Bentley A Fane

Bentley A Fane

Professor, Plant Sciences
Professor, Applied BioSciences - GIDP
Professor, Genetics - GIDP
Professor, Immunobiology
Professor, BIO5 Institute
Primary Department
Department Affiliations
Contact
(520) 626-6634

Work Summary

Upon infection, viruses must transport their genomes into cells and produce progeny, often under a strict time deadline. We study how the viral proteins interact with with each other and with host cell proteins to efficiently accomplish these processes.

Research Interest

Bentley A. Fane, PhD, is a Professor in the School of Plant Sciences, College of Agriculture and Life Sciences and holds a joint appointment in the Department of Immunobiology, Arizona College of Medicine. Dr. Fane has an international reputation for his research into virus structure, assembly and evolution. His research focuses on the viruses of the Microviridae, of which he is considered one of the leading experts. He has been instrumental in defining the biochemical and structural parameters that allow these viruses to replicate and produce progeny in as little as five minutes. The rapid lifecycle has facilitated in depth studies into how viruses evolved resistance mechanism to anti-viral proteins targeting particle assembly.He has published over 60 original research paper in leading scientific journals, including Nature, Molecular Cell, and Journal of Virology, in which his publications on the evolution of resistance mechanisms and kinetic traps have been selected by the journal editors as articles of “significant interest.” He is a frequent presenter at national and international meetings, and has been invited to State of the Art and plenary talks at give the American Society for Virology. He presently serves on the Editorial Boards of two leading virology journals: Virology and the Journal of Virology. At the University of Arizona, Dr. Fane has been actively involved in promoting undergraduate research has been honored with teaching awards on the department, college, and university levels. Keywords: Virus structure and assembly, Viral DNA translocation, Viral evolution

Publications

Morais, M. C., Fisher, M., Kanamaru, S., Przybyla, L., Burgner, J., Fane, B. A., & Rossmann, M. G. (2004). Conformational switching by the scaffolding protein D directs the assembly of bacteriophage φX174. Molecular Cell, 15(6), 991-997.

PMID: 15383287;Abstract:

The three-dimensional structure of bacteriophage φX174 external scaffolding protein D, prior to its interaction with other structural proteins, has been determined to 3.3 Å by X-ray crystallography. The crystals belong to space group P41212 with a dimer in the asymmetric unit that closely resembles asymmetric dimers observed in the φX174 procapsid structure. Furthermore, application of the crystallographic 41 symmetry operation to one of these dimers generates a tetramer similar to the tetramer in the icosahedral asymmetric unit of the procapsid. These data suggest that both dimers and tetramers of the D protein are true morphogenetic intermediates and can form independently of other proteins involved in procapsid morphogenesis. The crystal structure of the D scaffolding protein thus represents the state of the polypeptide prior to procapsid assembly. Hence, comparison with the procapsid structure provides a rare opportunity to follow the conformational switching events necessary for the construction of complex macromolecular assemblies.

Fane, B., Ruboyianes, M. V., Chen, M., Dubrava, M. S., Cherwa, J. E., & Fane, B. A. (2009). The expression of N-terminal deletion DNA pilot proteins inhibits the early stages of phiX174 replication. Journal of virology, 83(19).

The phiX174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (DeltaH). The expression of the DeltaH proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory DeltaH proteins, restoring both DNA and protein synthesis to wild-type levels. DeltaH proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the DeltaH proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled.

Cherwa Jr., J. E., & Fane, B. A. (2009). Complete virion assembly with scaffolding proteins altered in the ability to perform a critical conformational switch. Journal of Virology, 83(15), 7391-7396.

PMID: 19474099;PMCID: PMC2708623;Abstract:

In the φX174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30° kink to form in α-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rossmann, M. G., Dokland, T., Bemal, R., McKenna, R., Dag, L. L., & Fane, B. A. (1998). Structure of a viral procapsid with molecular scaffolding. FASEB Journal, 12(8), A1332.

Abstract:

Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Eschcrichia coli bacteriophage 4X174. The XI74 virion contains the proteins F, G, H and I. During assembly, two scaffolding proteins B and D are required for the formation of a 108S, 360-A diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturadon involves packaging of DNA andJ proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel -sandwich motif common to many plant and animal viruses. The structure of a procapsid-like particle at 3.5 A resolution will be described, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and the F protein undergoes conformational changes during capsid maturation.

Cherwa, J. E., Tyson, J., Bedwell, G. J., Brooke, D., Edwards, A. G., Dokland, T., Prevelige, P. E., & Fane, B. A. (2017). ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro. Journal of Virology (article was selected for the journal's "Spotlight" section), 91(1).

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context.