Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest.
Human immunodeficiency virus (HIV) Vpr contributes to nuclear import of the viral pre-integration complex and induces G(2) cell cycle arrest. We describe the production of synthetic Vpr that permitted the first studies on the structure and folding of the full-length protein. Vpr is unstructured at neutral pH, whereas under acidic conditions or upon addition of trifluorethanol it adopts alpha-helical structures. Vpr forms dimers in aqueous trifluorethanol, whereas oligomers exist in pure water. (1)H NMR spectroscopy allows the signal assignment of N- and C-terminal amino acid residues; however, the central section of the molecule is obscured by self-association. These findings suggest that the in vivo folding of Vpr may require structure-stabilizing interacting factors such as previously described interacting cellular and viral proteins or nucleic acids. In biological studies we found that Vpr is efficiently taken up from the extracellular medium by cells in a process that occurs independent of other HIV-1 proteins and appears to be independent of cellular receptors. Following cellular uptake, Vpr is efficiently imported into the nucleus of transduced cells. Extracellular addition of Vpr induces G(2) cell cycle arrest in dividing cells. Together, these findings raise the possibility that circulating forms of Vpr observed in HIV-infected patients may exert biological effects on a broad range of host target cells.
Henklein
P
P
Humboldt University, Institute of Biochemistry, 10115 Berlin, Germany.
Bruns
K
K
Sherman
M P
MP
Tessmer
U
U
Licha
K
K
Kopp
J
J
de Noronha
C M
CM
Greene
W C
WC
Wray
V
V
Schubert
U
U
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism* / Q000821:virology; D002942:Circular Dichroism; D019281:Dimerization; D016195:G2 Phase; D016336:Gene Products, vpr / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D021381:Protein Transport; D012542:Scattering, Radiation; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014270:Trifluoroethanol / Q000494:pharmacology; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; D010446:Peptide Fragments; D054325:vpr Gene Products, Human Immunodeficiency Virus; D014270:Trifluoroethanol