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Thomas Smith's Laboratory

 

Human Rhinovirus

Human rhinovirus is one of the major causes of the common cold. There are over 100 serotypes of this virus, making it unlikely that there will ever be a traditional vaccine for the common cold using conventional vaccine methods. We have examined the neutralization and dynamics of this virus to better understand how antibodies block infection and how to make more efficacious and novel vaccines.

 

    

 

HRV Organization

About each icosahedral 5-fold axis lies a canyon-like structure. From the atomic structures, the sites of natural escape mutations were found to lie at the top of these canyons. From this result, the dimensions of the canyon, and the conservation of some of the residues lying at the canyon floor, it was thought that the receptor might bind to the bottom of the canyon thus hiding important residues from the immune system. In this way, the virus might be allowed to change its surface residues, thwart antibody binding, but retain crucial residues necessary for viral infectivity.

 HRV structure

 

 

Drugs for the common cold

A series of compounds (WIN) were developed by the Sterling-Winthrop company. They bind to a cavity within VP1 of HRV and prevent uncoating. We have continued analyzing the mode of action of these compounds and have shown that they stabilize or prevent the conformational changes associated with receptor interactions thereby preventing the release of its RNA genome.

 drug picture

 

HRV14 capsid 'breathing'

In a series of studies, we have used MALDI and limited proteolysis to show that portions of the HRV capsid, buried in the crystal structure, are transiently expressed in a 'breathing' process. The first sites of the capsid clipped by matrix bound trypsin are actually buried next to the RNA (yellow spheres). This 'breathing' is shut down by the the addition of WIN compounds and is affected by mutations that fill that drug binding cavity with hydrophobic compounds.

 maldi

 

Mechanisms of antibody neutralization

When these studies were initiated, there were several proposed mechanisms for antibody mediated neutralization of viral infections. They could block several stages of viral infections; attachment, endocytosis, and uncoating. The attachment could be blocked by simply getting in the way with receptor attachment, they could rigidify the capsids, or they could induce conformational changes in the capsid. This latter was the dominant idea at the time. The other popular idea was that antibodies could aggregate the virions and decrease the number of independent particles. Again, the most dominant hypothesis was that antibodies (as few as one) could bind and distort the capsid, rendering it non-infectious.

 neutralization

 

 

 

Antibody-mediated neutralization

In order to better understand which of the above proposed mechanisms is most likely to be relevant in-vivo, our collaborators in the Reuckert lab analyzed the neutralization properties of several monoclonal antibodies. The strongest neutralizing antibodes (12 and 17) were the most straightforward - the more you add the better the neutralization. It was thought that if any antibodies were to cause conformational changes, it would be these since there is no apparent aggregation during the neutralization process. In contrast antibodies like 1 were weakly neutralizing and strongly aggregated the virions. Then there were antibodies with mix properties such as 23 and 14 that strongly aggregated leading to either strong or weak neutralization. Nevertheless, 12 and 17 clearly demonstrated that aggregation is not essential to in-vitro neutralization of HRV14.
antibody-response

 

Cryo-electron microscopy

The next step was to direct test the idea that antibodies induce large conformational changes in the virion. In collaboration with the Baker group, we determined the structures of strongly (12 and 17) and weakly (1) neutralizing antibodies. In all cases there was no evidence of large structural changes in the virion. However, the orientations of the bound Fabs of 12 and 17 suggested that they might be binding bivalently - confirmed by the structure of the IgG of mAb17 bound to HRV14. Even so, large structural changes were not observed. On the other hand, Fab1 binds to the same epitope as 12 and 17 and yet is a strongly aggregating and weakly neutralizing antibody. From the cryo-TEM structure of the complex, this is likely due to binding orientation differences that prevents bivalent attachment and promotes inter-particle cross-linking.

 cryo

 

Atomic structure of the Fab17/HRV14 complex.

The above cryo-TEM image reconstructions clearly suggested that the antibodies do not cause conformational changes in the virion upon binding. However, the resolution of these structures were limited and finer changes might have been missed. To this end, we crystallized the Fab17/HRV14 complex and determined its crystal structure. We found, again, that the virus is neutralized without associated conformational changes. Even more important, we found that the antibody penetrates the receptor binding site. This was important in that the popular idea at the time was that the receptor binding region was canyon shaped to prevent contact with the immune system. This is clearly not the case here. In the end, these studies demonstrated that the immune system is more plastic than previously thought and that vaccine development need only to focus on production of immunogenic pathogen mimics rather than what might happen to the virus after the antibody binds.

