Quantitative Analysis of Human Immunodeficiency Virus Type 1-Infected CD4⁺Cell Proteome: Dysregulated Cell Cycle Progression and Nuclear Transport Coincide with Robust Virus Production

Abstract

Relatively little is known at the functional genomic level about the global host response to human immunodeficiency virus type 1 (HIV-1) infection. Microarray analyses by several laboratories, including our own, have revealed that HIV-1 infection causes significant changes in host mRNA abundance and regulation of several cellular biological pathways. However, it remains unclear what consequences these changes bring about at the protein level. Here we report the expression levels of ϳ3,200 proteins in the CD4 ؉ CEMx174 cell line after infection with the LAI strain of human immunodeficiency virus type 1 (HIV-1); the proteins were assessed using liquid chromatography-mass spectrometry coupled with stable isotope labeling and the accurate mass and time tag approach. Furthermore, we found that 687 (21%) proteins changed in abundance at the peak of virus production at 36 h postinfection. Pathway analysis revealed that the differential expression of proteins was concentrated in select biological pathways, exemplified by ubiquitin-conjugating enzymes in ubiquitination, carrier proteins in nucleocytoplasmic transport, cyclin-dependent kinase in cell cycle progression, and pyruvate dehydrogenase of the citrate cycle pathways. Moreover, we observed changes in the abundance of proteins with known interactions with HIV-1 viral proteins. Our proteomic analysis captured changes in the host protein milieu at the time of robust virus production, depicting changes in cellular processes that may contribute to virus replication. Continuing analyses are expected to focus on blocking virus replication by targeting these pathways and their effector proteins.

HIV-1 remodels the nuclear pore complex

“…The results presented here using both HIV-1-infected T cells and pNL4-3-transfected CD4  HeLa cells, along with the biochemical and physical evidence for virion-associated Nup62, Quantitative correspondence between this study and those of Chan et al (2007Chan et al ( , 2009). …”

Dynamic Host Energetics and Cytoskeletal Proteomes in Human Immunodeficiency Virus Type 1-Infected Human Primary CD4 Cells: Analysis by Multiplexed Label-Free Mass Spectrometry

“…Surface CD4 expression was verified in aliquots of isolated cells by flow cytometry on an FC500 flow cytometer (Beckman-Coulter) via fluorescein isothiocyanate (FITC)-conjugated anti-CD4 labeling (Miltenyi Biotec). HIV-1 LAI (3, 52) virus inoculum was prepared as described previously (6). Enriched CD4 ϩ…”

“…In the first proteomic analyses of HIV-1 infection in the CEMx174 cell line, we reported on protein abundance changes that correlated with G 1 /G 0 cell cycle arrest and alterations in Ran-mediated nuclear transport (6). A two-dimensional (2D) fluorescence difference gel electrophoresis analysis of HIV infection in the Jurkat cell line (42) also revealed changes in the protein abundance of host factors involved in nuclear transport, carbohydrate metabolism, and cell cycle progression in virus-producing CD4 cells.…”

“…Recent proteomic characterizations of abundance changes in HIV-infected (6,42) and -transfected (9) cells offer yet another way to characterize the interplay between HIV and the inopportune host; arguably, proteins are the effectors of most cellular functions and may not be governed by transcriptomic changes. In the first proteomic analyses of HIV-1 infection in the CEMx174 cell line, we reported on protein abundance changes that correlated with G 1 /G 0 cell cycle arrest and alterations in Ran-mediated nuclear transport (6).…”

Cellular and viral determinants of retroviral nuclear entry

“…Hepatitis B virus, herpes simplex virus, influenza virus, and adenovirus also use NUPs for nuclear entry (Trotman et al 2001;König et al 2008König et al , 2010Copeland et al 2009;Schmitz et al 2010), whereas poliovirus and cardiovirus induce modifications of NUPs (Gustin and Sarnow 2001;Porter and Palmenberg 2009). Previous LC-MS/MS studies revealed that KTN1, NUP43, NUP358, NUP45, and NUP54 expression is downregulated on HIV-1 infection while NUP358, NUP98, and TPR expression is upregulated (Chan et al 2007; Monette et al 2011). …”