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 G2 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 (Vpr1-40) and smaller fragments thereof have been investigated. 1H NMR data indicate Vpr1-40 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 approximately 40% 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 (Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, P., Luban, J., Neidleman, J., Greene, W. C., and Schubert, U. (2003) J. Biol. Chem. 278, 43170-43181) of a functional interaction between the major cellular isomerase cyclophilin A and Vpr, both of which are incorporated into HIV-1 virions.
Highlights
The 96-residue human immunodeficiency virus (HIV) accessory protein Viral protein R (Vpr) serves manifold functions in the retroviral life cycle including augmentation of viral replication in non-dividing host cells, induction of G2 cell cycle arrest, and modulation of HIV-induced apoptosis
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, provides an explanation for the molecular heterogeneity observed in the full-length molecule and indicates that in vivo the folding and function of Vpr should depend on a cis/trans-proline isomerase activity, as two of the proline residues in positions 14 and 35 show considerable amounts of cis isomers
To obtain further details of the structure of the full-length molecule we analyzed the previously characterized synthetic peptide synthetic full-length Vpr (sVpr) [20] that comprises the sequence of a Vpr protein derived from the isolate HIV-1NL4–3 [48]. 1H NMR spectra of sVpr were recorded in both water alone and in 50% TFE as this latter solution tends to stabilize secondary structure and alleviates to some extent problems associated with intermolecular interactions at concentrations necessary for NMR investigations [49]
Summary
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 G2 cell cycle arrest, and modulation of HIV-induced apoptosis. The molecular heterogeneity observed for sVpr may arise, at least in part, from cis/trans isomerism of proline residues located in the N-terminal region of the molecule at positions 5, 10, 14, and 35 (Fig. 1).
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