Laboratory of Molecular Virology

Head of the lab: Dr. Penelope Mavromara,  Email: penelopm@hol.gr, Tel: +30-210-6478877

About

The research in the Molecular Virology Laboratory focuses on the molecular and cellular  mechanisms that control pathogenesis of HCV infection. We are interested in understanding how viral proteins interact with the host cell machinery to modulate virus replication and/or contribute to pathogenesis of HCV infection. Viral-host interactions implicated in  the regulation of host transcription and modulation of host signalling pathways involved in innate and adapted immune responses and carcinogenesis are investigated with the use of multiple approaches including DNA microarrays, qPCR, ChIP assays, confocal and live imaging. In addition, the Molecular Virology Laboratory has long standing expertise in the use of viral expression systems based on baculovirus, lentivirus and herpes simplex virus for heterologous gene expression, the development of oncolytic viruses and the production of VLPs and has established close links with National Hospitals and Industry.

The webpage is under construction

Members

Research programs

Research programs

Publications

2011

Boumlic, A., et al., Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b. Virus Res, 2011. 155(1): p. 213-20.

Budkowska, A., et al., Synonymous mutations in the core gene are linked to unusual serological profile in hepatitis C virus infection. PLoS One, 2011. 6(1): p. e15871.

Dalagiorgou, G., et al., High levels of HCV core+1 antibodies in HCV patients with hepatocellular carcinoma. J Gen Virol, 2011. 92(Pt 6): p. 1343-51.

Serti, E., et al., Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase. Cell Mol Life Sci, 2011. 68(3): p. 505-22.

2010

Arnaud, N., et al., Hepatitis C virus controls interferon production through PKR activation. PLoS One, 2010. 5(5): p. e10575.

Boleti, H., et al., ER targeting and retention of the HCV NS4B protein relies on the concerted action of multiple structural features including its transmembrane domains. Mol Membr Biol, 2010. 27(1): p. 50-74.

Boumlic, A., et al., Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein. FEBS J, 2010. 277(3): p. 774-89.

Foka, P., et al., Novel tumour-specific promoters for transcriptional targeting of hepatocellular carcinoma by herpes simplex virus vectors. J Gene Med, 2010. 12(12): p. 956-67.

Katsarou, K., et al., Endocytosis of hepatitis C virus non-enveloped capsid-like particles induces MAPK-ERK1/2 signaling events. Cell Mol Life Sci, 2010. 67(14): p. 2491-506.

Madesis, P., et al., A hepatitis C virus core polypeptide expressed in chloroplasts detects anti-core antibodies in infected human sera. J Biotechnol, 2010. 145(4): p. 377-86.

Njouom, R., et al., Evaluation of core and NS4B synthetic peptide-based immunoassays for the detection of hepatitis C virus antibodies in clinical samples from Cameroon, Central Africa. J Clin Virol, 2010. 49(1): p. 61-4.

2005-2009

Kalliampakou, K.I., M. Kalamvoki, and P. Mavromara, Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. J Gen Virol, 2005. 86(Pt 4): p. 1015-25.

Manservigi, R., et al., Immunotherapeutic activity of a recombinant combined gB-gD-gE vaccine against recurrent HSV-2 infections in a guinea pig model. Vaccine, 2005. 23(7): p. 865-72.

Mavromara, P., et al., The impact of HCV diversity on diagnosis tools for HCV infection. Med Mal Infect, 2005. 35 Suppl 2: p. S103-4.

Georgopoulou, U., et al., The protein phosphatase 2A represents a novel cellular target for hepatitis C virus NS5A protein. Biochimie, 2006. 88(6): p. 651-62.

Kalamvoki, M., U. Georgopoulou, and P. Mavromara, The NS5A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol. J Biol Chem, 2006. 281(19): p. 13449-62.

Tsitoura, E., U. Georgopoulou, and P. Mavromara, HSV-1 based amplicon vectors as an alternative system for the expression of functional HCV proteins. Curr Gene Ther, 2006. 6(3): p. 393-8.

Kouvatsis, V., et al., Characterization of herpes simplex virus type 1 recombinants that express and incorporate high levels of HCV E2-gC chimeric proteins. Virus Res, 2007. 123(1): p. 40-9.

Tsitoura, P., et al., Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped capsid-like particles. FEBS Lett, 2007. 581(21): p. 4049-57.

