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. 2018 Aug 29;92(18):e00679-18.
doi: 10.1128/JVI.00679-18. Print 2018 Sep 15.

Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

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Free PMC article

Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering

Michael Herren et al. J Virol. .
Free PMC article

Abstract

Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

Keywords: Morbillivirus; attachment protein; host cell invasion; linker module; membrane fusion activation; protein folding; stalk domain.

Figures

FIG 1
FIG 1
Mutagenesis screen in CDV H linker and connector region. (A) Schematic representation of the CDV H protein. CT, cytosolic tail; TM, transmembrane domain; CO, connector domain; uR, unresolved region in crystal structures; α, α-helix H1. Structural information derived from MeV H protein (PDB entries 2ZB5 and 4GJT) (24, 25). (B) Sequence comparison of the linker uR, α-helix, and uR regions in various morbilliviruses. Amino acid conservation is indicated by an asterisk. H-A75, CDV H of the A75/17 strain; H-OP, CDV H of the Onderstepoort vaccine strain; H-EDM, MeV H of the Edmonston strain; H-ICB, MeV H of the ICB strain; PPRV H-GHA, peste des petits ruminants virus H of the Ghana strain; RPV H-KAB, Rinderpest virus H of the Kabete strain; H-PDV, phocine distemper virus H protein. (C) Nonconservative mutagenesis scan results for surface expression and fusion promotion activity, both relative to the wild type (for naturally hydrophobic amino acids the alanine mutation also is shown). Surface expression results are derived from nine independent transfections. Fusion promotion capability was determined by quadruplicate measurements of three independent transfections. Mutants showing significant reduction in fusion promotion activity are color coded blue. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.0001). (D) Qualitative fusion assay of selected CDV H mutants (cotransfected with CDV F-wt) in receptor-positive Vero-cSLAM cells.
FIG 2
FIG 2
Detailed mutagenesis scan on CDV H-I146. (A) Surface expression, fusion promotion activity, and SLAM binding of various CDV H-I146 mutations relative to the wild type. Mutants showing significant reduction in fusion promotion activity are color coded blue. Surface expression and SLAM binding results are derived from nine independent transfections. Fusion promotion capability was determined by quadruplicate measurements of three independent transfections. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.0001). (B) Qualitative fusion assay of selected CDV H-I146 mutants (cotransfected with CDV F-wt) in receptor-positive Vero-cSLAM cells.
FIG 3
FIG 3
Detailed mutagenesis scan on MeV H-I146. (A) Surface expression, fusion promotion activity, and SLAM binding of various MeV H-I146 mutations relative to the wild type. Mutants showing significant reduction in fusion promotion activity are color coded blue. Surface expression and SLAM binding results are derived from nine independent transfections. Fusion promotion capability was determined by quadruplicate measurements of three independent transfections. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.0001). (B) Qualitative fusion assay of selected MeV H-I146 mutants (cotransfected with MeV F-wt) in receptor-positive Vero-hSLAM cells.
FIG 4
FIG 4
Reciprocal coimmunoprecipitation of the CDV surface glycoproteins H and F. (A) Total lysate of transfected cells is shown (input) as well as immune-precipitated H (IP) and coimmunoprecipitated F (coIP). (B) Total lysate of transfected cells is shown (input) as well as immune-precipitated F (IP) and coimmunoprecipitated H (coIP). The Western blot shows F0 (∼65 kDa), F1 (∼45 kDa), and various H-I146 mutants (∼75 kDa).
FIG 5
FIG 5
Qualitative fusion assay after cotransfection of CDV H-I146 mutants along with either F-wt or F-V447T (hyperfusogenic F mutant) in receptor-negative Vero cells or receptor-positive Vero-cSLAM cells at various time points after fusion-inhibitory drug (3G) removal. 3G was removed 24 h posttransfection, and pictures were taken at the indicated time points.
FIG 6
FIG 6
Bioactivity investigation of headless H-stalk 1-159 proteins harboring I146 mutation in different assays. (A) Schematic representation of the H-stalk 1-159 construct. CO, connector domain. (B) Qualitative fusion assay after cotransfection of indicated CDV H constructs along with F-V447T in receptor-positive Vero-cSLAM cells. (C) Oligomerization pattern in a nonreducing surface immunoprecipitation experiment using H-stalk 1-159 wild-type or I146D sequences. Three wells of a 6-well plate were transfected per mutant. IB, immunoblot. (D) Qualitative fusion restoration experiment with the cotransfection of various CDV H mutants that are individually fusion-triggering deficient in combination with F-wt in receptor-positive Vero-cSLAM cells. H2, H-stalk 1-159 dimer (∼36 kDa); H4, H-stalk 1-159 tetramer (∼70 kDa); HO, H-stalk 1-159 higher oligomers. Pictures were taken 30 h posttransfection.
FIG 7
FIG 7
