sequence analysis of ς1 cleavage products liberated during the generation of T3D ISVPs by using trypsin. (A) Isolation of a ς1 cleavage product by using anti-ς1 MAb G5. Purified ³⁵S-labeled virions of T3D at a concentration of 2 × 10¹³ particles per ml were treated with 100 μg of trypsin per ml at 15°C for 30 min. Cleavage products were purified by using MAb G5-conjugated Sepharose, resolved in an SDS–14% polyacrylamide gel, and visualized by autoradiography. Lane 1, 4 × 10¹¹ untreated viral particles; lane 2, 4 × 10¹¹ viral particles treated with trypsin; lane 3, supernatant from trypsin digest (shown in lane 2) after incubation with MAb G5-conjugated Sepharose; lane 4, trypsin-generated virion cleavage products (from a total of 2 × 10¹² viral particles) bound to MAb G5-conjugated sepharose. Viral proteins are labeled. Positions of molecular weight standards (in kilodaltons) are indicated. An arrow indicates the ς1 cleavage product (lane 4) isolated by using MAb G5-conjugated Sepharose. This band was used as a reference to identify the Coomassie blue-stained ς1 cleavage product (see Materials and Methods) subjected to amino-terminal sequence analysis. (B) Identification of the trypsin cleavage site in virion-associated T3D ς1 protein. Amino-terminal residues 1 through 8 of the trypsin-generated ς1 cleavage product are aligned with a region of sequence proposed to form the ς1 neck, amino acids 239 to 259 (47). Residues in boxes occur in the a or d position of a heptad repeat motif characteristic of α-helical coiled coils (42). This alignment indicates that trypsin cleaves ς1 between Arg²⁴⁵ and Ile²⁴⁶ during the generation of ISVPs. The cleavage site in ς1 primary sequence is indicated by an arrow.

FIG. 9

Analysis of viral structural proteins following generation of ISVPs by using a murine intestinal wash. Purified ³⁵S-labeled virions of T1L, T3D, T3C9, T3C31, or T3C84 at a concentration of 3.3 × 10¹² particles per ml were treated with various concentrations of a murine intestinal wash (int. wash) at 20°C for 3.5 h. Aliquots of 12 μl were heated at 100°C in sample buffer and subjected to electrophoresis in an SDS–10% polyacrylamide gel, followed by autoradiography to visualize viral proteins. Viral proteins are labeled. The ς1 protein is indicated by an arrow. , 0 to 83% (vol/vol) intestinal wash.