Vassilaki, N., H. Boleti, and P. Mavromara, Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells. The influence of the core protein and proteasomes on the intracellular levels of core+1. FEBS J, 2007. 274(16): p. 4057-74.

Nianiou, I., et al., Expression of an HCV core antigen coding gene in tobacco (N. tabacum L.). Prep Biochem Biotechnol, 2008. 38(4): p. 411-21.

Vassilaki, N., H. Boleti, and P. Mavromara, Expression studies of the HCV-1a core+1 open reading frame in mammalian cells. Virus Res, 2008. 133(2): p. 123-35.

Vassilaki, N., et al., Role of the hepatitis C virus core+1 open reading frame and core cis-acting RNA elements in viral RNA translation and replication. J Virol, 2008. 82(23): p. 11503-15.

Vassilaki, N., K.I. Kalliampakou, and P. Mavromara, Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system. J Gen Virol, 2008. 89(Pt 1): p. 222-31.

Katsarou, K., et al., Green fluorescent protein – Tagged HCV non-enveloped capsid like particles: development of a new tool for tracking HCV core uptake. Biochimie, 2009. 91(7): p. 903-15.

Oprisan, G., et al., Comparative methods for genotyping hepatitis C virus isolates from Romania. Roum Arch Microbiol Immunol, 2009. 68(3): p. 151-7.

Tsitoura, E., et al., Infection with herpes simplex type 1-based amplicon vectors results in an IRF3/7-dependent, TLR-independent activation of the innate antiviral response in primary human fibroblasts. J Gen Virol, 2009. 90(Pt 9): p. 2209-20.

Vassilaki, N. and P. Mavromara, The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product. IUBMB Life, 2009. 61(7): p. 739-52.

2000-2004

Miriagou, V., et al., The C-terminal cytoplasmic tail of herpes simplex virus type 1 gE protein is phosphorylated in vivo and in vitro by cellular enzymes in the absence of other viral proteins. J Gen Virol, 2000. 81(Pt 4): p. 1027-31.

Kalamvoki, M., et al., Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays. Arch Virol, 2002. 147(9): p. 1733-45.

Kalliampakou, K.I., L. Psaridi-Linardaki, and P. Mavromara, Mutational analysis of the apical region of domain II of the HCV IRES. FEBS Lett, 2002. 511(1-3): p. 79-84.

Tsitoura, E., et al., Expression of hepatitis C virus envelope glycoproteins by herpes simplex virus type 1-based amplicon vectors. J Gen Virol, 2002. 83(Pt 3): p. 561-6.

Varaklioti, A., et al., Alternate translation occurs within the core coding region of the hepatitis C viral genome. J Biol Chem, 2002. 277(20): p. 17713-21.

Georgopoulou, U., K. Caravokiri, and P. Mavromara, Suppression of the ERK1/2 signaling pathway from HCV NS5A protein expressed by herpes simplex recombinant viruses. Arch Virol, 2003. 148(2): p. 237-51.

Lucas, M., et al., Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus. J Gen Virol, 2003. 84(Pt 3): p. 545-54.

Vassilaki, N. and P. Mavromara, Two alternative translation mechanisms are responsible for the expression of the HCV ARFP/F/core+1 coding open reading frame. J Biol Chem, 2003. 278(42): p. 40503-13.

Kalamvoki, M. and P. Mavromara, Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein. J Virol, 2004. 78(21): p. 11865-78.

Lauterbach, H., et al., Protection from bacterial infection by a single vaccination with replication-deficient mutant herpes simplex virus type 1. J Virol, 2004. 78(8): p. 4020-8.

1990-1999

Georgopoulou, U., et al., Identification of a new transcriptional unit that yields a gene product within the unique sequences of the short component of the herpes simplex virus 1 genome. J Virol, 1993. 67(7): p. 3961-8.

Ho, L., et al., The genetic drift of human papillomavirus type 16 is a means of reconstructing prehistoric viral spread and the movement of ancient human populations. J Virol, 1993. 67(11): p. 6413-23.

Ong, C.K., et al., Evolution of human papillomavirus type 18: an ancient phylogenetic root in Africa and intratype diversity reflect coevolution with human ethnic groups. J Virol, 1993. 67(11): p. 6424-31.

Labropoulou, V., et al., Typing of human papillomaviruses in condylomata acuminata from Greece. J Med Virol, 1994. 42(3): p. 259-63.

