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Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in receptor binding and extended host range.

ABSTRACT: To study the process of spike (S)-receptor interaction during coronavirus entry, we evaluated the contributions of mutations in different regions of the murine hepatitis virus (MHV) S protein to natural receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (CEACAM1a) dependence and to the acquisition of extended host range. Extended-host-range variants of MHV strain A59 were previously obtained from persistently infected cells (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9504, 1997). These variant viruses contain several mutations in the S protein that confer to the viruses the ability to enter cells in a heparan sulfate-dependent manner (C. A. de Haan, Z. Li, E. te Lintelo, B. J. Bosch, B. J. Haijema, and P. J. M. Rottier, J. Virol. 79:14451-14456, 2005). While the parental MHV-A59 is fully dependent on murine CEACAM1a for its entry, viruses carrying the variant mutations in the amino-terminal part of their S protein had become dependent on both CEACAM1a and heparan sulfate. Substitutions in a restricted, downstream part of the S protein encompassing heptad repeat region 1 (HR1) and putative fusion peptide (FP) did not alter the CEACAM1a dependence. However, when the mutations in both parts of the S protein were combined, the resulting viruses became independent of CEACAM1a and acquired the extended host range. In addition, these viruses showed a decreased binding to and inhibition by soluble CEACAM1a. The observations suggest that the amino-terminal region of the S protein, including the receptor-binding domain, and a region in the central part of the S protein containing HR1 and FP, i.e., regions far apart in the linear sequence, communicate and may even interact physically in the higher-order structure of the spike.

SUBMITTER: de Haan CA

PROVIDER: S-EPMC1642182 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

Project description:We have analyzed the spike protein S1/S2 cleavage site of selected strains of MHV by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of the various MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We thus identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

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Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in receptor binding and extended host range.

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