Dr Fasseli Coulibaly

Senior Research Fellow

Email:    fasseli.coulibaly@med.monash.edu.au
Phone: +61 3 990 29225
Fax:       +61 3 990 29500
Office:   Room G29, Building 76, Clayton

Research interests

My research aims at understanding the molecular mechanisms underlying the infectious cycles of animal and human viruses. I am using a structural approach with X-ray crystallography as the main tool.

Research projects

Understanding how viruses assemble and interact with their cellular hosts not only helps fighting off viral pathogens but it also provides a powerful probe to analyse many fundamental processes in biology. The study of poxviruses has contributed to both of these aims. Poxviruses have been a paradigm in vaccination that culminated in the eradication of smallpox by the end of the 1970s. In addition, poxviruses have been model systems because of their complex infectious cycle and their remarkable ability to hijack and neutralize cellular defense mechanisms. In particular, the assembly of vaccinia has been extensively studied by electron microscopy of infected cells and, more recently, by tomography of infectious vaccinia virus particles. Building on this knowledge, our research aims at elucidating the molecular details of poxvirus assembly using X-ray crystallography.

Scaffolding proteins in Poxvirus assembly

The formation of immature virus particles (IV) is an early, mandatory intermediate in the poxvirus infectious cycle. IV are non-infectious spherical particles formed by wrapping the viral core into a membrane whose origin and structure remains controversial. To investigate the mechanisms underlying this essential maturation step, we focus on a scaffolding protein called ORFV075 in orf virus, a poxvirus of sheep. This protein is thought to play a central role in poxvirus assembly because point mutations of its vaccinia orthologue, D13, block morphogenesis before the formation of IV. Also the antibiotic rifampicin induces a similar arrest in vaccinia virus morphogenesis that is overcome by point mutations in D13.
We have determined the structure of ORFV075 by electron microscopy of 2-D crystals. This structure suggests that this protein is similar to capsid proteins of large icosahedral DNA viruses that adopt a typical double-barrel fold. To understand further the structure-function of this scaffolding protein, we are now working on the determination of the atomic structure of ORFV075 and on the identification of its viral and cellular partners.

Viral Microcrystals

Poxvirus are unusual in the virus world because they exhibit several infectious particles differing both by their structures and cellular localisations. Among these infectious forms, entomopoxvirus spheroids are probably the most striking. Spheroids are crystals forming inside infected cells and visible by light microscopy. They function as a dense matrix that protects virus particles from environmental insults so that they remain viable for years in soil, analogous to bacterial spores. Recently, we have developed methods allowing the structural analysis of such infectious crystals for an unrelated family of viruses, cypovirus polyhedra. We are now focusing on using these cutting-edge microcrystallography techniques to study spheroids with the aim of unravelling the evolutionary origin of this unique survival strategy and advancing our understanding of in vivo protein crystallization.
In addition, the protective function and the robustness of these crystals has significant applied outcomes, in that they form natural platforms for the development of strong and versatile nanocontainers for example to design novel vaccine vectors.

Past research

Structural analysis of birnaviruses.

Birnaviruses are ubiquitous animal viruses infecting hosts ranging from insects and molluscs to fishes and birds. The prototype virus is the Infectious Bursal Disease Virus (IBDV) that causes serious concerns to the poultry industry since the emergence of hypervirulent strains escaping vaccination. We have determined the structure of IBDV infectious particles by X-ray crystallography. This structure revealed an unusual single-layered architecture of the virus particles and suggested that birnaviruses constitute a missing link between simple positive-stranded RNA viruses (Tetraviridae) and complex double-stranded RNA viruses (Reoviridae; e.g. rotavirus). The knowledge of the atomic structure of the capsid protein VP2 has also been a precious tool to understand the molecular basis for the antigenicity and virulence of different viral strains in search for an improved vaccine.

Microcrystallography

Our structural analysis of the silkworm cypovirus-1 (CPV-1) polyhedra has allowed the determination of the first atomic structure of viral infectious crystals. These are among the smallest crystals used for solving a protein structure by X-ray crystallography (5-12 micrometers in diameter; data collected at beamline PX06SA of the Swiss Light Source). Our results suggest molecular bases for the outstanding stability of polyhedra in the environment as well as for the rapid dissolution at the alkaline pH encountered in the mid-guts of larvae. The structure of CPV-1 polyhedra also provides a platform for the design of protein nanocontainers protecting and delivering valuable cargos such as vaccine components, fluorescent probes or drugs.

Structure of the Pili of Streptococcus pyogenens

Hair-like appendages on the surface of many gram-negative bacteria, commonly called pili, participate to the process of invasion of target tissues. They constitute important virulence factors and their assembly and function have been well established. In contrast, gram-positive bacteria have long been considered as devoid of pili before the recent identification of pili in a virulence island of Streptococci species. We have initiated the structural analysis of the putative backbone pili subunit of Streptococcus pyogenens resulting in the 2Å structure of this protein. In addition to the two-domain fold of the pili subunit, the crystal packing suggests a model for the whole pili assembly. These pili are head-to-tail, covalent polymers of the backbone subunit with sortase-catalysed inter-molecular isopeptide bonds. Interestingly, we also identified two self-generated, intra-molecular isopeptides stabilising each subunit. This presumably allows the pili to resist intense mechanical stress when binding target tissues. Our subsequent 3-D template search of the PDB revealed that such isopeptide bonds could be a common feature of gram-positive bacterial surface proteins that has been overlooked until now.

Publications

Kang H.J., Coulibaly F., Clow, F., Proft T. and Baker E.N. (2007) Stabilizing isopeptide bonds revealed in gram-positive bacterial pilus structure. Science 318:1558-9.

Letzel T.§, Coulibaly F. §, Rey F.A., Delmas B., Jagt E., van Loon A.A. and Mundt E. (2007) Molecular and Structural Bases for the Antigenicity of VP2 of Infectious Bursal Disease Virus. J. Virol.81:12827-35  (§ joint first authors)

Hyun J.K. §, Coulibaly F. §, Turner A.P., Baker E.N., Mercer A.A. and Mitra A.K. (2007) The Structure of a Putative Scaffolding Protein of Immature Poxvirus Particles by Electron Microscopy Suggests Similarity with Capsid Proteins of Large Icosahedral DNA Viruses. J. Virol. 81:11075-83. (§ joint first authors)

Coulibaly F., Chiu E., Ikeda K., Gutmann S., Haebel P.W., Schulze-Briese C., Mori H. and Metcalf, P. (2007). The Molecular Organization of Cypovirus Polyhedra. Nature 446:97-101.

Coulibaly F., Chevalier C., Gutsche I., Pous J., Navaza J., Bressanelli S., Delmas B. and Rey F.A. (2005). The Birnavirus Crystal Structure Reveals Structural Relationships among Icosahedral Viruses. Cell 120:761-772.

Coulibaly, F., Chevalier, C., Galloux, M., Da Costa, B., Lepault, J., Delmas, B. and Rey, F.A. (2006). Les relations structurales entre birnavirus et autres virus icosaédriques à ARN. Virologie 10:233-235.