:: Laboratory :: Μembers :: Publications :: Funding ::

The main objectives of our research work are:

The HCV genotype 1a encodes for a novel protein, recently described independently by three research groups including our own laboratory and known as core+1(to indicate location), ARFP (alternative reading frame protein), or F protein for (frame shifting protein). The ARFP/F/core+1 protein is encoded by an open reading frame overlapping within the core-coding sequence and it is synthesised due to a frameshift event occurring at the beginning of the core sequence (Varaklioti et al, JBC 2002). However, we have also reported that expression studies carried out in transiently transfected cells result in the synthesis of a shorter form of the ARFP/F/core+1 protein expressed from internal translation initiation codon(s), independently of the synthesis of the HCV core protein (Vassilaki and Mavromara, JBC 2003). ARFP/F/core+1 is recognised by antibodies from the serum of chronically infected HCV patients, implying its expression during natural HCV infection in humans. However, the biological role of the novel ARFP/F/core+1 protein(s), as well as its possible contribution to some of the known HCV core functions, remains unknown. The objective of this project is to investigate the expression and functional properties of the core+1 protein. To this end a broad spectrum of experimental approaches are been used including functional genomics and proteomics.

Our research is mainly focused on signal transduction pathways which are directly or indirectly involved in interferon response and are implicated in virus replication.. The MAPKs pathway and its cross-talk with the interferon pathway, recently has been identified as a major pathway involved in the replication of many viruses.

1. HCV NS5A, core and core+1/ARFP/F protein interaction with MAPKs (ERKs) pathway. By using recombinant HSV viruses expressing NS5A and core proteins we have already showed) that NS5A protein interacts with the Grb-2 adaptor protein (Georgopoulou et al 2003). This interaction results in the suppression of ERK1/2 phosphorylation in the MAPKs pathway. Furhtermore we are currently studying the possible interaction between NS5A and and specific phosphatases involved in the dephosphorylation of ERK1/2 (Georgopoulou U. et al, J Gen Virol, submitted).
   In parallel we study the effect of core, core+1/ARFP/F protein in the MAPKs signalling. Additionally we have produced core particles by the useof  recombinant  baculoviruses (Tsitoura P. et al in preparation) in order to study the possible effect of the exogenously added core to the MAPK signaling pathway.
2. Effect of selected HCV proteins with ISRE (IFN α/β) and GAS (IFN γ) promoter gene elements. We are interested in studying the effect of core, core+1/ARFP/F and NS5A proteins in ISRE and GAS promoter elements in the presence and absence of exogenous interferons.
3. Cellular genes and proteins related with the interferon action in HCV, HIV and HCV/HIV infections. We study  the effect of HCV core, NS5A and HIV TAT protein alone or in combination on the expression of interferon induced genes such as such as 2’5’OAS.

The NS4B protein is the only HCV non-structural protein for which very little information concerning its biological function is available yet. Evidence exists that it is a component of the viral replication complex and that is involved in the formation of the membrane domains where recplication of HCV RNA takes place. NS4B, a membrane associated protein of 27 kDa is cleaved from the viral polyprotein by the viral NS3/NS4A protease. The exclusive localization of NS4B in the Endoplasmic Reticulum (ER) occurs cotranslationaly.

We are investigating the structural requirements for insertion and retention of the NS4B protein in the ER membrane and as well for its interaction with other HCV non-structural proteins or host cell proteins. We are also interested in understanding the molecular mechanism of the postulated role of NS4B in viral replication.

The translation initiation process of the HCV genome is mediated by an Internal Ribosome Entry Site (IRES) contained in the 5' non-translated region of the viral genome. This region folds upon itself to create several highly conserved stem/loop domains that contain binding sites for viral and/or cellular factors. This project is focused on the identification and characterization of RNA sequences/structures and protein factors implicated in the IRES function. In this context, using a site directed mutagenesis approach, we have identified a number of cis-acting elements (Varaklioti et al, BBRC 1998, Psaridi et al, FEBS 1999, Kalliampakou et al, FEBS 2002). Furhtermore, we have recently shown that the HCV NS5A proteinnegatively regulates HCV IRES activity (Kalliampakou et al, JGV 2005). Our long-term goal is to understand the mechanisms responsible for the regulation of expression of the HCV polyprotein during the life cycle of the virus.

Vectors based on herpes simplex virus type 1 (HSV-1) are potent gene delivery vehicles because they can efficiently infect many different cell types and have large transgene capacities. HSV-1 amplicon vectors are originated from bacterial plamids that contain in addition to sequences that allow their propagation in bacteria, the transgene of interest and cis-acting sequences required for replication, cleavage and packaging as a herpes virus. Furthermore, non-replicated, non-toxic recombinant HSV-1 vectors carrying multiple mutations in selected key viral genes are also available. Both viral vector systems have been extensively used to transfer/express foreign genes in the context of gene therapy studies but their use for the development of new generation DNA vaccines has just begun. This project aims to construct and characterize recombinant HSV viruses and amplicon vectors expressing selected HCV genes and examine their potential value for the generation of protective or therapeutic vaccines against HCV infection (Tsitoura et al, JGV 2002, Lucas et al JGV 2003). In addition we use this system for the production of viral like particles (VLPs) in eukaryotic systems