Modified oligomerization pattern of nonfunctional CDV and MeV H-I146 mutants. (A) Oligomerization pattern revealed by immunoblotting (IB) in a nonreducing surface immunoprecipitation experiment (IP) of various CDV H-I146 mutants. (B) Quantification of tetrameric and dimeric band intensities of CDV H-I146 mutants using the Aida Image Analysis software. (C) Oligomerization pattern revealed by IB in a nonreducing surface IP of various MeV H-I146 mutants. (D) Quantification of tetrameric and dimeric band intensities of MeV H-I146 mutants using the Aida Image Analysis software. H2, H dimer (∼170 kDa); H4, H tetramer (∼350 kDa); FPA, fusion promotion activity (red, nonfunctional; green, functional). For quantification shown in panels B and D, at least three independent experiments were analyzed per mutant H. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.005).
FIG 8
FIG 8
Investigation of H-I146 mutation-induced oligomeric changes and disulfide bond reduction experiment. (A) Model of wild-type H-linker (blue) contribution to oligomerization into dimer-of-dimers. Disulfide bonds between two natural dimers are indicated in green. Residue I146 is shown in pink. Mutation at position 146 of CDV and MeV H may force unnatural (indicated in red) disulfide bond formation to monomers belonging to another dimer via C139 (B) or C154 (C). Residue I146 mutated in either E or D is shown in orange. (D) Denaturation of potential noncovalent stable tetramers by increasing the boiling incubation time. H2, H dimer (∼170 kDa); H4, H tetramer (∼350 kDa). (E) Disulfide bond reduction experiment using DTT in combination with F-wt in receptor-positive Vero-cSLAM cells to evaluate if covalent oligomerization is the cause of fusion promotion deficiency in H-I146 mutants. H-L105C is the positive control for fusion promotion restoration after DTT treatment.
FIG 9
FIG 9
Validation of phenotypes observed for C139A or C154A harboring mutant H proteins. Qualitative fusion assay after cotransfection of CDV H-I146 mutants additionally having C139 or C154 mutated into alanine along with either F-wt (A) or F-V447T (B) in receptor-positive Vero-cSLAM cells. (C) Surface expression and fusion promotion (in combination with F-wt) of various CDV H mutants relative to the wild type. Surface expression results are derived from nine independent transfections. Fusion promotion capability was determined by quadruplicate measurements of three independent transfections. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.0001). (D) Oligomeric profile of cysteine-to-alanine mutants under nonreducing, denaturing conditions. (E) Quantification of tetrameric and dimeric band intensities of CDV H-I146 mutants using the Aida Image Analysis software. Note that this gel and the one presented in Fig. 10 are identical. For clarification and better interpretation of the data, the gel was divided into two parts. The black line indicates where the image has been cropped for better presentation. For quantification, at least three independent experiments were analyzed per mutant H. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.005).
FIG 10
FIG 10
Investigation of CDV H-I146G phenotypes. (A) Qualitative fusion assay of CDV H-I146G with F-wt in receptor-positive Vero-cSLAM cells. (B) Quantification of SLAM binding, fusion promotion activity, and surface expression of CDV H-I146G mutant proteins relative to the wild type. Surface expression results are derived from nine independent transfections, and fusion promotion capability was determined by quadruplicate measurements of three independent transfections. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.001). (C) Oligomeric patterns of CDV H-I146G under nonreducing but denaturing conditions. (D) Quantification of tetrameric and dimeric band intensities of the CDV H-I146G mutant using the Aida Image Analysis software. This gel and the one presented in Fig. 9 are identical. For clarification and better interpretation of the data, the gel was divided into two parts. For quantification, at least four independent experiments were analyzed. The means and the standard deviations from the means are shown. An unpaired two-tailed t test was performed to assess significant differences compared to the wild type (*, P value of ≤0.005).
FIG 11
FIG 11
Potential mode of action of morbillivirus linker domain in H-mediated F-triggering activity. For simplicity, only two out of four linkers are shown. The cysteines Cys188 and Cys602 form intramonomer disulfide bonds. (A) Model of H in prereceptor binding state showing the potential hydrophobic hinge region within the linker. The hydrophobic hinge may be essential in preserving the autorepressed state prior to receptor binding. (B) Model of H in postreceptor binding state where one potential hinge region was refolded during the fusion activation signal transduction. Repositioning of H heads with regard to the stalk may in turn enable stalk destabilization and refolding within the central region (not shown). Pm, plasma membrane.
FIG 12
FIG 12
Robetta tertiary structure prediction for the CDV H amino acids 129 to 607 based on the crystal structure of the measles virus hemagglutinin (PDB entry 2ZB6) by the Ginzu domain prediction method. (A) Amino acids 129 to 607 are shown. Cysteines naturally responsible for covalent dimer formation are shown as yellow sticks. (B) Predicted hydrophobic hinge region involving I146. Hydrophobic amino acids potentially involved in hydrophobic patch formation are shown as sticks.

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