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Cytoskeletal Remodeling of the HIV-infected Macrophage: Proteomics and Functional Immunochemical Analysis of Giant Cell Formation
Irena Kadiu*, P Ciborowski, M Ricardo-Dukelow, and H Gendelman
Univ of Nebraska Med Ctr, Omaha, US
Background: The cytoskeleton‑particularly
filamentous actin, myosin, and microtubules‑affects cell life and death
processes during progressive HIV-1 infection. Macrophage and T-cell shape,
maintenance, polarity, movement, and intra- and inter-cellular protein
trafficking, as well as binding and entry of virus are directed by remodeling of
cytoskeleton. Little is known of the interactions between HIV and the
cytoskeleton during giant cell formation; a critical component of the
macrophage-directed encephalitis seen associated with progressive viral
infection.
Methods: A proteomics
platform consisting of surface-enhanced laser desorption ionization time of
flight (SELDI-TOF), 1D SDS-PAGE, in-gel tryptic digestion, and liquid
chromatography tandem mass spectrometry (LC-MS/MS) were used to quantitatively
and qualitatively identify proteins in the secretome of HIV-1-infected human
monocyte-derived macrophages (MDM). MDM were infected with HIV-1ADA
at a multiplicity of 1 infectious viral particles/cell. Albumin-depleted cell
supernatants and lysates, were collected at days 3, 5, and 10. Sucrose-banded
virus and exosomes were also isolated. Quantitative immunoblotting and confocal
microscopy were performed for protein confirmation and co-localization of viral
and cellular proteins.
Results: SELDI-TOF and
LC/MS-MS demonstrated significant increases in actin and profiling 1 in the
secretome of infected cells over controls that were confirmed by
immunoblotting. Protein levels in cell lysates were unchanged. Serum starvation
with serial 8- by 48-hour secretome analyses showed that the cytoskeletal
proteins were contained in exosomes and secreted following the peaks of reverse
transcriptase activity but not packaged into progeny virions. Confocal analysis
demonstrated remodeling of the cytoskeleton to the plasma membrane during the
formation of giant cells.
Conclusions: The macrophage cytoskeleton is remodeled as a result of
progressive viral infection and appears to act as a cytoplasmic effector of the
virus, the host cell, and its ultimate fate.
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