Georgopoulou, U., et al., Characterization of the US8.5 protein of herpes simplex virus. Arch Virol, 1995. 140(12): p. 2227-41.

Kakkanas, A., et al., Escherichia coli expressed herpes simplex virus gG1 and gG2 proteins in ELISA and immunoblotting assays. Intervirology, 1995. 38(6): p. 346-51.

Miriagou, V., et al., Expression of the herpes simplex virus type 1 glycoprotein E in human cells and in Escherichia coli: protection studies against lethal viral infection in mice. J Gen Virol, 1995. 76 ( Pt 12): p. 3137-43.

Labropoulou, V., et al., Genital papillomavirus in Greek women with high-grade cervical intraepithelial neoplasia and cervical carcinoma. J Med Virol, 1996. 48(1): p. 80-7.

Labropoulou, V., et al., Type-specific prevalence of genital human papillomaviruses in benign, premalignant, and malignant biopsies in patients from Greece. Sex Transm Dis, 1997. 24(8): p. 469-74.

Papadopoulou, K., et al., Detection of human papillomaviruses in squamous cell carcinomas of the lung. Virchows Arch, 1998. 433(1): p. 49-54.

Varaklioti, A., et al., Mutational analysis of two unstructured domains of the 5′ untranslated region of HCV RNA. Biochem Biophys Res Commun, 1998. 253(3): p. 678-85.

Psaridi, L., et al., Mutational analysis of a conserved tetraloop in the 5′ untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function. FEBS Lett, 1999. 453(1-2): p. 49-53.

 

Funding

NIH R29: Regulation of 3’ coterminal HSV-1 gene expression (1990-1994) Total Direct Costs: $353,059

UNIDO: HPV and cervical cancer in Greece: Epidemiological studies, HPV typing, development of immunoreagents (1993-1995)

FP4: Life Science (1996-1998) BIOMED: Molecular approaches to analyze Hepatitis C virus replication for the development of effective therapy and vaccines Total Direct Costs: 45,000.00€,

FP4: (1996-2000) FMRX-CT-960053: European Union network for investigation of dendritic immunotherapy for induction of anti-viral and anti-tumor immunity and transplantation tolerance. Total Direct Costs: 200,000.00€

FP4: (1996-2000) BIOTECH PL960422: European Union Network for Evaluation of Nucleic Acid-Based Vaccines for Induction of Antiviral Immunity Total Direct Costs: 300,000.00€

INCO-COPERNICUS (1998-2000) IC15-CT98-0304: Improvement of surveillance and prevention of HCV infection: Total Direct Costs: 40,000.00€

FP5 (2004-2006) EUROAMP QLK2-CT-00055: European (EURO) Network for Development of Novel Safe Vaccines Based on New Generation Amplicons (AMP) and Other Defective HSV – 1 Derived Vectors as Foreign Antigen Delivery Systems Total Direct Costs: 300,000.00€

FP6 (2005-2009) COMPUVAC-LSHB-CT-2004-005246: Rational design and standardized evaluation of novel genetic vaccines, Total Direct Costs: 724,000.00€,

FP6 (2005-2009) THOVLEN-LSHB-CT-018649: Targeted herpes-virus derived oncolytic vectors for liver cancer European Network, Total Direct Costs: 289,260€

INSERM: ATC-Hépatite C: Vers la rèsolution de controverses concernant les mècanismes de la traduction chez le virus de l’hépatite C: conséquences en pathologie humaine (2004-2005) Total Direct Costs: 12,000.00€

Institut Pasteur International Network (RIIP) ACIP I: Etude Comparative d’epidemiologie moleculaire des infections par l’hepatitis C en Europe et l’Iran (1997 – 1999)

Institut Pasteur International Network (RIIP  ACIP II: Recherche des recombinants chez des virus ARN de polarite positive (enterovirus et virus de l’hepatite C) (1998 – 2000)

Institut Pasteur International Network (RIIP)  ACIP III: Etude des genotypes du virus de l’hepatite C en Asie du Sud Est (Cambodge et Vietnam) et developpement de methodes diagnostiques specifiques (2003-2005), Total Direct Costs: 10,000.00€,

Institut Pasteur International Network (RIIP) PTR Implications of HCV diversity in the diagnosis and pathogenesis of virus infection in Eastern Europe, SE Asia and Central Africa (2003 – 2005), Total Direct Costs: 40,000.00€

