Abstract
Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.
Highlights
From the ‡Division of Hepatology and Gene Therapy, Proteomics and Bioinformatics Unit, Centre for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain, §Universitede Lyon, UCB-Lyon 1, Lyon, F-69003, France; CNRS, UMR5534, Centre de Genetique Moleculaire et Cellulaire, 16 rue Dubois, Villeurbanne, F-69622, France, ¶Department of Experimental and Diagnostic Medicine - Section of Microbiology, University of Ferrara, Via Luigi Borsari 46, 44100 Ferrara, Italy, ʈDepartment of Structural Biology and Bioinformatics, Biomedical Proteomics Group, Geneva University, Geneva 4, Geneva 1211, Switzerland
Protein species from Non-POU domaincontaining octamer-binding protein, TAR DNA-binding protein 43, hnRNPC, hnRNPA2/B1, hnRNPH3, peptidyl-prolyl cis-trans isomerase E, and Pre-mRNA-splicing factor SPF27 were down-regulated in nuclear fraction of Herpesvirus type 1 (HSV-1) Cgalϩinfected Huh7 cells
By comparative cytosolic and microsomal proteome analysis we have previously identified deregulation of central intermediates targeted by HSV-1 Cgalϩ resulting in the impairment of apoptosis and cell survival pathways in human hepatoma cells [16]
Summary
Materials—The following reagents and materials were used: antiQKI-5 was kindly provided by Dra. 36 h after transfection, cells were infected as mentioned above with HSV-1 Cgalϩ at MOI of 5 pfu/cell for the indicated periods of time and processed for appropriate analysis. Alkylation, Digestion, and Labeling with 6-Plex TMT— Thirty micrograms of nuclear protein extracts from mock and HSV-1 Cgalϩ-infected Huh cells were dissolved in 300 l triethylammonium hydrogen carbonate buffer (TEAB) 0.1 M adjusted to pH 8 (Fluka). Off-gel Separation of Pooled TMT-labeled Sample, LC-ESI LTQOrbitrap MS/MS Analysis, and Database Search—After drying, the pooled TMT-labeled sample was dissolved in 1616.4 l H2O with 172.8 l glycerol 50% (Agilent Technologies, Santa Clara, CA) and 10.8 l of carrier ampholytes IPG buffer pH 3–10 (GE Healthcare). Immunofluorescence Microscopy—HSV-1 Cgalϩ-infected or Mock-infected Huh cells were grown in chamber slides for the indicated periods of time, washed twice with PBS, fixed for 10 min in 3.7– 4% formaldehyde, and made permeable by Triton-X100 5% for 30 min. The immunofluorescence figures are representative of the overall effects observed under each set of conditions
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