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Virus Structure and Function

J.E. Johnson, A. Schneemann, V. Reddy, T. Lin, G. Cingolani, H. Giesing, P. Natarajan, E. Sabini, J. Tang, L. Tang, D. Taylor, H. Walukiewicz, W. Wikoff

We investigate the structure and function of viruses from a variety of perspectives. We are interested in modes of viral entry, disassembly, assembly, and maturation and in the recognition events associated with each of these processes. By investigating a variety of viruses with genomes stored as single-stranded and double-stranded RNA and as double-stranded DNA, we have developed evolutionary relationships in these functional areas based on structure, genotype, and phenotype. The various virus systems that we study and recent discoveries associated with them are described in the following sections of our report.

We use a variety of physical methods to investigate structure-function relationships, including single-crystal and solution x-ray diffraction, electron cryo-microscopy and image reconstruction, mass spectrometry, structure-based computational analyses, and methods associated with thermodynamic characterization of viral particles and their transitions. Biological methods we use include genetic engineering of viral genes and their expression in Escherichia coli, mammalian cells, insect cells, and yeast and the characterization of these gene products and their assemblies by the physical methods mentioned previously. For cytological studies of viral entry and infection, we use fluorescence and electron microscopy and particles assembled in heterologous expression systems. Our studies depend on extensive collaborations with others at TSRI, including the groups led by C.L. Brooks, M.G. Finn, M.R. Ghadiri, M. Manchester, R.A. Milligan, V. Reddy, A. Schneemann, G. Siuzdak, K.F. Sullivan, and M. Yeager, and a variety of groups outside Scripps.

SINGLE-STRANDED RNA VIRUSES

We investigate single-stranded RNA viruses of animals (nodavirus, tetravirus, and picornavirus) and plants (comovirus). High-resolution crystallographic studies of 4 nodaviruses led to a detailed description of protein-protein and protein-RNA interactions, structure-based hypotheses of modes of entry, RNA translocation into the cytoplasm, and events associated with assembly and maturation. The T = 3 icosahedral particles of nodaviruses consist of 180 subunits of a single type; each subunit is composed of approximately 400 amino acids. In vivo studies currently under way with virus and viruslike particles are designed to test a number of our hypotheses.

The T = 4 icosahedral particles of tetraviruses consist of 240 subunits of a single type; each subunit is composed of approximately 650 amino acids. We solved the high-resolution structure of one virus in this family (Fig. 1), and we are currently investigating a second. Unexpectedly, we found that the tetraviruses are closely related to the nodaviruses at the structural level despite no detectable sequence similarity. Nodaviruses and tetraviruses undergo autocatalytic cleavage via a similar mechanism after assembly; however, the tetraviruses undergo a dramatic structural transition from a 500-Å-diameter procapsid to a 400-Å-diameter capsid. We are using electron cryo-microscopy, solution x-ray scattering, and high-resolution pH titrations to investigate this pH-dependent transition. The transition has been rendered reversible by a site-directed mutation that stops cleavage.

Our crystallographic studies of insect picornaviruses indicated a remarkable similarity between mammalian and insect viruses in this group but also revealed a striking example of a domain swap in the insect structure compared with mammalian viruses. In collaboration with M.G. Finn, Department of Chemistry, we recently developed a novel area of virus study in which we use single-stranded RNA plant viral particles as platforms for chemical, biomaterial, and biological applications based on covalently attached functional groups that range in size from a few atoms to entire proteins.

BACTERIAL VIRUSES

We solved the structure of a 650-Å-diameter, viruslike, mature capsid of the double-stranded DNA phage HK97. The structure revealed a T = 7l capsid that contains 420 subunits of a single type with a novel subunit fold and a remarkable physical cross-linking of the subunits into concatenated hexamers and pentamers. Recently, we used electron cryo-microscopy and crystallography to determine the structure of the 450-Å-diameter HK97 procapsid at moderate resolution. The subunit structure, determined in the mature capsid, was modeled into the density of the procapsid. The dramatic change in particle size from procapsid to capsid is due mostly to rigid body subunit rotations with large changes in subunit dihedral angles. Time-resolved studies of the transition are under way.

DOUBLE-STRANDED RNA VIRUSES

Recently we solved the first structure of a virus that infects yeast. The L-A viral particle is a specialized compartment for the transcription and replication of double-stranded RNA. The particle is 390 Å in diameter and is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, with approximately 2 copies of an RNA-directed RNA polymerase and a 4.6-kb linear duplex RNA. The capsid is organized like the inner cores of reovirus and blue tongue virus but lacks the outer shells found in these mammalian viruses.

L-A virus mRNA synthesized by RNA polymerase is extruded from the shell into the cytoplasm as uncapped mRNA. Compared with capped mRNA, uncapped mRNA is rapidly degraded by cellular enzymes. However, the Gag protein of L-A virus has a 5´-decapping activity that results in a covalent linkage between 7-methyl guanine and His154 of the capsid protein. The structure revealed a cleft where the cleavage occurs, and a proposed mechanism for the chemical reaction is being tested by using mutagenesis.

PUBLICATIONS
Broo, K., Wei, J., Marshall, D., Brown, F., Smith, T., Johnson, J., Schneemann, A., Siuzdak, G. Viral capsid mobility: A dynamic conduit for inactivation. Proc. Natl. Acad. Sci. U. S. A. 98:2274, 2001.

Conway, J., Wikoff, W., Cheng, N., Duda, R., Hendrix, R., Johnson, J., Steven, A. Virus maturation involving large subunit rotations and local refolding. Science 292:744, 2001.

Naitow, H., Canady, M., Lin, T., Wickner, R., Johnson, J. Purification, crystallization and preliminary x-ray analysis of L-A virus. J. Struct. Biol., in press.

Qu, C., Liljas, L., Opalka, N., Brugidou, C., Yeager, M., Beachy, R., Fauquet, C., Johnson, J., Lin, T. 3D domain swapping modulates the stability of members of an icosahedral virus group. Struct. Fold. Des. 8:1095, 2000.

Tang, L., Johnson, K., Ball, L., Lin, T., Yeager, M., Johnson, J. The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA. Nat. Struct. Biol. 8:77, 2001.

Tsuruta, H., Johnson, J. Low-angle x-ray scattering. In: International Table of Crystallography, Vol. F. Rossmann, M., Arnold, E. (Eds.). Kluwer Academic, New York, in press.

Wikoff, W., Liljas, L., Duda, R., Tsuruta, H., Hendrix, R., Johnson, J. Topologically linked protein rings form the dsDNA bacteriophage HK97 capsid. Science 289:2129, 2000.

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