ΚΕΕΛΠNO-HCDCP: Research on the biological functions of the  core+1/F protein of the HCV virus: new prospects on the treatment of HCV infection. (2003-2004), Total Direct Costs: 30,000.00€,

Greek General Secretariat of Science and Technology, PENED I: Production of recombinant antigens –for diagnostic purposes- of the Human Pappiloma Virus in eukaryotic cells with the use of viral vector systems of expression. Development of immunodiagnostic systems.  (1996 – 1998) Total Direct Costs: 8.000.000Drs

Greek General Secretariat of Science and Technology, PENED 99 II: Gene cloning of regulatory proteins and biotechnological development-application of systems for the production of heterologous peptides. (11/2000 – 9/2001) Total Direct Costs: 14.800.000Drs

Greek General Secretariat of Science and Technology, Program for a carrier opportunity in Greece for Greek-speaking researchers abroad. Research on the molecular pathogenesis and treatment of viral infections and carcinogenesis in humans. (1999 – 2001) Total Direct Costs: 33.000.000Drs

Greek General Secretariat of Science and Technology, Bilateral S&T Collaboration Program Greece – Italy: Recombinant HSV expression viral vectors for the study of selected viral proteins: Vaccine development against the HCV virus. C (1999-2001) Total Direct Costs: 4.200.000Drs

Greek General Secretariat of Science and Technology, Bilateral S&T Collaboration Program Greece – France PLATO 1: Development and use of amplicons (viral expression systems) for the expression of the structural proteins of the HCV virus. (1999­2001) Total Direct Costs: 3.150.000Drs

Greek General Secretariat of Science and Technology, Bilateral S&T Collaboration Program Greece – France PLATO 2: Construction of HSV-based viral vectors (amplicon) for the development of vaccines against HCV: Development of strategy and improvement of antigen presentation to the immune system. (2002-2004) Total Direct Costs: 12,325.75€

Greek General Secretariat of Science and Technology, Bilateral S&T Collaboration Program Greece – France PLATO 3: HCV – dependent hepatocellular cancer. Development of a suppression system with RNA mediation (RNAi) that suppresses the reproduction of the HCV virus with the use of HSV-based viral vectors (amplicon) (2004 – 2006), Total Direct Costs: 11,800.00€

Greek General Secretariat of Science and Technology, PENED: Study of the immunoregulatory function of the core protein and the –until recently unknown- core+1 protein of the HCV virus: Use of novel technology – prospects for novel therapeutic approaches. (2006-2008) Total Direct Costs: 124,000€

Bodosakis Foundation: Donation for laboratory equipment (2009) Total Direct Costs: 80,000€

 

 

Technologies

Overview of the Microarray Gene  Expression platform operating in the Molecular Virology Laboratory

The Microarray Gene Expression facility currently operates the CodeLinkTM Microarray platform (Applied Microarrays) for the determination of mRNA transcriptomes of human, mouse and rat samples. It provides quality assessment of starting RNA sample, labeled cRNA preparation, hybridization and analysis of the transcriptomic data. In addition, the facility can provide custom gene expression arrays and protein arrays through the Applied Microarray systems. RT2-PCR for the validation of microarray data and other gene expression analyses is available.

The instruments integrated in this facility are:

i. AXON GenePix personal 4100A scanner (Molecular Devices) currently used for CodeLinkTM whole genome arrays, that can also scan various commercial and custom cDNA, protein and formalin fixed tissue microarrays. The GenePix 4100A scanner can detect up to 4 custom fluorophores in addition to the two standard ones.
ii. Forma Orbital hybridization Shaker (Thermo Scientific, model 420) used for the hybridisation of the CodeLink microarrays under constant temperature.
iii. Agilent bioanalyzer for RNA sample quality control assessment, DNA fragment analysis and SDS-PAGE analysis of protein samples.
iv. NanoDrop 2000 1 position spectrophotometer that requires only 1 µl of a sample to provide a very accurate and fast measurement of sample concentration, solvent contamination and purity. It measures concentrations of nucleic acids, proteins, and bacterial cultures. One of its unique features is monitoring dye incorporation rate after labeling and before microarray hybridization.
v. Rotor Gene 6000 Real Time PCR machine (Corbett Applied Science) utilised for flexible real-time PCR quantification and High Resolution Melting curve (HRM) analysis.

