Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation.
vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.
Mahalingam
S
S
Institute of Biotechnology and Advanced Molecular Medicine, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Khan
S A
SA
Murali
R
R
Jabbar
M A
MA
Monken
C E
CE
Collman
R G
RG
Srinivasan
A
A
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D004355:Drug Stability; D016336:Gene Products, vpr / Q000737:chemistry* / Q000378:metabolism*; D016334:Genes, vpr; D018698:Glutamic Acid; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014771:Virion / Q000378:metabolism*; D054325:vpr Gene Products, Human Immunodeficiency Virus
D016336:Gene Products, vpr; D011994:Recombinant Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D001224:Aspartic Acid; D018698:Glutamic Acid