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Journal of Virology, December 2003, p. 12699-12709, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12699-12709.2003

Quantitation of HLA Class II Protein Incorporated into Human Immunodeficiency Type 1 Virions Purified by Anti-CD45 Immunoaffinity Depletion of Microvesicles

Charles M. Trubey,1 Elena Chertova,1 Lori V. Coren,1 Joanne M. Hilburn,1 Catherine V. Hixson,1 Kunio Nagashima,2 Jeffrey D. Lifson,1 and David E. Ott1*

Basic Research Program,1 Research Technology Program, SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, Maryland 21702-12012

Received 15 April 2003/ Accepted 26 August 2003

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-1MN 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 ß 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.

* Corresponding author. Mailing address: Basic Research Program SAIC-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201. Phone: (301) 846-5723. Fax: (301) 846-5588. E-mail: ott{at}ncifcrf.gov.

Journal of Virology, December 2003, p. 12699-12709, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12699-12709.2003




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