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Measles virus protein interactions in yeast: new findings and caveats
M Chen1, J C Cortay, D Gerlier
1Immunité et Infections Virales, CNRS-UCBL UMR5537, IFR 62 Laennec, 69372 Cedex 08, Lyon, France.
Abstract:
Complementary DNA clones of measles virus N, N (S228Q; L229D), Ncore (N1-400), Ntail (N401-525), P, PNT (P1-230), PCT (P231-507), L, MEL (L800-2183) and EL (L1300-2183) were fused in frame downstream of the Gal4 binding domain (BD) or activating domain (AD). All but BD-L, BD-MEL and BD-EL, were detected by western blot, with additional C- and/or N-terminal truncated products in the case of BD-N, and BD-P. BD-P and BD-PNT directly activated the reporter genes, indicating that the PNT domain displays transactivating properties. In yeast two-hybrid assays, PNT and PCT domains bind to Ncore and Ntail domains, respectively, indicating that N and P interact in a head to tail orientation via two independent binding sites. BD-N (S228Q; L229D) and AD-N displayed no or poor interaction with P proteins possibly because they may not be properly folded. L binding site on P lies within the PCT domain, and two PCT binding sites lie within the L1-799 and L800-1300 regions. Thus, N to P and P to L protein interactions in measles virus shared many features with other related Paramyxoviridae. From a human cDNA library, several candidate partners of N protein were identified which all reacted with BD-Ncore, and RNA was found to bridge the N protein with one partner.
Insights
Measles virus nucleocapsid (N) and phosphoprotein (P) interact head-to-tail via specific domains. RNA bridges the N protein with potential partners, revealing new insights into measles virus assembly.
Area of Science:
- Virology
- Molecular Biology
- Protein-Protein Interactions
Background:
- Measles virus is a significant human pathogen.
- Understanding the interactions between measles virus proteins is crucial for developing antiviral strategies.
- The nucleocapsid (N) and phosphoprotein (P) are key components of the viral ribonucleoprotein complex.
Purpose of the Study:
- To investigate the interaction domains between measles virus N and P proteins.
- To identify binding sites for the L polymerase protein on the P protein.
- To explore potential interactions of the N protein with host factors.
Main Methods:
- Yeast two-hybrid assays were employed to study protein-protein interactions.
- Complementary DNA (cDNA) clones encoding various measles virus protein domains were generated.
- Western blotting was used to confirm protein expression and integrity.
Main Results:
- The PNT and PCT domains of the P protein interact with the Ncore and Ntail domains of the N protein, respectively, indicating a head-to-tail orientation.
- Specific binding sites within the P protein (PCT domain) and L protein (L1-799 and L800-1300 regions) for L polymerase interaction were identified.
- Mutated N proteins showed reduced interaction with P proteins, suggesting potential folding issues.
- RNA was identified as a bridging molecule between the N protein and a host cell partner.
Conclusions:
- Measles virus N and P proteins interact in a head-to-tail manner through distinct binding sites.
- The identified interaction interfaces between N, P, and L proteins share similarities with other Paramyxoviridae viruses.
- The discovery of RNA bridging the N protein to a host factor opens new avenues for understanding viral replication and assembly.
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