Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages

Abstract

Human immunodeficiency virus type 1 (HIV-1) infects CD4(+) T lymphocytes and monocytes/macrophages, incorporating host proteins in the process of assembly and budding. Analysis of the host cell proteins incorporated into virions can provide insights into viral biology. We characterized proteins in highly purified HIV-1 virions produced from human monocyte-derived macrophages (MDM), within which virus buds predominantly into intracytoplasmic vesicles, in contrast to the plasmalemmal budding of HIV-1 typically seen with infected T cells. Liquid chromatography-linked tandem mass spectrometry of highly purified virions identified many cellular proteins, including 33 previously described proteins in HIV-1 preparations from other cell types. Proteins involved in many different cellular structures and functions were present, including those from the cytoskeleton, adhesion, signaling, intracellular trafficking, chaperone, metabolic, ubiquitin/proteasomal, and immune response systems. We also identified annexins, annexin-binding proteins, Rab proteins, and other proteins involved in membrane organization, vesicular trafficking, and late endosomal function, as well as apolipoprotein E, which participates in cholesterol transport, immunoregulation, and modulation of cell growth and differentiation. Several tetraspanins, markers of the late endosomal compartment, were also identified. MDM-derived HIV contained 26 of 37 proteins previously found in exosomes, consistent with the idea that HIV uses the late endosome/multivesicular body pathway during virion budding from macrophages.

FIG. 1.

Analysis of CD45-depleted HIV. (A) Immunoblots of CD45-depleted virion preparations (equal amounts by volume) isolated from parallel uninfected (−) and infected (+) cell cultures are presented. Samples from the immunoaffinity-depleted supernatant (Sup) and anti-CD45 bead fractions are identified above their respective lanes. Antibody or antiserum used is indicated above each blot. IgL, immunoglobulin light chain. (B) An analytical Coomassie blue-stained SDS-PAGE gel of CD45-depleted uninfected (−) and HIV-infected (+) preparations (equal amounts by volume) produced from MDM is shown.

FIG. 2.

Electron microscopy of HIV-1. Thin-section transmission electron micrographs of untreated and CD45-depleted virus preparations are shown. Samples are identified above their respective micrographs. A white 200-nm bar has been added to each micrograph for reference.

FIG. 3.

Fractionated preparative SDS-PAGE gel. The Coomassie blue-stained preparative SDS-PAGE (4 to 20%) gel of the CD45-depleted virus sample is shown. Gel fractions taken are indicated at the right and the positions of molecular mass markers are denoted at the left.

FIG. 4.

CD63 and Tsg101 immunoblots of MDM-derived HIV-1. Immunoblots of virion preparations (equal amounts by volume with approximately 200 ng p24^CA in the virion sample) isolated from parallel uninfected and HIV-1_NLAD8-infected MDM cultures are shown. The samples are identified above their respective lanes. The antibody or antiserum used is indicated above each blot. For the CD63 immunoblot, a lane containing a cell lysate control from uninfected MDM (lysate from 5 × 10⁵ cells) is included. frg, fragment.

FIG. 5.

Env content analysis of MDM-derived HIV-1. (A) An HPLC chromatogram (A₂₀₆) of HIV-1_ADA produced from MDM is shown. Reversed-phase HPLC of denatured virions was performed as described in Materials and Methods. Viral protein peaks (identified by SDS-PAGE gels) are labeled above the chromatograph, and Coomassie blue-stained SDS-PAGE gel analysis of pertinent corresponding fractions is shown below, with positions of the molecular mass markers denoted at the left. (B) A two-dye SYPRO-stained SDS-PAGE gel is shown. Samples are identified above their respective lanes. For the recombinant gp120^SU (rgp120^SU) and purified p24^CA standards, the amount of material loaded in each lane (in ng) is indicated above each lane. The relative loads, by volume, for the HPLC fractions are indicated in parentheses on the lane labels. The images of these standards have been digitally overlaid and merged for presentation purposes. The relative intensities of these images have not been altered. The positions of molecular mass markers are denoted at the left. p6^f, an HIV protease cleavage fragment of p6; Fr., fraction.