Bringing Molecules to Life: novel blends of crystallography and electron cryo microscopy (CryoEM) to study virus particle dynamics in
John E. Johnson
Department of Molecular Biology , The Scripps Research Institute , La Jolla, CA
jackj@scripps.edu
Programmed movement at the molecular level is the essence of living systems. Energy sources to drive these actions have evolved with both straightforward and sophisticated mechanisms. Complex viruses occupy an interesting and informative niche in the arena of large-scale molecular motion and associated driving forces. They require the formation of assembly intermediates due to the intricate and convoluted nature of the final nucleoprotein products. These intermediates organize a complete or partial inventory of particle components through an initial equilibrium reaction that favors assembly. At a defined stage of this process elements of the transitional complex (often a virally encoded protease or auto-catalytic subunit processing) are activated to create an irreversible assembly state that is, itself, metastable. This intermediate is referred to as a procapsid and normally matures shortly after assembly and does not accumulate unless maturation is blocked. The procapsids are imbued with an energy landscape that, when raised from the local minima, drives programmed subunit trajectories of exceptional length and rotary motion leading to dramatic changes in the contacts between subunits. The termination of this reorganization achieves a global energy minimum and a structure that defines the mature virion. Procapsids were first observed among the dsDNA bacteriophages (1) , but have now also been observed for herpesviruses (2) , retroviruses (3) , dsRNA phages (4) , and an insect virus (5) . To date our observations of Procapsid to Capsid transitions have agreed remarkably well with the concept of "autostery" described 25 years ago by Don Caspar (6) . In this presentation examples of capsid maturation in two virus systems will be described. A molecular motor encoded within the viral subunit drives the maturation in one case (a single stranded RNA insect virus) and this motor is controlled by pH and is reversible in the absence of auto-proteolysis. Maturation in the second case (the capsid of a dsDNA bacteriophage) is driven by the creation of a metastable intermediate created by hydrolysis of a portion of capsid protein by a virally encoded protease. This metastable particle is driven from its local minima by DNA packaging causing dramatic expansion and morphological change.
Nudaurelia Capensis omega virus (N w V)
N w V infects larvae of the Pine Emperor moth. The virus is icosahedral in shape, 410Å in its maximum dimension and is formed by 240 chemically identical subunits distributed with T=4 quasi symmetry. The subunit is 70kD and undergoes a post assembly, autocatalytic, cleavage at residue 570 (7) in the authentic virus. The subunit has three domains, a canonical virus subunit b -sandwich, an Ig domain inserted between two strands of the b sandwich, and an internal helical domain formed by residues at the N and C terminal portions of subunit. The internal helices are the molecular switches that determine if the subunits are located at pentamers or hexamers (8) . Expression of the N w V subunit in a baculovirus system leads to spontaneous assembly into a spherical procapsid (when purified at pH 7.0 or above) with T=4 symmetry, a diameter of 480Å, large pores and no autocatalytic cleavage (5) . The procapsid spontaneously transforms into an icosahedral-shaped virion that undergoes autocatalytic cleavage and is indistinguishable from the authentic virion described above when the pH is lowered to 5.0 (9) . The fully mature particle after cleavage does not expand when the pH is raised to 7 or above. Changing ASN 570 to THR prevents cleavage and makes the morphological change reversible (10) . The presentation describes recent experiments that indicate the procapsid is an organizational event that positions all of the subunits in their proper locations, but in a weakly interacting state. Lowering the pH protonates a cluster of acidic residues that eliminates electrostatic repulsion and allows the particle to compact. Cleavage locks the particle in this state by uncoupling the electrostatic "motor" from the "cargo" and eliminates the reversibility.
Bacteriophage HK97
HK97 is a temperate, l -like coliophage with a 40Kbp, dsDNA, genome. Near the 5' end of the genome are 3 contiguous genes compromising less than 4Kbp that encode the proteins found in the naturally occurring capsid (11) . Two (gp3, gp5) are present in whole or in part throughout morphogenesis, the third (gp4) is a protease that is transient component found only in the initial assembly product (12) . The initially assembled particle has T=7 quasi symmetry containing 415 identical proteins (gp 5) with one pentamer replaced by 12 portal proteins (gp 3) and roughly 60 copies of the protease (gp 4) packaged within the particle. Upon assembly the protease is activated and digests the first 103 amino acids from the head protein and then digests itself, with all the polypeptide fragments exiting the capsid, leaving a particle composed of a 31kD head protein and a portal, now competent to package DNA. This is the natural occurring, metastable, Procapsid. The 40Kbp DNA genome is next inserted into the Procapsid through the portal and this triggers a remarkable reorganization of the particle quaternary structure and local refolding of the subunit. The overall shape of the particle changes from a corrugated, round shell with protuberances at the pentamer and hexamer axes to an icosahedrally shaped particle with thin, smooth walls and flat faces between the 5-fold axes. As described below, we have shown that expansion results from a rapid first stage and a slower second stage when the autocatalytic cross-linking of the subunits occurs in which the side chains of LYS169 and ASN356 are joined with the release of NH 4 +. This reaction chemically joins subunits to each other, forming hexamers and pentamers and also physically concatenates hexamers and pentamers to each other resulting in a “chain-mail” association of proteins that chemically and physically associates the subunits (13) . Either following, or simultaneously with the cross-linking, the tail assembly is added to the portal to complete the formation of the mature particle.
