Abstract
The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.
Highlights
HIV-1 incorporates cellular proteins from the host cell during assembly and budding [1]. These proteins can provide important information about virus-cell interactions, yet biochemical analyses are greatly hindered by the presence of protein-laden vesicles in virion preparations, especially those produced by hematopoeitic cells
Vesicles can come from two sources: microvesicles that bud from the plasma membrane [4,5] and exosomes that form in late endosomal bodies and are released by exocytosis [6,7]
The SDS-PAGE gel results showed that the HIV-1 preparations from the SupT1/CCR5 cells contained a large amount of cellular proteins compared with the Jurkat E61 preparation (Figure 1B)
Summary
HIV-1 incorporates cellular proteins from the host cell during assembly and budding [1]. While we have consistently observed this in our experiments [12,13,14], two papers report that "exosomes" produced by Jurkat T cells, i.e. dense particles isolated from culture supernatants, do not contain CD45 [15,16], apparently excluding this protein during vesicle formation. The results showed that, similar to the CD45 finding, actin was not detectable in the depleted samples, but was present as an intense band in all three untreated samples as well as the bead fractions (Figure 1A).
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