Equipment of DNA Microarray facility


Personnel:
The facility is currently run by two post-doctoral fellows Eliza Tsitoura (Molecular Biologist) and Dorothea Kazazi (Molecular Biologist/Bioinformatician).

Until recently the facility was organized to serve the purposes of the FP6 Integrated European Project CompuVac. The overall objective of this proposal was to characterise the initial events mediated by the interaction of dendritic cells with vaccine delivery viral-based vectors using a mouse model, with the long term goal to identify innate immune response or dendritic cell gene expression signatures associated with successful, adaptive immune responses. Such signature(s) could be used for predicting performance of experimental vaccines. The molecular signatures of more than 20 vaccine vectors have been determined following multiple experiments and the results from this work have been deposited at the GeVads database (http://gevads.cs.put.poznan.pl/) as of June 2009. The analysis of the large amount of dendritic cell data generated during the CompuVac project is ongoing in collaboration with Dr. D. Klatzman and Dr. A. Six, at the I3 Immunology-Immunopathology-Immunotherapy group, Pitié-Salpêtrière Hospital, Paris.

 Clustering of the vaccine vectors according to the differential expression of at least 2-fold affected genes


Participation in Scientific meetings

 

Dendritic cell signatures of virus-based vaccine delivery vehicles. Dorothea Kazazi, Eliza Tsitoura, Shirin Khalili, Devrim Oz-Arslan, Niki Vassilaki, Elina Aslanoglou, Manos Kochlios, Luca Beltrame, Duccio Cavalieri, Vladimir Benes, Thomas Brocker, David Klatzmann  and Penelope Mavromara. Invited talk at the DC-THERA Network meeting, May 2010, Athens, Greece.

 

Gene expression profiling of dendritic cells upon in vivo exposure to candidate vaccine vectors. Dorothea Kazazi, Eliza Tsitoura, Niki Vassilaki, Elina Aslanoglou, Manos Kochlios, Shirin Khalili, Devrim Oz-Arslan, Florian Kreppel, Stephan Kochanek, Gary Jennings, François-Loic Cosset, Alberto Epstein, Tina Dalianis, Anna-Lise Williamson, Bertrand Bellier, Adrien Six, Thomas Brocker, Tomi Ivacevic, Vladimir Benes, David Klatzmann and Penelope Mavromara. Poster presentation at the 5th International Greek Biotechnology Forum, July 2009, Athens, Greece.

Gene expression profiling of dendritic cells upon in vivo exposure to recombinant viral vaccine vectors and virus-like particles (VLPs). Khalili, S., Oz-Arslan, D., Kazazi, D., Ivacevic, T., Tsitoura, E., Kreppel, F., Kochanek, S., Jennings, G., Bellier, B., Brocker, T., Benes, V., Klatzmann, D. and Mavromara, P. Poster presentation at the 33rd FEBS Congress & 11th IUBMB Conference, 28 June – 3 July, 2008, Athens, Greece (second best poster award).

Host Responses–Molecular Signatures of vector vaccines. HEVAR (FP6) course, invited talk by Eliza Tsitoura in Montevideo, April 2008, Uruguay.

Characterisation of dendritic cell related mRNA expression profile elicited by HSV-1 and HSV-1-based amplicon vectors. Oz-Arslan, D., Tsitoura, E., Kazazi, D., Foka, P. and Mavromara, P. Poster presentation at the XIV, International Congress of Virology, 11-15 August 2008, Istanbul, Turkey.

 

Gene expression profiling of dendritic cells upon in vivo exposure to recombinant viral vaccine vectors. Khalili, S., Oz-Arslan, D., Kazazi, D., Ivacevic, T., Tsitoura, E., Kreppel, F., Kochanek, S., Jennings, G., Bellier, B., Brocker, T., Benes, V., Klatzmann, D. and Mavromara, P. Poster at the Challenges of Global Vaccine Development, Keystone Symposia, 8-13, October 2007, Cape Town, South Africa.

 


Contact Information

Eliza Tsitoura, PhD
Molecular Virology Laboratory
Hellenic Pasteur Institute
Athens, Greece
tel +30 210 6478878
fax +30 210 6478877
elizatsitoura@pasteur.gr

Dorothea Kazazi, PhD
Molecular Virology Laboratory
Hellenic Pasteur Institute
Athens, Greece
tel +30 210 6478878
fax +30 210 6478877
dkazazi@pasteur.gr