Digestion pattern of reovirus outer capsid protein sigma3 determined by mass spectrometry

Abstract

Reovirus is an enteric virus comprising eight structural proteins that form a double-layered capsid. During reovirus entry into cells, the outermost capsid layer (composed of proteins sigma3 and mu1C) is proteolytically processed to generate an infectious subviral particle (ISVP) that is subsequently uncoated to produce the transcriptionally active core particle. Kinetic studies suggest that protein sigma3 is rapidly removed from virus particles and then protein mu1C is cleaved. Initial cleavage of mu1C has been well described and generates an amino (N)-terminal delta peptide and a carboxyl (C)-terminal phi peptide. However, cleavage and removal of sigma3 is an extremely rapid event that has not been well defined. We have treated purified reovirus serotype 1 Lang virions with a variety of endoproteases. Time-course digestions with chymotrypsin, Glu-C, pepsin, and trypsin resulted in the initial generation of two peptides that were resolved in SDS-PAGE and analyzed by in-gel tryptic digestion and MALDI-Qq-TOFMS. Most tested proteases cut sigma3 within a "hypersensitive" region between amino acids 217 and 238. In addition, to gain a better understanding of the sequence of subsequent proteolytic events that result in generation of reovirus subviral particles, time-course digestions of purified particles were performed under physiologic salt conditions and released peptide fragments ranging from 500 to 5000 Da were directly analyzed by MALDI-Qq-TOFMS. Trypsin digestion initially released a peptide that corresponded to the C-terminus of mu1C, followed by a peptide that corresponded to amino acids 214-236 of the sigma3 protein. Other regions of mu1C were not observed until protein sigma3 was completely digested. Similar experiments with Glu-C indicated the hypersensitive region of sigma3 was cut first when virions were treated at pH values of 4.5 or 7.4, but treatment of virions with pepsin at pH 3.0 released different sigma3 peptides, suggesting acid-induced conformational changes in this outer capsid protein. These studies also revealed that the N-terminus of sigma3 is acetylated.

Fig. 1

Kinetics of virus digestion. Aliquots of gradient-purified T1L virions were diluted to concentrations of 7.3–8.6 × 10¹¹ particles/μl in appropriate buffers (pH 7.4 for α-chymotrypsin, trypsin, and S. aureus V8 (Glu-C); or pH 4.5 for Glu-C; or pH 3.0 for pepsin), digested with either (A) 200 μg/ml α-chymotrypsin, or (B) 10 μg/ml α-chymotrypsin, (C) 10 μg/ml trypsin, (D) 10 μg/ml pepsin, or (E) 10 μg/ml Glu-C for the indicated periods of time (minutes for (A–D); hours for (E)) at 37°C. Reactions were stopped by chilling and addition of phenylmethylsulfonyl fluoride to a final concentration of 5 mM to α-chymotrypsin reactions, by addition of soybean trypsin inhibitor to a final concentration of 125 μg/ml to trypsin reactions, by adjustment of pepsin reactions to a pH of 7.5, or by chilling Glu-C reactions. One-quarter volume of 5× electrophoresis sample buffer was added and peptides from about 1 × 10¹¹ particles were resolved in either a 4–16% exponential gradient (A) or a 6–16% linear gradient (B–E) mini-SDS–PAGE (8 × 10 × 0.04 cm) at 150–180 V for 60 min. Gels were fixed and stained with Coomassie brilliant blue R-250. V: gradient-purified virions; P: 150 ng of indicated protease; C: gradient-purified core particles, separately prepared. Molecular weights, determined from coelectrophoresis of Kaleideoscope Rainbow Marker, are indicated to left of each gel. Virion proteins (λ, μ, and σ1–σ3), μ1C digestion product δ, and potential σ3 digestion products (indicated by arrowheads) are indicated to the right of each gel.

