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Peptide-based Identification of Functional Motifs and their Binding Partners
Published on: June 30, 2013
Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix
Lillian S Kuo1, Laura L Baugh, Sarah J Denial
1Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Y9.206, Dallas, TX 75390, USA.
Retrovirology
|June 2, 2012
Summary
HIV-1 Nef protein interactions are crucial for viral pathogenicity. Mutations in its polyproline segment disrupt interactions with SH3 domain proteins, affecting viral infectivity and MHC Class I downregulation.
Area of Science:
- Virology
- Molecular Biology
- Protein-Protein Interactions
Background:
- HIV-1 Nef is essential for viral pathogenicity and functions via protein-protein interactions.
- A key interaction site involves the polyproline segment (P69VRPQVPLRP78) containing the PXXPXR motif, which binds SH3 domains.
- The study investigates the role of this motif and surrounding residues in Nef's interactions.
Purpose of the Study:
- To determine the impact of mutations within the Nef polyproline segment, specifically the PXXPXR motif and adjacent residues, on protein interactions.
- To elucidate the specific roles of different Nef residues in mediating interactions with SH3 domain proteins like Hck and PAK2.
- To investigate the contribution of these interactions to viral functions such as enhancement of virion infectivity (EVI) and MHC Class I downregulation.
Main Methods:
- Site-directed mutagenesis was used to alter specific proline, arginine, and non-motif residues within the Nef polyproline segment.
- Nef-SH3 domain binding was assessed by examining interactions with Hck and PAK2.
- Viral functions, including enhancement of virion infectivity (EVI) and MHC Class I downregulation, were measured in response to these mutations.
Main Results:
- Mutations in the PXXPXR motif (prolines and arginine) impaired Nef binding to Hck, Nef/PAK2 complex formation, and EVI.
- Mutations of non-motif 'X' residues (Q73, V74, L75) affected Nef/PAK2 complex formation and EVI differently than Hck binding, suggesting involvement of other SH3 proteins.
- A novel interaction locus (GFP/F: G67, F68, P69, F90) was identified for Nef/PAK2 complex formation and EVI.
- MHC Class I downregulation was partially inhibited by PXXPXR mutations but fully inhibited by C-terminal P78 mutations, requiring G67 and F68, and did not involve SH3 domain proteins.
Conclusions:
- Nef utilizes its polyproline helix to interact with multiple host cell SH3 domain proteins.
- Multifunctional protein interaction sites exist on both N- and C-terminal sides of the polyproline helix.
- Nef employs a non-canonical binding surface on its polyproline segment for MHC Class I downregulation, independent of SH3 domain proteins.
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