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Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations
J W Bess1, R J Gorelick, W J Bosche
1AIDS Vaccine Program, SAIC, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
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.
Insights
Cellular microvesicles, not HIV-1, often contaminate virus samples. These microvesicles contain cellular proteins like HLA DR and beta 2-M, complicating HIV-1 protein analysis.
Area of Science:
- Virology
- Cell Biology
- Biochemistry
Background:
- Accurate identification of HIV-1 proteins is hindered by copurifying cellular proteins.
- Cellular microvesicles share density properties with retroviruses, complicating purification.
- Microvesicles contain cellular proteins, RNA, and DNA, mimicking viral components.
Purpose of the Study:
- To characterize microvesicles produced by HIV-1-infected and uninfected human T-cells.
- To differentiate microvesicles from HIV-1 particles.
- To investigate the protein content of microvesicles and their impact on HIV-1 analysis.
Main Methods:
- Propagation of HIV-1-infected and uninfected human T-cell lines.
- Purification of virus and microvesicles using sucrose density gradient centrifugation.
- Quantification of HIV-1 p24CA, HLA DR, and beta 2-microglobulin (beta 2-M) via radioimmunoassay.
- Electron microscopic analysis of microvesicles.
Main Results:
- Microvesicles contain cellular proteins (e.g., HLA DR, beta 2-M) and nucleic acids.
- Ratios of HIV-1 p24CA to HLA DR and beta 2-M varied based on HIV-1 isolate and host cell.
- Microvesicles from HIV-1 infected cells showed minimal HIV-1 gp120SU.
Conclusions:
- Microvesicles are a significant source of cellular protein contamination in HIV-1 preparations.
- Distinguishing microvesicles from HIV-1 is crucial for accurate viral protein quantitation.
- Understanding microvesicle composition aids in refining methods for studying HIV-1 biology.