Objectives
• Interactions of viral proteins with proteins from host hemocytes
• Functional characterization of polydnavirus CcBV proteins.
• The role of ankyrin repeat proteins (IκΒα-like) in transcriptional regulation of immune related genes and signalling pathways.
• Identification of new agents for control of insect pest populations
1. Viral protein interactions with host proteins.
Our previous results on the investigation of the viral CcV1 protein showed that this protein interacts with the host protein, hemolin. Hemolin belongs to the immunoglobulin-like protein family and is the most abundant immune induced protein after microbial infection in Manduca sexta. The binding of CcV1 to hemolin was shown to inhibit known hemolin functions including binding to lipopolysaccharides (LPS) and its ability to cause bacterial agglutination. The viral protein and hemolin co-localized on the cell surface suggesting interference with the function of hemolin as a pattern recognition molecule. CcV1 protein was also found to inhibit phagocytosis of E.coli bacteria by lepidopteran hemocytes or hemocyte-like cells (Figure1). Current investigation focuses on the role of CcV1 protein in the melanization process, another important defence mechanism in insects and the interaction of viral gene products with serine protease inhibitors, serine protease homologues and transferrin molecules of the host, found to interact with CcV1 in the yeast two hybrid system.
2. The effect of CcBV Ank proteins on antimicrobial gene transcription.
Among the CcBV gene families, there is a family of ank genes, which comprises members encoding proteins with characteristic ankyrin repeats and similarities to the eukaryotic IκBα protein, the inhibitor of NF-κB transcription factor, and Cactus protein (IκBα homologue) of Drosophila melanogaster. The NF-κB/Rel transcription factors are known to control the expression of many immune related genes in mammals and flies.
CcBV ank genes were cloned into mammalian expression vectors and examined for their ability to suppress TNFα–mediated induction of a luciferase reporter gene placed under the control of an NFκB-responsive promoter. Two of them, Ank 4 and 7, inhibited TNFα induced transcription by 40% suggesting an IκΒα-like role for the viral proteins.
In insects two important pathways are known to govern the gene transcription of immune related genes, the Toll and Imd pathways. We have developed a lepidopteran-based system to study the effect of CcBV Ank proteins on the transcription of immune related genes in insects. Expression constructs for the Bombyx mori transcription factors BmRelA, BmRelB, BmRelish1 and the constitutively active BmRelish1-d2, along with the reporter constructs based on promoters of the B.mori cecropin and attacin antimicrobial peptide genes are employed in transiently transfected lepidopteran cell lines to study the effects of the virally encoded proteins on the transcription of antimicrobial peptide gene promoters (Figure2). In our studies, six out of the nine cloned members of the CcBV ank gene family were examined and showed different degree of inhibition in transcriptional activation on both Toll and Imd pathways.
Relevant publications
1. Labropoulou, V., Douris, V., Stefanou D., Magrioti C., Swevers L., and Iatrou, K. (2008). "Endoparasitoid wasp bracovirus-mediated inhibition of hemolin function and lepidopteran host immunosuppression". Cell. Microbiol. 10: 2118-28.
2. Douris, V., Swevers, L., Labropoulou, V., Andronopoulou E., Georgoussi, Z. and Iatrou, K. (2006). Stably transformed insect cell lines: tools for expression of secreted and membrane-anchored proteins and high throughput screening platforms for drug and insecticide discovery. Adv. in Virus Res., 68: 113-156
3. Lapointe, R, Wilson, R., Vilaplana, L., O'Reilly, D.R., Falabella, P., Douris, V., Bernier?Cardou, M., Pennacchio, F., Iatrou, K., Malva, C., and Olszewski J.A. (2005). Expression of a Toxoneuron nigriceps polydnavirus (TnBV) encoded protein, TnBV1, causes apoptosis?like programmed cell death in lepidopteran insect cells. J. Gen. Vir. 86, 963?971.
4. Espagne, E., Douris, V., Lalmanach, G., Provost, B., Cattolico, L., Iatrou, K., Drezen, J?M., and Huguet, E. (2005). A virus required for hymenopteran parasite survival into lepidopteran host expresses genes encoding cystatins. J Virol.;79(15):9765?76.
