Roger M. Burnett, Ph.D.

Professor
Immunology Program
215-898-2201, Office
burnett@wistar.org

Introduction

The laboratory of Roger M. Burnett, Ph.D., studies the molecular structures of viruses to gain insights into their biological functions. One of their main objects of research is human adenovirus, an important vehicle for delivering human gene therapy treatments and vaccines.

Research Summary

The Burnett laboratory studies large, spherical viruses to understand how their molecular structures relate to their various functions. This is not only relevant to understanding how human viruses cause disease, but also to how the viruses can be used therapeutically in human gene therapy or vaccine delivery. Some viruses, called bacteriophages, target bacteria and so are natural bacteriocides. As viruses are comparatively simple examples of biological architecture, they also provide models for how proteins and nucleic acids organize and assemble into large complexes. The researchers use X-ray crystallography to obtain atomic resolution images of the molecules forming the virus particle - or virion - and electron microscopy and image analysis to determine its overall architecture. Human adenovirus, an important vector for human gene therapy and vaccine delivery, has been a major focus of their work. Another is PRD1, an unusual lipid-containing bacteriophage that infects antibiotic-resistant strains of bacteria such as E. coli. The research team discovered that PRD1 has many structural and functional similarities to adenovirus. This shows that animal and bacterial viruses are related and strongly suggests that PRD1 and adenovirus share a primitive ancestor. This similarity is important in that one virus may be used to understand the other.

Recent Scientific Advances

Adenovirus: Adenoviruses cause human ailments such as respiratory infections, conjunctivitis, and enteric dysentery by infecting cells. However, modified adenoviruses can be used as vectors to deliver genes for combating sickness (Farina et al., 2001). The ~1000 Å virion is characterized by its icosahedral shape and the ~300 Å fibers projecting from its 12 vertices (Stewart et al., 1991). The ~150x106 Da virion is complex, containing about 2700 polypeptides from at least 10 different protein species (Burnett, 1997). As it is very large, the Burnett laboratory is using a novel combination of two imaging methods - X-ray crystallography and electron microscopy (EM) - to reveal its structure (Stewart et al., 1993; San Martín & Burnett, 2003; Rux & Burnett, 2004).

The virion architecture is known from a three-dimensional EM image reconstruction at 35 Å resolution (Stewart et al., 1991). This revealed how hexons form the facets and showed the interaction of penton base and fiber at the vertex. A novel difference-imaging approach was devised for the minor protein components. The X-ray hexon image was fitted to its 240 positions in the EM virion image and then subtracted out to disclose the minor proteins (Stewart et al., 1993). Three proteins were found at key positions in the virion where they weld the major components together to form a stable capsid. It is likely that other large macromolecular assemblies contain similar "cementing" proteins. EM imaging of wild-type and mutant adenovirus is currently being performed at very high resolution (15 Å) (Scheres et al., 2005). An improved virion model will more precisely delineate the minor proteins and reveal how they play their important stabilizing role.

X-ray structures of the major coat protein have been obtained for the 967-residue type 2 (Ad2) hexon and the related 951-residue Ad5 hexon (Rux & Burnett, 2004). Hexon's very long polypeptide chains form two 8-stranded "viral" barrels. Hexon is a trimer so its six barrels form a pseudo-hexagonal base that closely packs with neighbors in the capsid to encase the viral genome. The hexon barrels are topologically similar to each other and to all other known viral coat barrels. Three sets of loops rise from the barrels and intertwine to form a triangular hexon top with three towers. This topology makes the molecule highly stable. The towers lie on the outside of the virion to give it the bumpy external surface seen with EM. A comparison of different adenovirus species shows that the towers have highly variable sequences (Rux & Burnett, 2000). The immune system recognizes the adenovirus virion through antibodies that bind to the hexon molecule at these "hypervariable" regions. These have been defined more precisely by mapping all known full-length hexon sequences onto the crystal structures of ad2 and ad5 hexon (Rux et al., 2003).

