HSV-1 Cgal+ Infection Promotes Quaking RNA Binding Protein Production and Induces Nuclear-Cytoplasmic Shuttling of Quaking I-5 Isoform in Human Hepatoma Cells

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.

CDK1 and PLK1 co-ordinate the disassembly and re-assembly of the Nuclear Envelope in vertebrate mitosis

“…However, the NPCs require the additional phosphorylation by PLK1 for their full dispersal at onset of mitosis, and PLK1 inactivation for the re-assembly at mitotic exit. These observations are consistent with the fact that mitotic phosphorylation of several nucleoporins depends on PLK1 as suggested by published proteomic studies [27][28][29] and supported by previous observations linking lack of PLK1 to delays of NEBD in human cells [30], C. elegans [31] [32] and in mouse oocytes maturation [33]. However this is the first demonstration that NPC and Lamin A/C are behaving in an independent manner.…”

Methylthioadenosine (MTA) Regulates Liver Cells Proteome and Methylproteome: Implications in Liver Biology and Disease

“…Protein electrophoresis and blotting were done as described previously (31). Primary antibodies were used at dilutions as indicated by the manufacturer.…”

“…QKI-5 is a member of signal transduction activator of RNA metabolism proteins that induces cell cycle arrest by increasing p27 kip1 levels (84) and has a relevant participation in cell differentiation in the CNS (85) while its function in the liver is largely unknown. We have recently demonstrated that modulation of QKI-5 levels, as well as its nucleus-cytoplasm shuttling capacity constitutes an essential intermediate mechanism orchestrating the response of liver cells to an oncolytic viral vector (31). Enhanced p27 kip1 in SK-Hep1ϩ cells upon MTAP expression points out to methylation mediated by PRMT4/CARM4 (53) as the regulatory mechanism responsible of QKI-5 activity regulation, considering that no changes at the protein level were observed.…”

Investigating the biology of alpha herpesviruses with MS‐based proteomics

“…In another study, Sanchez‐Quiles et al. analyzed nuclear isolates from HSV‐1‐infected hepatoma cells by two separate approaches, 2D‐DIGE and tandem mass tag isobaric labeling (TMT) . The two approaches identified 24 and 38 proteins, respectively, with significant changes.…”

“…The expression levels of 12 cytosolic and 6 microsomal proteins involved in apoptosis, signal transduction, and ER-associated degradation pathway, were altered. In another study, Sanchez-Quiles et al analyzed nuclear isolates from HSV-1-infected hepatoma cells by two separate approaches, 2D-DIGE and tandem mass tag isobaric labeling (TMT) [65]. The two approaches identified 24 and 38 proteins, respectively, with significant changes.…”