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Journal of Virology, September 2009, p. 9283-9295, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00814-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Eric Y. Chan,1 Jennifer N. Sutton,2 Jon M. Jacobs,3 Andrey Bondarenko,4 Richard D. Smith,3 and Michael G. Katze1,5*

Department of Microbiology,1 Washington National Primate Research Center, University of Washington, Seattle, Washington,5 Thermo Fisher Scientific, Cambridge, Massachusetts,2 Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington,3 Rosetta Biosoftware, Seattle, Washington4

Received 22 April 2009/ Accepted 26 June 2009

We report on a proteomic analysis of ex vivo human immunodeficiency virus (HIV) type 1 infection in human primary CD4 cells by shotgun liquid chromatography-tandem mass spectrometry analysis, revealing two distinct proteomic profiles at two phases of virus replication. Relative to mock-infected cells, 168 signature proteins exhibited abundance changes at the first sign of Gag p24 production (8 h postinfection [p.i.]) or the peak of virus replication (24 h p.i.); interestingly, most of the changes were exclusive to only one phase of virus replication. Based on characterization by functional ontology and known human-HIV protein interactions, we observed the enrichment for protein abundance increases pertaining to protein synthesis and nucleasomal reorganization amid an otherwise placid cellular proteome at the first sign of HIV replication. In contrast, we observed indications of decreased protein turnover, concomitant with heightened DNA repair activities and preludes to apoptosis, in the presence of robust virus replication. We also observed hints of disruptions in protein and small molecule trafficking. Our label-free proteomic strategy allowed us to perform multiplexed comparisons—we buttressed our detection specificity with the use of a reverse transcriptase inhibitor as a counterscreen, enabling highlighting of cellular protein abundance changes unique to robust virus replication as opposed to viral entry. In conjunction with complementary high-throughput screens for cellular partners of HIV, we put forth a model pinpointing specific rerouting of cellular biosynthetic, energetic, and trafficking pathways as HIV replication accelerates in human primary CD4 cells.

* Corresponding author. Mailing address: Department of Microbiology, University of Washington, P.O. Box 358070, Seattle, WA 98195-8070. Phone: (206) 732-6135. Fax: (206) 732-6056. E-mail: honey{at}u.washington.edu

{triangledown} Published ahead of print on 8 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, September 2009, p. 9283-9295, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00814-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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