The Role of Nef-mu1 Interactions in the Modulation of MHC I
Colleen Noviello, Veterans Medical Research Foundation, San Diego
Biomedical and Clinical Sciences
Background: HIV-1 evades cellular adaptive immunity by Nefmediated removal of class I major histocompatibility complex (MHC I) from the cell surface. Nef binds the clathrin adaptor protein-1 complex (AP-1), both through the hemi-complex of gamma-sigma subunits and mu1 subunit alone. While the gamma-sigma hemi-complex interaction is dependent upon the acidic-dileucine motif found in the unstructured C-terminus of Nef, the mu1 interaction is independent of this motif. Recently, a ternary interaction has been revealed between the AP-1 complex, Nef, and the MHC I molecule itself. Since the dileucine motif is not required for the modulation of MHC I, we propose that the Nef-mediated interaction with the mu1 subunit of AP1 is responsible for this effect. This idea is strengthened by the existence of a putative "cryptic" tyrosine-based sorting signal in the cytoplasmic tail of MHC I; the tyrosine in this sequence is required for the Nef effect, and similar tyrosine-based signals in cellular proteins bind to the mu subunits of AP complexes. Thus, we hypothesized that the MHC I cytoplasmic tail binds to the ? subunit of AP-1 in the presence of Nef, which binds to both mu1 and the MHC I tail.
Methods: We tested this hypothesis by measuring protein-protein interactions using yeast two- and three-hybrid assays. We also utilized the GST-pulldown system, expressing GST-tagged proteins in E. coli to use as the "bait", and using HeLa cell lysate as the source for cellular proteins.
Results: We have not detected an interaction between the cytoplasmic tail of MHC I and Nef or between the tail and mu1 in the yeast two-hybrid system. We have also not detected a ternary interaction between the MHC-I tail, Nef and mu1 using the three-hybrid system. However, we observed that the MHC-I tail, when fused to Nef, increases the efficiency of interaction with intact AP-1 as measured in GST pull-down experiments. Interestingly, the MHC-1 tail does not render the interaction between Nef and AP-1 independent of the Nef dileucine motif. We are currently determining whether the contribution of the MHC-I tail to the interaction with AP-1 requires the tyrosine residue necessary for the modulation of MHC-I by Nef.
Conclusions: We will use the MHC-I tail/Nef chimera in our yeast two- and three-hybrid assays to determine whether the contribution of the tail to the interaction with AP-1 is due to an increased interaction with the mu1 subunit or with the hemi-complex of gamma-sigma subunits. These data should allow us to elaborate a more precise model for the ternary interaction between MHC-I, Nef, and AP-1.