Trypsin cleavage stabilizes the rotavirus VP4 spike

Abstract

Trypsin enhances rotavirus infectivity by an unknown mechanism. To examine the structural basis of trypsin-enhanced infectivity in rotaviruses, SA11 4F triple-layered particles (TLPs) grown in the absence (nontrypsinized rotavirus [NTR]) or presence (trypsinized rotavirus [TR]) of trypsin were characterized to determine the structure, the protein composition, and the infectivity of the particles before and after trypsin treatment. As expected, VP4 was not cleaved in NTR particles and was cleaved into VP5(*) and VP8(*) in TR particles. However, surprisingly, while the VP4 spikes were clearly visible and well ordered in the electron cryomicroscopy reconstructions of TR TLPs, they were totally absent in the reconstructions of NTR TLPs. Biochemical analysis with radiolabeled particles indicated that the stoichiometry of the VP4 in NTR particles was the same as that in TR particles and that the VP8(*) portion of NTR, but not TR, particles is susceptible to further proteolysis by trypsin. Taken together, these structural and biochemical data show that the VP4 spikes in the NTR TLPs are icosahedrally disordered and that they are conformationally different. Structural studies on the NTR TLPs after trypsin treatment showed that spike structure could be partially recovered. Following additional trypsin treatment, infectivity was enhanced for both NTR and TR particles, but the infectivity of NTR remained 2 logs lower than that of TR particles. Increased infectivity in these particles corresponded to additional cleavages in VP5(*), at amino acids 259, 583, and putatively 467, which are conserved in all P serotypes of human and animal group A rotaviruses and also corresponded with a structural change in VP7. These biochemical and structural results show that trypsin cleavage imparts order to VP4 spikes on de novo synthesized virus particles, and these ordered spikes make virus entry into cells more efficient.

FIG. 1

Three-dimensional reconstructions, viewed along the icosahedral threefold axis, of SA11 4F TLPs grown in the presence (top) and absence (bottom) of trypsin and treated with 0 (A and D), 25 (B and E), 50 (C), and 75 (F) μg of trypsin/ml. Fivefold axial positions defining one of the icosahedral facets of the T=13 lattice are indicated in panel A. Reconstructions are radially colored according to the chart. Reconstructions were carried out from electron cryo-images of individual specimens embedded in a thin layer of vitreous ice as described in Materials and Methods. Figures 3 to 5 show biochemical analyses of these particles.

FIG. 2

Structural changes in the TLPs induced by exogenous trypsin. Radial cutaways at ∼405 Å (A), ∼385 Å (B), and ∼360 Å (C) of TLP structures grown in the presence of trypsin treated with 0 (top) and 25 (bottom) μg of trypsin/ml. Red arrows point to the location where significant and reproducible changes from three independent reconstructions are observed in the trypsin-treated structure in relation to the particles without added trypsin. An interior portion of VP7, which is tucked inside between the VP7 trimers, swivels out (shown by a white arrow in bottom panel C) when virions are treated with exogenous trypsin. Identical structural changes are also seen in NTR particles upon exogenous trypsin treatment.

FIG. 3

Determination of infectivity of SA11 4F TLPs by fluorescent focus assay. Preparations of NTR and TR SA11 4F TLPs were treated with 0, 25, or 50 μg of trypsin/ml for 30 min at 37°C, and the titer was determined by fluorescent focus assay. Significant differences in titer between mock- and trypsin-treated preparations are indicated (^∗; Student's t test [P < 0.05]). Error bars represent the standard error of the mean. FFU, focus-forming units.

FIG. 4

SDS-PAGE and Western blot analysis of SA11 4F TR and NTR particles. SA11 4F TLPs grown in the presence (A) and absence (B) of trypsin were purified and treated with increasing concentrations of trypsin as indicated. The proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected with a hyperimmune anti-SA11 4F TLP mouse serum. The location of the individual proteins is indicated on the right of each panel. The approximate molecular weights for the new bands representing previously uncharacterized cleavage products from viruses exposed to trypsin after purification are indicated. The arrows highlight two new bands representing previously uncharacterized VP5^∗ cleavage products of 24K and 22K, and the asterisk highlights a new VP8^∗-specific cleavage product of 25K from viruses exposed to trypsin.

FIG. 5

SDS-PAGE and Western blot analysis of NTR and TR SA11 4F virus. NTR or TR SA11 4F TLPs or DLPs were purified and treated with increasing concentrations of trypsin as indicated. The proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected with a hyperimmune anti-SA11 4F TLP mouse serum (A), a VP8^∗ (amino acids 160 to 186) peptide antiserum (B and D), or monoclonal antibodies against VP5^∗ (C). The location of the individual proteins is indicated on the right of each panel. The approximate molecular weights for the new bands representing previously uncharacterized cleavage products from viruses exposed to trypsin after purification are indicated.

FIG. 6

SDS-PAGE and Western blot analysis of NTR SA11 4F virus. The medium from SA11 4F-infected MA104 cells grown in the absence of trypsin with aprotinin was clarified, and the virus was concentrated and then treated with increasing concentrations of trypsin for the time indicated. The proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected with a hyperimmune anti-SA11 4F TLP mouse serum (A) or a VP8^∗ (amino acids 160 to 186) peptide antiserum (B). The location of the individual proteins is indicated on the right. The new VP8^∗-specific band [VP8^∗∗(25K)] representing a previously uncharacterized cleavage product from NTR exposed to trypsin is indicated.

FIG. 7

SDS-PAGE and fluorography of ³⁵S-labeled NTR and TR SA11 4F virus. Radiolabeled NTR and TR SA11 4F TLPs were purified and treated with increasing concentrations of trypsin as indicated. The proteins were separated by SDS–10% PAGE (A) or SDS–8% PAGE (B), and the gel was dried and exposed to X-ray film. The location of the individual proteins is indicated on the right of each panel. The approximate molecular weight for the new VP8^∗-specific band [VP8^∗∗(25K)] representing a previously uncharacterized cleavage product from viruses exposed to trypsin after purification is indicated.