Robert L. Garcea

Professor M.D., University of California at San Francisco

Current Research

Our understanding of how large macromolecular structures are assembled has been greatly advanced in recent years by the crystallization of viruses. In particular, the polyoma and papillomaviruses (e.g. SV40, human papillomaviruses or HPV) represent some of the largest biological structures yet determined at atomic resolution. Polyoma is a dsDNA virus comprised of 72 pentamers (capsomeres) of the VP1 coat protein, arranged in a T=7 icosahedral shell (capsid; 500 Å in diameter) around a viral minichromosome. Two minor coat proteins, VP2 and VP3, appear to serve as bridges between the viral minichromosome and the capsid. VP1, purified after expression in E. coli, self-assembles in vitro into capsid-like structures upon the addition of calcium. This property has been used as an assay in the structure-function analysis of capsid assembly by introducing mutations in domains of VP1 (identified in the 3.1 Å resolution crystal structure of the virus) involved in capsid formation. A second goal is to analyze the structure and assembly of the related papillomavirus capsid. We have recently solved the structure of a T=1 assembly of the HPV16 L1 (analogous to VP1) capsid protein to 3.5 Å resolution, and are now studying HPV neutralizing epitopes and interpentamer bonds.

Polyomavirus virion assembly occurs in the nucleus of the infected cell. The mechanisms used to both prevent cytoplasmic assembly and promote nuclear assembly are of particular interest. These controls appear to involve cellular chaperone proteins as assembly inhibitors, and calcium ions as assembly promoters. We have recently reconstituted capsid assembly in vitro with both prokaryotic and eukaryotic hsc70 chaperones. We are now attempting to couple chaperone-mediated capsid assembly with the well-characterized SV40 DNA replication system to form complete virions in vitro.

Recombinant HPV proteins are also being studied as potential vaccine candidates to prevent the development of cervical cancer. In particular, we have found that capsomeres are equally as effective in eliciting a protective immune response as fully assembled “virus-like particles” (VLPs). We are now developing a capsomeric vaccine as a next generation prophylactic vaccine for use in underdeveloped countries. The HPV capsid proteins are intrinsically very immunogenic, and fusion of unrelated epitopes to L1 enhances the immunogenicity of the fusions. We are now testing fusions of the early HPV proteins, E6 and E7, to L1 in order to generate a “therapeutic” vaccine that will eradicate already infected cells.

Recent Publications

Click Here For An Updated List Of Dr. Garcea's Publications

Velupillai, P., Garcea, R.L., and Benjamin, T.L. Polyoma Virus-like particles elicit polarized cytokine responses in antigen presenting cells from tumor-susceptible and –resistant mice. J. Immunol. 2006; 176:1148-1153.

Chromy, L.R., Oltman, A., Estes, P.A., and Garcea, R.L. Chaperone-mediated in vitro disassembly of polyoma and papillomaviruses. J.Virol. 2006; 80:5086-5091.

Chromy LR, Oltman A, Estes PA, Garcea RL. Chaperone-mediated in vitro disassembly of polyoma- and papillomaviruses. J Virol. 2006 May;80(10):5086-91.

Garcea RL, Gissmann L. Virus-like particles as vaccines and vessels for the delivery of small
molecules. Curr Opin Biotechnol. 2004 Dec;15(6):513-7. Review.

Chromy LR, Pipas JM, Garcea RL. Chaperone-mediated in vitro assembly of Polyomavirus capsids.
Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10477-82.

Finnen, R.L., Erickson, K.D., Chen, X.S., and Garcea, R.L. Interactions between papillomavirus L1 and L2 capsid proteins. J. Virol. 2003; 77:4818-4826.

Öhlschläger, P., Osen, W., Dell, K. Faath, S., Garcea, R. L., Hochmus, I., Müller, M., Pawlita, M. Schäfer, K. Sehr, P, Staib, C., Sutter, G, Gissmann, L. Human papillomavirus (HPV) type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice. J. Virol. 2003; 77:4635-4645.
Chen, X., Casini, G. Harrison, S.C., and Garcea, R.L. Papillomavirus capsid protein expression in Escherichia coli: Purification and assembly of HPV11 and HPV16 L1 J. Mol. Bio. 2001: 307: 173-182.

Szomolanyi-Tsuda, E., Brien, J.D., Dorgan, J.E., Garcea, R.L., Woodland, R.T., and Welsh, R.M. Antiviral T cell-independent type 2 antibody responses induced in vivo in the absence of T and NK cells. Virology 2001; 280:160-168.

Yuan, H., Estes, P.A., Chen, Y., Newsome, J., Olcese, V.A., Garcea, R.L., and Schlegel, R. Immunization with a pentameric L1 fusion protein protects against papillomavirus infection. J. Virol. 2001; 75: 7848-7853.

Chen, X., Garcea, R.L., Casini, G., Goldberg, I., and Harrison, S.C. Structure of small virus-like particles assembled from the L1 protein of human papillomavirus 16. Mol. Cell 2000; 5:557-567.

Schwartz, R., Garcea, R.L., and Berger, B. "Local Rules" theory applied to polyomavirus polymorphic capsid assemblies. Virology 2000; 268:461-470.

Szomolanyi-Tsuda, E., Brien, J.D., Dorgan, J.E., Welsh, R.M., and Garcea, R.L. The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses. J. Immunol. 2000; 164:5877-82.

Rose, R.C., White, W., Li, M., Suzich, J., Lane, C., and Garcea, R.L. Human papillomavirus type 11 (HPV-11) recombinant L1 capsomeres induce virus-neutralizing antibodies. J. Virol. 1998; 72:6151-6154.