 Fab

 

Our relevant publications:

  • Katpally, U., Smith, T. J. (2007) Pocket factors unlikely play a major role in the life cycle of human rhinovirus.  J. Virol. 81:6307-6315.
  • Goncalves, R. B., Mendes, Y. S., Soared, M. R., Katpally, U., Smith, T. J., Silva, J. L., Oliveira, A. C. (2007) VP4 protein from human rhinovirus 14 is released by pressure and locked in the capsid by the antiviral compound WIN.  J. Mol. Biol. 366:295-306.
  • Thomas, J., Katpally, U., Chase, E., Harris, K., Siuzdak, G., Smith, T. J., (2003) Human Rhinovirus dynamics is controlled by canyon flexibility. Virol. 314:34-44.
  • Smith, T. J., (2003) Structural studies on antibody-virus complexes. In Advances in Protein Chemistry (Chiu, W., ed.) 64:445-490.
  • Smith, T. J., (2002) Antibody recognition of human rhinovirus. In Molecular Biology of Picornaviruses (Wimmer, E., and Semler, B., eds) ASM Press, Washington, D.C. pp 39-49.
  • Smith, T., J. (2001) Antibody interactions with rhinovirus; lessons for mechanisms of neutralization and the role of immunity in viral evolution. In Current topics in microbiology and immunology; Antibodies in Viral Infection.  Burton, D. R. ed. (Springer, New York) pp 1-28.
  • Broo, K., Wei, J., Marshall, D., Brown, F., Smith, T. J., Johnson, J. E., Schneeman, A., Siuzdak, G. (2001) Viral capsid mobility: a dynamic conduit for inactivation. PNAS 98:2274-2277.
  • Belnap, D. M., Kumar, A., Folk, J. T., Smith, T. J., Baker, T. S. (1999) Low-resolution density maps from atomic models: how stepping 'back' can be a step 'forward.' J. Struc. Biol. 125:166-175.
  • Smith, T. J. and Baker, T. (1999) Picornaviruses: epitopes, canyons, and pockets. Adv. Virus Res. 52: 1-23.
  • Oliveira, A. C., Ishimaru, D., Goncalves, R. B., Smith, T. J., Mason, P., Sá-Carvalho, D., Silva, J. L. (1999) Low temperature and pressure stability of picornaviruses: implications for virus uncoating. Biophysical Journal 76:1270-1279.
  • Lewis, J. K., Bendahmane, M., Smith, T. J., Beachy, R. N. Siuzdak, G. (1998) Identification of Viral Mutants by Mass Spectrometry. Proc. Natl. Acad. Sci. USA 95:8596-8601.
  • Lewis, J. K., Bothner, B., Smith, T. J., Siuzdak, G. (1998) Antiviral agent blocks breathing of the common cold virus. Proc. Natl. Acad. Sci. 95:6774-6778.
  • Che, Z., Olson, N. H., Leippe, D., Lee, W.-M., Mosser, A., Rueckert, R. R., Baker, T. S., Smith, T. J. (1998) Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryo-electron microscopy and X-ray crystallography of virus-Fab complexes. J. Virol. 72:4610-4622.
  • Thomas J. Smith, Elaine S. Chase, Timothy J. Schmidt, Norman H. Olson, Timothy S. Baker. (1996) Neutralizing antibody to human rhinovirus penetrates the receptor-binding canyon. Nature (London), 383:350-354.
  • Thomas J. Smith, Norman H. Olson, Zhiwei Che, Elaine Chase, Anne Mosser, Donna Leippe, Holland Cheng, Roland R. Rueckert, and Timothy S. Baker. (1996) Structural Studies on Virus-Antibody Interactions. Cold Spring Harbor Symposium Proceedings -Vaccines96. 161-167.
  • Rossmann, M.G., Greve, J.M., Kolatkar, P.R., Olson, N.H., Smith, T.J., McKinlay, M.A., and Rueckert, R.R. (1997) Rhinovirus attachment and cell entry. In Chiu, W., Burnett, R., and Garcea, R. (eds.), Structure Biology of Viruses. Oxford University Press, New York. pp.105-133.
  • Smith, TJ, Mosser, AG, and Baker, TS. (1995) Structural Studies on the Mechanisms of Antibody-Mediated Neutralization of Human Rhinovirus. Seminars in Virology 6:233-242.
  • Smith, TJ, and Mosser, AG. (1997) Antibody-mediated neutralization of picornaviruses. In Chiu, W., Burnett, R., and Garcea, R. (eds.), Structure Biology of Viruses. Oxford University Press, New York pp. 134-156.
  • Chiu, W., and Smith, T.J. (1994) Structural studies of virus-antibody complexes by electron cryomicroscopy and X-ray crystallography. Cur. Opin. in Struc. Biol. 4:219-224.
  • Liu, H., Smith, T.J., Lee, W.M., Leippe, D, Mosser, A., and Rueckert, R.R. (1994) The purification, crystallization, and structure determination of an Fab fragment that Neutralizes Human Rhinovirus 14 J. Mol. Biol. 240:127-137.
  • Rossmann, M.G., Smith, T.J., and Rueckert, R.R. (1993) The structure of human rhinovirus 14. Structure Introductory Issue: xxiv-xxv.
