Epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: amount of Vpr in relation to Gag in HIV-1.
We used an epitope-tagging approach to determine the ratio of Gag (structural) to Vpr (nonstructural) in the virus particles directed by human immunodeficiency virus type 1. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK), and the sequences corresponding to the chimeric protein were introduced into human immunodeficiency virus type 1 proviral DNA (NL4-3) to determine the ratio in the virus particles when these proteins are expressed in cis. In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL when it is expressed in trans through a heterologous promoter. The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated that (1) the ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis (in the context of proviral DNA), is in the range of 150-200:1 (14-18 molecules of Vpr per virion) and (2) the expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1 (392-550 molecules of Vpr). These results suggest that the presence of the same epitope on different viral proteins may provide an accurate comparison of these proteins in the virus particles.
Singh
S P
SP
Department of Microbiology, Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, Philadelphia, Pennsylvania, 19107, USA.
Lai
D
D
Cartas
M
M
Serio
D
D
Murali
R
R
Kalyanaraman
V S
VS
Srinivasan
A
A
D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000378:metabolism*; D016336:Gene Products, vpr / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000138:chemical synthesis / Q000276:immunology; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D016336:Gene Products, vpr; D016655:HIV Core Protein p24; D009842:Oligopeptides; D010455:Peptides; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C085706:FLAG peptide