The assembly of HK97 can be recapitulated in an expression system if only the capsid protein and the protease are expressed. Treating the particles with urea can mature the procapsid. The mature icosahedral particle is fully crosslinked, contains no portal and no tail and it was crystallized and the structure solved at 3.4Å (14) . The chain mail organization of the subunits was confirmed. The procapsid structure was also crystallized and the structure recently solved (15) . The lecture will describe recent studies of the particle maturation using a variety of biophysical methods (16, 17) . HK97 is like a molecular "mouse trap" that is caught in a local energy minima in Prohead II. The energy landscape drives it into the fully stable mature form.
1. Simon, L. D. 1972. Infection of Escherichia coli by T2 and T4 bacteriophages as seen in the electron microscope: T4 head morphogenesis. Proc Natl Acad Sci U S A69:907-11.
2. Trus, B. L., Booy, F. P., Newcomb, W. W., Brown, J. C., Homa, F. L., Thomsen, D. R., and Steven, A. C. 1996. The herpes simplex virus procapsid: structure, conformational changes upon maturation, and roles of the triplex proteins VP19c and VP23 in assembly. J Mol Biol263:447-62.
3. Turner, B. G., and Summers, M. F. 1999. Structural biology of HIV. J Mol Biol285:1-32.
4. Butcher, S. J., Dokland, T., Ojala, P. M., Bamford, D. H., and Fuller, S. D. 1997. Intermediates in the assembly pathway of the double-stranded RNA virus phi6. Embo J16:4477-87.
5. Canady, M. A., Tihova, M., Hanzlik, T. N., Johnson, J. E., and Yeager, M. 2000. Large conformational changes in the maturation of a simple RNA virus, nudaurelia capensis omega virus (NomegaV). J Mol Biol299:573-84.
6. Caspar, D. L. 1980. Movement and self-control in protein assemblies. Quasi-equivalence revisited. Biophys J32:103-38.
7. Agrawal, D. K., and Johnson, J. E. 1995. Assembly of the T = 4 Nudaurelia capensis omega virus capsid protein, post-translational cleavage, and specific encapsidation of its mRNA in a baculovirus expression system. Virology207:89-97.
8. Munshi, S., Liljas, L., Cavarelli, J., Bomu, W., McKinney, B., Reddy, V., and Johnson, J. E. 1996. The 2.8 A structure of a T = 4 animal virus and its implications for membrane translocation of RNA. J Mol Biol261:1-10.
9. Canady, M. A., Tsuruta, H., and Johnson, J. E. 2001. Analysis of rapid, large-scale protein quaternary structural changes: time-resolved X-ray solution scattering of Nudaurelia capensis omega virus (NomegaV) maturation. J Mol Biol311:803-14.
10. Taylor, D. J., Krishna, N. K., Canady, M. A., Schneemann, A., and Johnson, J. E. 2002. Large-scale, pH-dependent, quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis. J Virol76:9972-80.
11. Duda, R. L., Martincic, K., and Hendrix, R. W. 1995. Genetic basis of bacteriophage HK97 prohead assembly. J Mol Biol247:636-47.
12. Duda, R. L., Martincic, K., Xie, Z., and Hendrix, R. W. 1995. Bacteriophage HK97 head assembly. FEMS Microbiol Rev17:41-6.
13. Duda, R. L. 1998. Protein chainmail: catenated protein in viral capsids. Cell94:55-60.
14. Wikoff, W. R., Liljas, L., Duda, R. L., Tsuruta, H., Hendrix, R. W., and Johnson, J. E. 2000. Topologically linked protein rings in the bacteriophage HK97 capsid. Science289:2129-33.
15. Wikoff, W. R., Che, Z., Duda, R. L., Hendrix, R. W., and Johnson, J. E. 2003. Crystallization and preliminary analysis of a dsDNA bacteriophage capsid intermediate: Prohead II of HK97. Acta Crystallogr D Biol Crystallogr59:2060-4.
16. Gan, L., Conway, J. F., Firek, B. A., Cheng, N., Hendrix, R. W., Steven, A. C., Johnson, J. E., and Duda, R. L. 2004. Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation. Mol Cell14:559-69.
17. Lee, K. K., Gan, L., Tsuruta, H., Hendrix, R. W., Duda, R. L., and Johnson, J. E. 2004. Evidence that a Local Refolding Event Triggers Maturation of HK97 Bacteriophage Capsid. J Mol Biol340:419-33.