  • Smith, T.J., Olson, N.H., Cheng, R.H., Chase, E.S., and Baker, T.S. (1993) Structure of a human rhinovirus-bivalent antibody complex: Implications for virus neutralization and antibody flexiblity. Proc. Natl. Acad. Sci. USA 90:7015-7018.
  • Smith, T.J., Olson, N.H., Cheng, R.H., Liu, H., Chase, E., Lee, W.M., Leippe, D.M., Mosser, A.G., Ruekert, R.R., and Baker, T.S. (1993) Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody J. Virol. 67: 1148-1158.
  • Smith, T.J., and Chase, E.S. (1992) Purification and crystallization of intact human rhinovirus complexed with a neutralizing Fab Virology 191:600-606.
  • Smith, T. (1992) Purification of mouse antibodies and Fab fragments. In Asai, D. (ed.), Methods in Cell Biology Chapter 4, pp 75-93.
  • Olson, N.H., Smith, T.J., Kolatkar, P.R., Oliveira, M.A., Rueckert, R.R., Greve, J.M., Rossmann, M.G., and Baker, T.S. (1992) Cryoelectron microscopy of complexes of human rhinovirus with a monoclonal Fab and the viral cellular receptor. Proc. Elect. Microsc. Soc. of Amer. 50:524-525.
  • Smith, T.J., Badger, J., Kremer, M., Oliveira, M., Rossmann, M.G., McKinlay, M.A., Diana, G.D., Pavear, D.C., Dutko, F.J., Rueckert, R.R., Heinz, B., and Shepard, D. (1990) Crystallographic and pharmacological studies of antiviral agents against human rhinovirus. In Bugg, C.E., and Ealick, S.E., (eds.), Crystallographic and modelling methods in molecular design Springer-Verlag, New York, pp. 9-28.
  • Kim, S., Smith, T.J., Chapman, M.S., Rossmann, M.G., Peavear, D.C., Dutko, F.J., Felock, P.J., Diana, G.D., and McKinlay, M.A. (1989) The crystal structure of human rhinovirus serotype 1A (HRV1A) J. Mol. Biol. 210:91-111.
  • Heinz, B.A., Rueckert, R.R., Shepard, D.A., Dutko, F.J., McKinlay, M.A., Francher, M., Rossmann, M.G., Badger, J., and Smith, T.J. (1989) Genetic and molecular analysis of spontaneous mutants of human rhinovirus 14 resistant to an antiviral compound. J. Virol. 63:2476-2485.
  • Badger, J., Minor, I., Oliveira, M.A., Smith, T.J., and Rossmann, M.G. (1989) Structural analysis of antiviral agents that interact with the capsid of human rhinoviruses. Proteins 6:1-19.
  • Diana, G.D., Pevear, D.C., Otto, M.J., McKinlay, M.A., Rossmann, M.G., Smith, T.J., and Badger, J. (1989) Inbitors of viral uncoating Pharmacol. Therapeut. 42:289-305.
  • Smith, T.J. (1989) Approaches to antiviral drug design. In Perun, T.J., and Propst, C.L., (eds.), Computer-aided drug design. Methods and applications Mercel Dekker, New York, pp. 371-403.
  • Badger, J., Minor, I., Kremer, M.J., Oliveira, M.A., Smith, T.J., Griffith, J.P., Guerin, M.A., Krishnaswamy, S., Luo, M., Rossmann, M.G., McKinlay, M.A., Dlana, G.D., Kutko, F.J., Fancher, M., Rueckert, R.R., and Heinz, B.A. (1988) Structural analysis of a series of antiviral agents complexed with human rhinovirus 14 Proc. Natl. Acad. Sci. U.S. 85:3304-3308.
  • Diana, G.D., Otto, M.J,. Treasurywala, A.M., McKinlay, M.A., Oglesby, R.C., Maliski, E.G., Rossmann, M.G., and Smith, T.J. (1988) Enantiomeric effects of homologues of disoxaryil on the inhibitory activity against human rhinovirus-14 J. Med. Chem. 31:540-544.
  • Rossmann, M.G., Arnold, E., Griffith, J.P., Kamer, G., Luo, M., Smith, T.J., Vriend, G., Rueckert, R.R., Sherry, B., McKinlay, M.A., Diana, G., and Otto, M. (1987) Common cold viruses Trends Biochem. Sci. 12:313-318.
  • Rossmann, M.G., Arnold, E., Kamer, G., Kremer, M.J., Luo, M., Smith, T.J., Vriend, G., Rueckert, R.R., Mosser, A.G., Sherry, B., Boege, U., Scraba, D.G., McKinlay, M.A., and Diana, G.D. (1987) Viral particles at atomic resolution. In Bercoff, R.P. (ed.), The molecular basis of viral replication Plenum Press, New York, pp.25-44.
  • Rossmann, M.G., Arnold, E., Kamer, G., Kremer, M.J., Luo, M., Smith, T.J., Vriend, G., Rueckert, R.R., Mosser, A.G., Sherry, B., Boege, U., Scraba, D.G., McKinlay, M.A., and Diana, G.D. (1987) Structure and function of human rhinovirus 14 and Mengo virus: Neutralizing antigenic sites, putative receptor binding site, neutralization by drug binding. In Brinton, M.A., and Rueckert, R.R., (eds.), Positive strand RNA viruses Alan R. Liss, New York, pp. 59-77.
  • Smith, T.J., Kremer, M.J., Luo, M., Vriend, G., Arnold, E., Kamer, G., Rossmann, M.G., McKinlay, M.A., Diana, G.D., and Otto, M.J. (1986) The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating Science 233:1286-1293.

 

 
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