A major problem in using adenovirus vectors for human gene therapy or vaccine delivery is that most people already have been exposed to common types of human adenovirus, such as Ad2 and Ad5. A promising avenue is to develop novel adenovirus vectors from non-human hosts, such as the chimpanzee (Farina et al., 2001). The research team has crystallized the 933-residue AdC68 chimpanzee hexon (80% sequence identity to Ad5) and is determining its molecular structure (Xue & Burnett, 2006). Artificial variants on the basic adenovirus vectors can be made by using molecular design strategies to vary the hexon towers, while leaving intact the invariant parts of the molecule.

Bacteriophage PRD1: The Burnett laboratory began studies on PRD1 in collaboration with Dr. Dennis H. Bamford (University of Helsinki, Finland) because its architecture is very unusual. PRD1, which infects Gram-negative bacteria, has a lipid membrane within its protein capsid. This study produced the surprising result that PRD1 is remarkably similar to adenovirus. Both have icosahedral virions with vertex fibers, trimeric major coat proteins, and linear double-stranded DNA with terminal proteins. The PRD1 major coat protein, P3, is analogous to adenovirus hexon. The vertex comprises P31, P5, and the receptor-binding protein, P2.

The 1.65 Å resolution X-ray structure of the 394-residue P3 shows the molecule's striking similarity to hexon (Benson et al., 1999). EM image reconstructions show that the virion architecture is the same as that in adenovirus (San Martín et al., 2001). As with hexon, the P3 barrel axes are normal to the facets, ascending loops form "towers" creating protuberances on the viral surface, and loops overlap neighboring subunits to increase molecular stability. As P3 is far smaller than hexon, the size and complexity of its loop region are more modest and its top is much smaller. Both molecules have N-termini underneath, where they can interact with the internal viral membrane in P3 (Benson et al., 1999; San Martín et al., 2001) or with DNA in adenovirus. Most recently, the quasi-atomic model of the PRD1 capsid (San Martín et al., 2002) was used to solve the crystal structure of the entire virion at 4 Å resolution (Abrescia et al., 2004) and reveal its organization in greater detail.

The strong similarities between P3 and hexon show that PRD1 and adenovirus are related and establish the first direct structural link between viruses from the animal and bacterial kingdoms (Benson et al., 1999; Bamford et al., 2002). The architecture of the PRD1 virion shows that it is intermediate between simple and complex spherical viruses (San Martín et al., 2002). PRD1 is stabilized by C-termini from P3, like the simpler polyomaviruses, and also by minor cementing proteins, like adenovirus. The analogy between PRD1 and adenovirus suggests interesting research directions, such as the role that the PRD1 fiber proteins play in entry to host cells. A recent X-ray structure of the 590-residue receptor-binding protein, P2, at 2.4 Å resolution (Xu et al., 2003) is being used to delineate peptides to target the receptor. PRD1 assembly is being studied by combining EM and X-ray images to show the structural changes in maturation (San Martín et al., 2002). As DNA is packaged, the capsid-membrane separation decreases and contacts between the P3 N-terminal helix and the membrane increase fourfold.

The increased prevalence of antibiotic-resistance has aroused renewed interest in using bacteriophages to combat bacterial infections. PRD1 has potential as a novel bactericide as it infects certain strains of Escherichia coli and Salmonella typhimurium that carry genes for antibiotic resistance. An intimate knowledge of PRD1's structure and its infective mechanism will be very helpful in informing the development of targeting and delivery strategies.

Viral Evolution: In recent work, evidence has been found that many other spherical viruses have virions constructed with an architecture similar to that used by adenovirus and bacteriophage PRD1. All have the same key feature – large facets formed by arrays of trimeric coat proteins with a characteristic “double-barrel” fold. As many viral features reflect traits picked up from the host or other microorganisms, these structural similarities provide the only remaining evidence for their shared lineage. A recent hypothesis proposes that these viruses, despite significant differences in genomic size, genetic complexity, and host, evolved from a common ancestor billions of years ago (Benson et al., 2004). There is now firm experimental evidence that the lineage includes viruses infecting hosts from all three domains of life: Eukarya; Bacteria; and Archaea (Burnett, 2006).

Research in this area may ultimately aid the discovery of anti-viral drugs. For example, in two of the viruses studied, one vertex of the apparently symmetric coat is different and used for DNA packaging. If human viruses in the lineage also have this unique vertex, new anti-virals could be developed to target the packaging mechanism.

