Related Experiment Video
Updated: Jun 4, 2026
A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein
Kathryn A Gravel1, Lori W McGinnes, Julie Reitter
1Department of Microbiology and Physiological Systems/Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Abstract:
The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity.
Insights
The transmembrane domain of Newcastle disease virus fusion protein is crucial for its structure and function. Specific sequences within this domain regulate protein interactions and membrane fusion activity.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- The Newcastle disease virus (NDV) fusion (F) protein mediates viral entry into host cells.
- The transmembrane (TM) domain of viral fusion proteins plays a critical role in their function.
- Understanding the specific sequence requirements of the NDV F protein TM domain is essential for elucidating viral entry mechanisms.
Purpose of the Study:
- To investigate the role of specific sequences within the NDV F protein's transmembrane domain.
- To determine how substituting the TM domain affects F protein structure, cell surface expression, and function.
- To assess the impact of TM domain alterations on interactions with the NDV hemagglutinin-neuraminidase (HN) protein and membrane fusion.
Main Methods:
- Site-directed mutagenesis to replace the NDV F protein TM domain with those from Sendai virus (SV) F protein, measles virus (MV) F protein, or vesicular stomatitis virus (VSV) G protein.
- Analysis of mutant protein expression, cell surface transport, and proteolytic cleavage.
- Assessment of membrane fusion and hemifusion activities.
- Investigation of complex formation between mutant F proteins and NDV HN protein using co-immunoprecipitation or similar assays.
- Conformational analysis of ectodomains using conformation-sensitive antibodies.
Main Results:
- Mutant F proteins with SV or MV F TM domains showed comparable expression and cleavage to wild-type, unlike those with the VSV G TM domain, which had reduced expression and cleavage.
- All TM domain substitution mutants were defective in membrane fusion and hemifusion.
- Mutant F proteins failed to form detectable complexes with the NDV HN protein.
- Conformation-sensitive antibody binding indicated altered ectodomain conformations in mutant proteins.
Conclusions:
- The specific sequence of the NDV F protein TM domain is critical for maintaining the pre-activation ectodomain conformation.
- The TM domain sequence influences the interaction between the NDV F and HN proteins.
- Proper TM domain sequence is essential for the fusion activity of the NDV F protein.
Related Concept Videos
01:26Insertion of Single-pass Transmembrane Proteins in the RER
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...
01:29Insertion of Multi-pass Transmembrane Proteins in the RER
The multipass transmembrane proteins are the type IV integral membrane proteins with multiple topogenic sequences determining their spatial arrangement in the ER membrane. Nearly all multipass proteins lack a cleavable signal sequence and use...
02:28Leaky Scanning
01:09Multi-pass Transmembrane Proteins and β-barrels
α-Helix containing multi-pass transmembrane proteins
Multi-pass transmembrane proteins such as G-protein-linked receptors (GPCRs) and...
01:25Single-pass Transmembrane Proteins
01:43SNAREs and Membrane Fusion
SNAREs exist in pairs that symmetrically interact and catalyze the fusion of the lipid bilayers in vesicle and target organelle. v-SNARE in the vesicle membrane are single polypeptide chains that bind to a complementary t-SNARE, composed of 2...