Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis.
The product of the vpr open reading frame of human immunodeficiency virus type 1 (HIV-1) is a 15-kDa, arginine-rich protein that is present in virions in molar quantities equivalent to that of Gag. We report here the results of our investigations into the mechanism by which Vpr is incorporated into virions during assembly in infected cells. For these studies we used an expression vector encoding a Vpr molecule fused at its amino terminus to a nine-amino-acid peptide from influenza virus hemagglutinin. The tagged Vpr expression vector and a vpr mutant HIV-1 provirus were used to cotransfect COS cells, and the resulting virions were tested for the presence of the tagged protein on immunoblots probed with monoclonal antibody against the hemagglutinin peptide. The COS-produced virions were found to contain readily detectable amounts of tagged Vpr and smaller amounts of a putative tagged Vpr dimer. Infectivity of the particles was not altered by incorporation of tagged Vpr. Our results using this system in combination with mutant HIV-1 proviruses suggested that incorporation of Vpr into virions requires the carboxy-terminal Gag protein of HIV-1 (p6) but not gp160, Pol, or genomic viral RNA. In addition, analysis of mutated, tagged Vpr molecules suggested that amino acids near the carboxy terminus (amino acids 84 to 94) are required for incorporation of Vpr into HIV-1 virions. The single cysteine residue near the carboxy terminus was required for production of a stable protein. Arginine residues tested were not important for incorporation or stability of tagged Vpr. These results suggested a novel strategy for blocking HIV-1 replication.
Paxton
W
W
Aaron Diamond AIDS Research Center, New York, New York.
Connor
R I
RI
Landau
N R
NR
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics / Q000254:growth & development*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009024:Morphogenesis; D011533:Proviruses / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000254:growth & development*; D054325:vpr Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D016336:Gene Products, vpr; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D011993:Recombinant Fusion Proteins; D054325:vpr Gene Products, Human Immunodeficiency Virus; D001120:Arginine; D003545:Cysteine