Fig. 2

Identification of large and small σ3 peptide fragments. (A) Electrophoretic separation of 2.5 × 10¹¹ virions digested with 10 μg/ml of indicated proteases for 30 s (α-chymotrypsin, trypsin, and pepsin) or for 1.5 h (Glu-C) and resolved in a 6–16% linear gradient SDS–PAGE (16 × 16 × 0.1 cm) at 6.0 W for 5 h. Molecular weights, determined from coelectrophoresis of Kaleidoscope Rainbow Marker, are indicated to left of gel. Virion proteins, enzymes (∼250 ng), and inhibitors (P: pepsin; G: Glu-C; T: trypsin/chymotrypsin; S: soybean trypsin inhibitor), and potential σ3 digestion products (indicated by arrowheads and subsequently analyzed by mass spectrometry) are indicated to right of gel. (B–E) MALDI-Qq-TOF mass spectra obtained from tryptic in-gel digestions of (B) α-chymotrypsin-generated large fragment (indicated by large arrow head in A), (C) α-chymotrypsin-generated small fragment (indicated by small arrow head in A), (D) trypsin-generated large fragment, and (E) trypsin-generated small fragment. Values above peaks indicate measured m/z values of peaks identified as corresponding to virion protein σ3 (shown in Table 1). Peak in B with m/z value of 2918.329 (subsequently shown to correspond to amino acid sequence 214–238) and peak in c with m/z value of 1069.651 (subsequently shown to correspond to amino acid sequence 239–247) are expanded in insets. (F) Diagrammatic representation of strategy to determine where each protease initially cleaves virion-associated σ3. Rectangles above sequence scale correspond to MS-predicted tryptic fragments found in large (∼27-kDa polypeptide) and rectangles below sequence scale correspond to MS-predicted tryptic fragments found in small (∼12.5-kDa polypeptide) as listed in Table 1. (G) Detailed sequence of σ3 protein between amino acid residues 201 and 260, indicating potential cleavage sites for trypsin (▿), α-chymotrypsin (▾), and Glu-C (↓). Underline corresponds to cleavage sites identified as initially used. (H) Diagrammatic representation of entire σ3 protein with initial cleavage sites of tested proteases indicated.

Fig. 3

Peptide mapping of tryptic T1L virion digests. 5 mg/ml purified T1L virions were digested with 5 μg/ml trypsin at 37°C. At various time intervals, aliquots were collected and analyzed by MALDI-Qq-TOFMS. Each peak was then selected and subjected to MS/MS analysis (see Fig. 4) to identify each peptide fragment (compiled in Table 2A). Each peak is labeled in the time frame at which it is first detected.

Fig. 4

MS/MS spectra of selected tryptic ions. (A) The mass spectrum of the first tryptic ion detected, at m/z 2684.23, was acquired at a laser repetition rate of 7 Hz. The ion was dissociated using argon as the collision gas. The collision energy was set to 140 eV and then slightly adjusted to obtain optimum fragmentation of the parent ion. Spectra data acquisition was performed using software developed in-house (Tofma, University of Manitoba, MB). Sequence analysis indicates the ion corresponds to σ3 peptide 214–236. (B) The mass spectrum of the ion at m/z 2511.21 was acquired and dissociated as above. Sequence analysis indicates the N-terminal acetylation of σ3 peptide 1–22.

Fig. 5

Diagrammatic representation of time-course enzymatic digests of σ3 and μ1 outer capsid proteins. (A) Time-course digestion of T1L σ3 and μ1 by trypsin at pH 7.4. (B) Time-course digestion of T1L σ3 and μ1 by Glu-C at pH 7.4 or 4.5 (results identical at both pH values). (C) Time-course digestion of T1L σ3 by pepsin at pH 3.0. White bar: undigested protein; gray bar: peptides identified by MS or MS/MS; hatched bar: nonidentified peptides; black bar: too many fragments to identify. Initial peptides cleaved are indicated with numbers below bars, with the exception of 144–162 in A (see Discussion).

Fig. 6

X-ray crystallographic structure of the σ3 monomer. The orientation of the σ3 monomer (adapted and modified from Olland et al., 2001 was examined with Molscript (Kraulis, 1991) and rendered with Raster3D (Merritt and Bacon, 1997). With the exception of selected regions shown in red (A–D) or olive (C–D), helices and turns are in light blue and β-sheets are in dark blue. (A) The selected red region corresponds to residues Ser₂₁₄-Arg₂₃₆, the initial tryptic fragment at pH 7.4. (B) The selected red region corresponds to residues Gly₁₉₉-Glu₂₁₇, the initial Glu-C protease fragment at pH 7.4. (C) and (D) The red region corresponds to residues Ser₂₁₄-Arg₂₃₆ from trypsin digestion at pH 7.4 and the olive region corresponds to residues Asn₁₁₁-Leu₁₂₈ from pepsin digestion at pH 3.0. In (D) the σ3 monomer is rotated 90° clockwise about the y-axis. The large green dot near the bottom of each image corresponds to the previously determined zinc ion (Olland et al., 2001).

Fig. 7

X-ray crystallographic structure of the σ3/μ1 heterohexamer. The orientation of the σ3/μ1 heterohexamer (adapted and modified from Liemann et al., 2002) using PyMol (DeLano, 2002) is shown in space-filling mode. Panels (A), (C), (E), and (G) show top views and panels (B), (D), (F), and (H) show side views of the structure. Individual proteins are identified in (A) and (B); μ1 are depicted in forest, olive, and lime; σ3 are depicted in wheat, pink, and salmon. Initial cleavage sites (red residues) and entire peptide fragments initially released from σ3 during time-course digestions (denoted in yellow) are indicated for trypsin (C, D), Glu-C (E, F), and pepsin (G, H).