Structural Characterization of the HIV-1 Vpr N Terminus

Abstract

The 96-residue human immunodeficiency virus (HIV) accessory protein Vpr serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G 2 cell cycle arrest, and modulation of HIV-induced apoptosis. Using a combination of dynamic light scattering, circular dichroism, and NMR spectroscopy the N terminus of Vpr is shown to be a unique domain of the molecule that behaves differently from the C-terminal domain in terms of self-association and secondary structure folding. Interestingly, the four highly conserved proline residues in the N terminus are predicted to have a high propensity for cis/trans isomerism. Thus the high resolution structure and folding of a synthetic N-terminal peptide (Vpr 1-40 ) and smaller fragments thereof have been investigated. 1 H NMR data indicate Vpr 1-40 possesses helical structure between residues 17-32, and for the first time, this helix, which is bound by proline residues, was observed even in aqueous solution devoid of any detergent supplements. In addition, NMR data revealed that all of the proline residues undergo a cis/ trans isomerism to such an extent that ϳ40% of all Vpr molecules possess at least one proline in a cis conformation. This phenomenon of cis/trans isomerism, which is unprecedented for HIV-1 Vpr, not only provides an explanation for the molecular heterogeneity observed in the full-length molecule but also indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, particularly as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers. This prediction correlates well with our recent observation

Cyclophilin A Interacts with HIV-1 Vpr and Is Required for Its Functional Expression

“…Recombinant CypA (Sigma) was immobilized at a concentration ranging from 4200 to 11200 response units using standard amine-coupling chemistry in three flow cells, and a further flow cell without CypA was used as a control. The peptides Vpr , Vpr [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , Vpr [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] , and respective proline mutants were dissolved at concentrations ranging from 1 to 250 M in a running buffer (10 mM Hepes, 150 mM NaCl, 50 M EDTA, 0.005% Tween 20, pH 7.4) and were injected over the flow cells at a flow rate of 5 l/min. Data were collected at a rate of 2.5 Hz during the 120-s association and dissociation phase.…”

“…in Vivo-Our recent finding (19) of unusually large amounts of cis proline conformations about the imidic bond for two of the proline residues in the N terminus of Vpr suggested that Vpr folding may require a PPIase enzyme activity. Since both Vpr and CypA are present in HIV-1 virions, and CypA binds to HIV-1 CA and promotes virus infectivity, we investigated whether Vpr and CypA also interact.…”

“…In initial experiments we were unable to obtain reliable BIAcore data using fulllength sVpr under a variety of experimental conditions, presumably because of the intrinsic tendency of Vpr to self-aggregate (13,20,49). Hence, in subsequent BIAcore studies we focused on the analysis of the N-terminal domain of Vpr, exemplified by the peptide Vpr 1-40 that exists as a monomer in solution and contains two proline residues in positions 14 and 35 with a high content of the cis conformation of the imidic bond (19).…”

“…These studies were performed in aqueous solution that required the presence of either detergents or micelles to detect the most structured forms of Vpr. The secondary structures in Vpr emerging from these analyses (summarized in the accompanying paper (19)) suggest the presence of an ␣-helix-turn-␣-helix motif between residues 17 and 48 and an amphipathic ␣-helix between amino acids 53-55 and 78 -83 (12,15). These helices most likely play a key role in self-association and the interaction of Vpr with heterologous proteins (13,20).…”

“…3). Furthermore, we have recently focused our attention on the N-terminal domain of Vpr (19) and found that two of the four conserved prolines at residues 14 and 35 in this domain exhibit an unusually high content of cis-conformations of the imidic bond. Based on the cis/trans phenomenon that could be important for the folding of Vpr, it was projected that a potential interaction between Vpr and a host peptidyl-prolyl isomerase (PPIase) might regulate the interconversion of the imidic bond of these N-terminal proline residues of Vpr in vivo.…”