Abstract
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.
Highlights
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
Since CD45 appears to be incorporated onto microvesicles and is apparently excluded from HIV-1 virions, microvesicles should be removed from virus preparations by a CD45 immunoaffinity approach
The study of cellular proteins incorporated into retroviruses is significantly hindered by the presence of microvesicles and their associated proteins in virus preparations purified by conventional methods
Summary
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. 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. 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. Human immunodeficiency virus type 1 (HIV-1) and other retroviruses incorporate a variety of cellular proteins in the process of assembly and budding (reviewed in reference 26). Microvesicles, which contain HLA-II but not Env, did not produce this effect, indicating a role for the CD4-Env interaction in this process Together, these results support a role for HLA-II on HIV-1 as a potential contributor to virus-mediated immunodeficiency. While microvesicles and virions have similar cellular protein compositions, the incorporation of host plasma membrane proteins into virions appears to have some specificity as some proteins appear to be excluded from the virion (reviewed in reference 26)
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