2006 Baum, Rebecca

							                               Baum, Rebecca
            Three-Dimensional Modeling of Two Forms of the
                          Hepatitis B Virus
                          David M. Belnap, Chemistry and Biochemistry


The hepatitis B virus (HBV) is a pathogen that causes liver disease and death in over one million
individuals per year. It is an enveloped retrovirus that is composed of a protein capsid
surrounding a nucleic acid core. This nucleic acid core, depending upon the maturation state of
the virus, consists of either RNA or DNA. The protein capsid of HBV is composed of four
quasiequivalent subunits (subunits that have identical polypeptide (amino acid) sequences but
slightly different tertiary structures). Each subunit forms an alpha-helical hairpin structure and
dimerizes with another subunit. The result is the appearance of spikes on the surface of the viral
capsid. 1

In 1997 Conway et al. and Bottcher et al. separately published results concerning the protein
capsid structure of the hepatitis B virus.2,3 Though the structures were nearly identical, the
orientation of the protein dimers differed. Other studies have also shown that the protein capsid
of HBV is quite flexible and can assume slightly different conformations based on the type of
nucleic acid that resides inside the capsid.4 In our study we have shown via cryo-electron
microscopy and handedness experiments that RNA-filled capsids and capsids that lack nucleic
acid are not only flexible, but exist in two slightly different conformations. And we are currently
involved in modeling the two structures in order to assess their residue interaction distinctions,
but have not yet obtained concrete conclusions.

The three-dimensional capsid reconstructions of HBV took more time than I had anticipated.
Thousands of particles were imaged by our collaborators at NIH so that we could create
relatively high resolution reconstructions (under 10 angstroms) of the viral capsids. The
handedness tests were less involved but provided us with unexpected results. We had
hypothesized that the two capsid conformations were based upon the nucleic acid interior. We
isolated and reconstructed RNA-filled capsids assuming that they would exist in only one
conformation. Yet when a handedness test was applied to our data two conformations were
shown to exist. This caused me to critically analyze our data and forced me to brainstorm for
new hypotheses.

To assess factors that could contribute to the two different capsid conformations and to model
dimer-dimer interactions between the two forms, we used fitting programs to dock the X-ray
crystal structure of the viral capsid into our three-dimensional reconstructions. By using higher
resolution reconstructions of the two forms, the crystal structure should fit with an error of  2 A
resulting in pseudo-atomic detail. We used multiple fitting programs in order to assess the
interactions that lead to the two forms of HBV, yet none accurately fit our crystal coordinates
into our density maps. We are still in the process of this modeling and hope to have more
concrete results by the summer.
The overall experience of this research revealed the frustration of unexpected results, the
excitement of new thoughts and hypotheses, the investment of time required for data collection,
and the depth of understanding acquired in an environment that applies classroom instruction. I
have found that what I thought to be a simple capsid reconstruction has resulted in a plethora of
questions and in multiple opportunities for further investigation into this capsid phenomenon.
The discovery and analysis of multiple capsid forms for HBV may instigate further research into
what roles these two protein forms play in its replication cycle. While the knowledge of the true
structure and pathway of the viral capsid will be valuable information for those developing drugs
against the virus that has caused agony and death in millions of individuals.

                                      References

1. Steven, A. C., Conway, J. F., Cheng, N., Watts, N. R., Belnap, D. M., Harris, A., Stahl,
   S. J., Wingfield, P. T. Structure, Assembly, and Antigenicity of Hepatitis B Virus
   Capsid Proteins. Advances in Virus Research 64: 125-164.

2. Conway, J. F., N. Cheng, A. Zlotnick, P. T. Wingfield, S. J. Stahl, and A. C. Steven.
   1997. Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron
   microscopy. Nature 386:91-94.

3. Böttcher, B., S. A. Wynne, and R. A. Crowther. Determination
   of the fold of the core protein of hepatitis B virus by electron cryomicroscopy.
   Nature 386:88-91.

4. Roseman, A. M., Berriman J. A., Wynne, S. A., Butler, J. G., Crowther R. A. A
   Structural Model for Maturation of the Hepatitis B. Virus Core. PNAS 102: 15821-
   15826.