Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles.
Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1(MN) by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of alpha and beta heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.
Trubey
Charles M
CM
Basic Research Program. Research Technology Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA.
Chertova
Elena
E
Coren
Lori V
LV
Hilburn
Joanne M
JM
Hixson
Catherine V
CV
Nagashima
Kunio
K
Lifson
Jeffrey D
JD
Ott
David E
DE
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000276:immunology* / Q000302:isolation & purification / Q000472:pathogenicity / Q000648:ultrastructure; D000949:Histocompatibility Antigens Class II / Q000032:analysis* / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000276:immunology*; D008854:Microscopy, Electron; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D014771:Virion / Q000276:immunology* / Q000302:isolation & purification / Q000472:pathogenicity / Q000648:ultrastructure
D000949:Histocompatibility Antigens Class II; D017493:Leukocyte Common Antigens; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1