Mechanisms of HIV-1 Nef function and intracellular signaling

Abstract

Advances in the last several years have enhanced mechanistic understanding of Nef-induced CD4 and MHCI downregulation and have suggested a new paradigm for analyzing Nef function. In both of these cases, Nef acts by forming ternary complexes with significant contributions to stability imparted by non-canonical interactions. The mutational analyses and binding assays that have led to these conclusions are discussed. The recent progress has been dependent on conservative mutations and multi-protein binding assays. The poorly understood Nef functions of p21 activated protein kinase (PAK2) activation, enhancement of virion infectivity, and inhibition of immunoglobulin class switching are also likely to involve ternary complexes and non-canonical interactions. Hence, investigation of these latter Nef functions should benefit from a similar approach. Six historically used alanine substitutions for determining structure-function relationships of Nef are discussed. These are M20A, E62A/E63A/E64A/E65A (AAAA), P72A/P75A (AXXA), R106A, L164A/L165A, and D174A/D175A. Investigations of less-disruptive mutations in place of AAAA and AXXA have led to different interpretations of mechanism. Two recent examples of this alternate approach, F191I for studying PAK2 activation and D123E for the critical residue D123 are discussed. The implications of the new findings and the resulting new paradigm for Nef structure-function are discussed with respect to creating a map of Nef functions on the protein surface. We report the results of a PPI-Pred analysis for protein-protein interfaces. There are three predicted patches produced by the analysis which describe regions consistent with the currently known mutational analyses of Nef function.

Figure 1. PPI-Pred analysis of Nef

(A) Three dimensional representation of Nef (PDB 1AVZ, Chain B) highlighting consensus regions of predicted protein-protein interaction resulting from PPI-Pred analysis. The patch analysis returns three regions exhibiting properties of protein-protein interfaces ranked by probability. All five Nef molecules from three separate crystal structures (PDBs 1AVZ, 1AVV and 1EFN) were analyzed. In addition, three individual models from the Nef NMR structure, 2NEF, were analyzed (Grzesiek et al., 1996b; Lee et al., 1996; Arold et al., 1997; Grzesiek et al., 1997; Trible et al., 2007). The three models were selected by having the lowest, highest or median RMS scored when aligned with the 1AVZ, Chain 1 crystal structure. Overlapping regions identified in each analysis were compared, and consensus residues were identified by occurrence in a specific region in at least four of the eight structures analyzed. The consensus regions were mapped onto 1AVZ, Chain B to be able to show residues 71, 72, and 73, which are missing in 1AVZ, Chain A. Residues of interest from biochemical data are shown in white ball and stick under a transparent surface. Residues of interest in the red region-L100, R106, I109, L112, W113, H116- correspond to six of eight residues identified in homodimerization docking experiments by ClusPro analysis. P72, Q73, V74, P75, L76, and R77 in the blue region represent the six residues in the proline helix of the Nef SH3 binding domain. Two residues, V85 and F191, of the green region are two of the four residues of a proposed protein interaction interface critical for PAK2 activation. F139, F191, H193, and R196 are found in a crystallization interface in cubic crystals (Arold et al., 2000). (B) Residues of each region are listed. A few residues are common between the regions. These are P78, H116 and F121.