Related Experiment Videos
Two amino acid substitutions in the SIV Nef protein mediate associations with distinct cellular kinases
S A Barber1, M F Maughan, J W Roos
1Division of Comparative Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland, 21205, USA.
Abstract:
A functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity.
Insights
Simian immunodeficiency virus (SIV) Nef protein enhances viral replication and infectivity by interacting with cellular kinases. This study identifies specific Nef amino acids crucial for these kinase interactions but finds no direct link to increased virion infectivity.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- A functional Nef protein is critical for Simian Immunodeficiency Virus (SIV) replication and pathogenesis in macaques.
- Both HIV and SIV Nef proteins are known to enhance virion infectivity, potentially by incorporating cellular kinases into viral particles.
- Previous studies identified distinct kinase associations for Nef proteins from different SIV clones (SIV/Fr-2 and SIV/17E-Fr).
Purpose of the Study:
- To identify specific amino acid differences in SIV Nef proteins responsible for kinase interactions and virion infectivity.
- To investigate the role of identified Nef amino acids in Nef/kinase interactions and Nef phosphorylation during SIV infection.
- To determine if specific Nef kinase associations correlate with enhanced virion infectivity.
Main Methods:
- Construction and analysis of a panel of SIV clones with specific amino acid reversions in the Nef protein.
- In vitro kinase assays to detect Nef-associated kinase activity.
- A novel infectivity assay using CEMX174 reporter cells and an SIV/LTR-luciferase construct.
Main Results:
- Previously uncharacterized amino acids in the N terminus and conserved core domain of SIV Nef are essential for Nef/kinase interactions and Nef phosphorylation.
- Despite differences in kinase associations between SIV/Fr-2 and SIV/17E-Fr Nef proteins, no direct correlation was found between specific Nef kinase associations and enhanced virion infectivity.
- The study identified key amino acids in Nef critical for kinase binding but not necessarily for infectivity enhancement.
Conclusions:
- Specific amino acid residues in the N-terminal and core domains of SIV Nef are indispensable for mediating Nef/kinase interactions and phosphorylation.
- The observed differences in kinase associations between SIV Nef variants do not directly translate to enhanced virion infectivity.
- Further research is needed to elucidate the precise mechanisms by which SIV Nef enhances virion infectivity independently of specific kinase associations.