Microvesicles Are a Source of Contaminating Cellular Proteins Found in Purified HIV-1 Preparations

Abstract

Identification and quantitation of cellular proteins associated with HIV-1 particles are complicated by the presence of nonvirion-associated cellular proteins that copurify with virions. Many cellular proteins are associated with nonviral particles that bud from the surface of cells called microvesicles. Microvesicles band in sucrose gradients in a range of densities that includes the same density as retroviruses. To characterize these microvesicles, HIV-1-infected and uninfected human T-cell lines were propagated and virus and microvesicles were purified from clarified cell culture supernatants by sucrose density gradient centrifugation or centrifugation through 20% sucrose pads. Microvesicles were found to contain various proteins, including HLA DR and beta 2-M, and a substantial amount of RNA and DNA. The concentrations of HIV-1 p24CA, HLA DR and beta 2-microglobulin (beta 2-M) were determined by radioimmunoassay. The ratios of HIV-1 p24CA to HLA DR and beta 2-M were found to vary with respect to the HIV-1 isolate, host cell, and other factors. Electron microscopic analysis of microvesicles revealed that they consisted of particles of various sizes and morphologies. Although HIV-1 particles are known to contain some cellular proteins, microvesicles from HIV-1 infected H9 cells appeared to contain little or no HIV-1 gp120SU.

Evidence That HIV Budding in Primary Macrophages Occurs through the Exosome Release Pathway

“…We have not been able to separate exosomes from virions produced by these cells. Our experience is consistent with that of others using cell lines (55) in which microvesicles (most likely exosomes) co-purify with virions.…”

“…Gag, HIV core antigen. (55). 2 This makes interpretation of Western blotting data obtained with such preparations problematic.…”

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands

“…Purified LPS from Escherichia coli 055:B5 (TLR4 agonist) was purchased from Sigma (St Louis, MO). Noninfectious Aldithriol-2 (AT-2) treated HIV-1 Ada (R5-tropic) and HIV-1 MN (X4-tropic) viral preparations along with matched control microvesicles, SUPT1-CCR5 (MV-CCR5) and CEMX174 (T1) (MV-X4), isolated from uninfected cell cultures (34) were kindly provided by Dr. Jeff Lifson from the AIDS Vaccine Program, National Cancer Institute (Frederick, MD).…”

HIV Type 1 Nef Is Released from Infected Cells in CD45+Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

“…Cells infected with HIV commonly release both virions and microvesicles that copurify with the virions due to similarity in size and density. 49,50 Some studies have separated microvesicles from HIV-1 virions using (Optiprep) iodixonal gradients clearly showing the capacity to discriminate between microvesicles, virions, and cell debris. 51,52 CD45 (human leukocyte antigen) is highly expressed on the surface of vesicles derived from hematopoietic cells but is not incorporated into HIV-1 virions.…”