HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells

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

HSV-1 Cgal+ infection promotes quaking RNA binding protein production and induces nuclear-cytoplasmic shuttling of quaking I-5 isoform in human hepatoma cells

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

Characterization of herpes simplex virus 1 strains as platforms for the development of oncolytic viruses against liver cancer

BACKGROUND: Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. AIMS: To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. METHODS: The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. RESULTS: The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. CONCLUSIONS: The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV