Gomez CY, Hope TJ; Conference on Retroviruses and Opportunistic Infections (11th : 2004 : San Francisco, Calif.).
Program Abstr Conf Retrovir Oppor Infect 11th 2004 San Franc Calif. 2004 Feb 8-11; 11: abstract no. 66.
Univ. of Illinois at Chicago, USA
BACKGROUND: Studies have shown that several cellular factors play key roles in the assembly and budding processes in HIV. In addition, the assembly of viral proteins varies among different cell types. We have shown that the HIV matrix protein accumulates in actin-dependent structures and we hypothesize that this may represent an intermediate in the assembly process. To further develop this hypothesis we have observed the assembly of full-length Gag polyprotein in live cells by deconvolution microscopy.METHODS: We have used live cell imaging of cells expressing p55-GFP. DNA encoding a p55-GFP fusion protein was microinjected into the nuclei of cells plated on glass. This led to the expression the fusion protein 2 to 4 hours after microinjection. We could then follow the behavior of newly expressed p55-GFP in live cells by time-lapse microscopy. This was done in several different cell types including Helas, Hos, primary human fibroblasts, and monocyte derived macrophages.RESULTS: Approximately 4 to 5 hours after injection the first aggregates of p55-GFP on the cell membrane appear at the glass surface and are primarily immobile and likely represent budding virus. As time progresses, dynamic blooms of p55-GFP aggregates appear on the outer edges of the cell membrane. These blooms appear to move toward the center of the cell and often pile up in a small region of the cell. This trend is held in all of the cell types we have mentioned, however, p55-GFP expressed in monocyte-derived macrophages exhibits atypical behavior. In these cells, VLP are seen in unique vesicles throughout the cell, and quite often these particles are moving inside intracellular compartments by Brownian motion.CONCLUSIONS: Our observations reveal that the blooms of p55-GFP assembly are taking place in membrane ruffle structures, which are known to form at the cell periphery and migrate toward the center of the cell. These results suggest that areas of dynamic membrane increase localized concentration of Gag molecules, thereby facilitating the assembly process. Facilitation of assembly in this way would cause virus release at regions of cell-cell contact. The observations of p55-GFP expressed in macrophages support this hypothesis. In addition, the presence of freely moving p55-GFP particles inside of an undefined cellular compartment suggests that there is an alternate mechanism for assembly, budding, and release specific to monocyte derived macrophages.
Publication Types:
Keywords:
- AIDS Vaccines
- Actins
- Blood Proteins
- Cell Membrane
- Gene Products, gag
- Genes, gag
- Green Fluorescent Proteins
- HIV
- HIV Protease
- Hela Cells
- Humans
- MPP1 protein, human
- Macrophages
- Membrane Proteins
- Recombinant Fusion Proteins
- genetics
- reverse transcriptase, Human immunodeficiency virus 1
Other ID:
UI: 102270792
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