Abstract

The aim is to identify and exploit viral modifiers of apoptosis and cytokin responses (particularly TNFRs) as a potential source of novel health care pharmaceuticals for manipulation of immune responses and treatment of certain diseases. Such virus genes will be identified by nucleotide sequence and functional analysis of cloned viral ORFs and cDNA libraries of three large DNA viruses (African swine fever, Ectromelia and Mouse herpes). Possible human homologue of novel sequences will be characterised. The cellular targets of these viral evasion genes and their impact on apoptosis and virus pathogenesis will be determined. Determination of minimal active sequences will permit development of low molecular weight peptoid drugs, starting with the calcineurin phosphatase inhibitor A238L gene of ASFV.

Objectives

The principal objective of this proposal is to exploit viral modifiers of apoptosis and cytokine responses (particularly TNFRs), in order to (1) Design low molecular weight, peptoid analogues for pharmaceutical intervention and (2) Identify as yet undiscovered viral analogues or antagonists potentially capable of manipulating apoptosis and the immune system in health and disease. Potential host modifying virus genes of African swine fever virus (ASFV), mouse y-herpes virus (MHV) and Ectromelia virus (EV) will be identified by sequence and functional screening of cloned viral ORFs, and cDNA libraries, introduced into murine cells in vitro by retrovirus technology, and their cellular targets and molecular and biological consequence will be determined. Possible human homologues will be defined and determination of minimal active sequences will permit the development of low molecular weight peptoid drugs, starting with the calcineurin phosphatase inhibitor A238L gene of ASFV.

Description of the work

Potential apoptosis and cytokine response modifying genes (particularly TNFRs) of three large DNA viruses (ASFV, MHV and EV) will be identified by sequence analysis and functional screening of cloned viral ORFs and cDNA libraries. Possible human homologue of novel viral sequences will be defined. An important strength of the project is that MHV and EV are natural pathogens of the mouse, thereby facilitating functional investigations.

We will start with the ASFV IkB homologue A238L, which performs the dual functions of inhibiting NFkB gene transcription and inhibiting calcineurin phosphatase activity. Analysis of deletion mutants will define these functional sequences and permit the design of low molecular weight peptoid analogues inhibiting immune responses, either by blocking NFkB or inhibiting calcineurin. Stable cell lines transfected with entire or partial sequence of A238L will provide an in vitro model, particularly for the study of apoptosis and perhaps for long lived cell lines useful for fermentation.

A similar strategy will be pursued with other apoptosis inhibitors encoded by ASFV, MHV and the newly sequenced EV. To detect novel apoptosis modifying viral molecules without obvious sequence homologues in the database, selected EV and MHV ORFs will be cloned into a retrovirus vector and their impact on the induction of apoptosis will be assessed after transfection of B- and T-lymphocytes. The cellular targets of these viral gene products will be defined with the yeast two hybrid system.

Sequencing of EV and virus expression libraries for novel immune modulators will provide further opportunities for exploitation. As TNF is critical in inflammation and immunity, viral TNFRs will be selected from cDNA expression libraries of MHV and EV. Finally, the role of these viral evasions molecules in pathogenesis will be explored through the construction and comparative testing of virus deletion mutants.

Milestones and Expected Results

• Define NFkB and calcineurin binding sequences of ASFV gene A238L and develop peptoid analogues.
• Sequence EV and identify novel evasion molecules.
• Identify novel virus modifiers of apoptosis and TNF responses through functional screening of virus genes.
• Identify cellular targets of viral modifiers of apoptosis.
• Transgenic expression of virus evasion genes.
• Develop long-lived transgenic cell lines.
• Construct virus deletion mutants.
• Production of models for assaying in vitro and in vivo effects of viral modulators of cytokines and apoptosis.

Contacts

Coordinator

• R M E PARKHOUSE / Gulbenkian de Ciencia Department of Immunology / PORTUGAL

Participant

• C. ALONSO / INIA Department of Genetics & Biotechnology / SPAIN
• L. K. DIXON / Institute for Animal Health Department of Molecular Biology / UNITED KINGDOM
• A. ALCAMI / University of Cambridge Department of virology / UNITED KINGDOM