Selected Publications

Stewart, P.L., Burnett, R.M., Cyrklaff, M. and Fuller, S.D. (1991). Image reconstruction reveals the complex molecular organization of adenovirus. Cell 67, 145-154.

Stewart, P.L., Fuller, S.D. and Burnett, R.M. (1993). Difference imaging of adenovirus: Bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO J. 12, 2589-2599.

Burnett, R.M. (1997). The structure of adenovirus. In: Structural Biology of Viruses (W. Chiu, R.M. Burnett and R.L. Garcea, eds.), Chap. 8, pp. 209-238, Oxford University Press, New York.

Benson, S.D., Bamford, J.K.H., Bamford, D.H. and Burnett, R.M. (1999). Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures. Cell 98, 825-833.

Rux, J.J. and Burnett, R.M. (2000). Type-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexon. Molecular Therapy 1, 18-30.

San Martín, C., Burnett, R.M., de Haas, F., Heinkel, R., Rutten, T., Fuller, S.D., Butcher, S.J. & Bamford, D.H. (2001). Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1, and shows key capsid and membrane interactions. Structure 9, 917-930.

Farina, S.F., Gao, G.-P., Xiang, Z.Q., Rux, J.J., Burnett, R.M., Alvira, M.R., Marsh, J., Ertl, H.C.J. and Wilson, J.M. (2001). Replication-defective vector based on a chimpanzee adenovirus. J. Virology 75, 11603-11613.

Bamford, D.H., Burnett, R.M. and Stuart, D.I. (2002). Evolution of viral structure. Theoretical Population Biology 61, 461-470.

San Martín, C., Huiskonen, J.T., Bamford, J.K.H., Butcher, S.J., Fuller, S.D., Bamford, D.H. and Burnett, R.M. (2002). Minor proteins, mobile arms, and membrane-capsid interactions in the bacteriophage PRD1 capsid. Nature Structural Biology 9, 756-463.

San Martín, C. and Burnett, R.M. (2003). Structural Studies on Adenoviruses. In: Adenoviruses: Model and Vectors in Virus Host Interactions (Current Topics in Microbiology and Immunology Series) (W. Doerfler & P. Böhm, eds.), Chap. 3, pp. 57-94, Springer-Verlag, Berlin.

Xu, L., Benson, S.D., Butcher, S.J., Bamford, D.H. and Burnett, R.M. (2003). The receptor-binding protein P2 of PRD1, a virus targeting antibiotic-resistant bacteria, has a novel fold suggesting multiple functions. Structure 11, 309-322.

Rux, J.J., Kuser, P.R. and Burnett, R.M. (2003). Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution X-ray crystallographic, molecular modeling, and sequence-based methods. J. Virology 77, 9553-9566.

Rux, J.J. and Burnett, R.M. (2004). Adenovirus Structure. Human Gene Therapy 15, 1167-1176.

Abrescia, N.G.A., Cockburn, J.J.B., Grimes, J.M., Sutton, G.C., Diprose, J.M., Butcher, S.J., Fuller, S.D., San Martín, C., Burnett, R.M., Stuart, D.I., Bamford, D.H. and Bamford, J.K.H. (2004). Insights into assembly from structural analysis of bacteriophage PRD1. Nature 432, 68-74.

Benson, S.D., Bamford, J.K.H., Bamford, D.H. and Burnett, R.M. (2004). Does Common Architecture Reveal a Viral Lineage Spanning all Three Domains of Life? Molecular Cell 16, 673-685.

Scheres, S.H.W., Marabini, R., Lanzavecchia, S., Cantele, F., Rutten, T., Fuller, S.D., Carazo, J.M., Burnett, R.M. and San Martín, C. (2005). Classification of single projection reconstructions for cryo-electron microscopy data of icosahedral viruses. J. Struct. Biol. 151, 79-91.

Burnett, R.M. (2006). More barrels from the viral tree of life. Proc. Natl. Acad. Sci. USA. 103, 3-4.

Xue, F. and Burnett, R.M. (2006). Capsid-like arrays in crystals of chimpanzee adenovirus hexon. J. Struct. Biol. In Press.