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Dennis T. Brown
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BA Chemistry/Biology, University of Pennsylvania,
1964 Email: dennis_brown@ncsu.edu
Brown/Hernandez Research Group
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Research Interests
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My laboratory investigates the structure, function and assembly of the model
membrane containing virus Sindbis. Sindbis virus is the prototype of the alpha viruses,
a group of infectious agents which are transmitted in nature by blood sucking
insects. The alphaviruses are simple in composition (composed of multiple copies
of three proteins) but complex in structure. The virus is made up of two structurally
identical icosahedral protein shells between which is sandwiched a membrane
bilayer. The two protein shells are connected to one another by
protein-protein interactions which span the membrane bilayer. The protein associations
result in a very strong but metastable structure capable of rapidly disassembling
to release its genetic material (a single strand of RNA) into a host cell upon
contacting an appropriate receptor.
The structure of the mature virion is being determined at high resolution using Electron Cryo-Microscopy. This technology uses massively parallel computers to analyze images of viruses obtained in high voltage electron microscopes equipped with liquid nitrogen stages and field emission guns. Data obtained by morphological studies are verified by biophysical and biochemical analysis of protein configuration and protein/protein associations in the virus structure. Our goal is to produce an image of the structure of this membrane containing virus at atomic resolution.
We are determining the pathway by which virus components are folded and associate with one another inside of the infected cell as the infectious virion is assembled from its component parts. We have determined that the two membrane glycoproteins which make up the outer icosahedral shell pass through a number of complex disulfide bridged intermediates as they are combined into the functional virion. The techniques of molecular and cellular biology together with molecular cloning and site directed mutagenesis are employed to elucidate the pathway of virus assembly. [description of Sindbis virus assembly]
Because the alphaviruses are vectored by insects they must be able to replicate in cells of both vertebrate and invertebrate origin. This has provided us with the opportunity to examine the expression of a single piece of genetic material (the virus RNA) in two phylogeneticaly unrelated biochemical and genetic environments. The expression of the virus genome has dramatically different consequences in the two host cell types in both the manner in which the cells respond to infection (vertebrate cells are killed, insect cells are not killed), and in the manner in which the virus is assembled within the two hosts. We are presently examining alterations in the pattern of cellular gene expression as the insect cell responds to virus infection by establishing a persistently infected state.
Images were provided by Dr. Angel Paredes Baylor College of Medicine. See the article "Parades, A.M., D.T. Brown, W. Chiu, R. Rothnagel, R. Johnston, R. Schoep, and B.V.V. Prasad (1993). Three-dimensional structure of a Membrane-containing virus. Proc. Nat. Acad. Sci. USA.90:9095-9099. [abstract]
Carlton, M. and D.T. Brown. (1996) Events in the Endoplasmic Reticulum Abrogate the Temperature Sensitivity of Sindbis Virus Mutant TS23. J. Virol. 70:952-959 [abstract]
Mulvey,M. and D.T. Brown. (1996) Assembly of the Sindbis Virus Spike Protein Complex. Virology. 219: 125-132 [abstract]
Liu N., H. Lee, R. Hernandez, and D.T. Brown. (1996) Mutations in the Endo Domain of Sindbis Glycoprotein E2 Block Phosphorylation, Reorientation of the Endo Domain and Nucleocapsid Binding. Virology 222: 226-246 [abstract]
Carlton, M. and D.T. Brown. (1996) Disulfide Bridge Mediated Folding of Sindbis Virus Glycoproteins. J. Virol. 70: 5541-5547 [abstract]
Lee, S., K. E. Owen, H-K. Choi, H. Lee, G. Lu, G. Wengler. D. T. Brown, M. Rossmann and R. J. Kuhn. (1996) Identification of a protein binding site on the surface of the alphavirus nucleocapsid and its implication in virus assembly. Structure. 4: 531-541
Carleton M., H. Lee, M. Mulvey and D.T. Brown. (1997) The Role of PE2 in Formation and Maturation of the Sindbis Virus Spike. J. Virol.71: 1558-1566 [abstract]
Karpf, A.R., J.M. Blake and D. T. Brown. (1997)Charactorization of the infection of Aedes albopictus cell clones by Sindbis virus. Virus Research. 50 : 1-13. [abstract]
Karpf, A. R., E. Lenches, E. G. Strauss, J.H. Strauss and D.T. Brown. (1997) Superinfection Exclusion of Alphaviruses in Three Mosquito Cell lines Persistently Infected with Sindbis Virus. J. Virol. 71:7119- 7123 [abstract]
Carleton M.C. and D.T. Brown. (1997) The Formation of Intra molecular Disulfide Bridges is Required for the Induction of Sindbis Virus Mutant ts23 Phenotype. J. Virol. 71: 7696-7703 [abstract]
Karpf, A.R. and D.T. Brown. (1998) Comparison of Sindbis virus-Induced pathology in mosquito and vertebrate cell cultures. Virology. 240: 193-201 [abstract]
Hernandez, R., H. Lee, C. Nelson and D.T. Brown (2000) A Single Deletion in the Membrane-Proximal Region of the Sindbis Virus Glycoprotein E2 Blocks Virus Assembly. J.Virol. 74: 4220-4228 [abstract]
Phinney, B. P., K. Blackburn and D.T. Brown (2000) The Surface Conformation of the Sindbis Virus Glycoproteins E1 and E2 at Neutral and Low pH as Determined by Mass-Spectroscopy Based Mapping. J. Virol. 74: 5667-5678 [abstract]
Brett S. Phinney and Dennis T. Brown (2000) Sindbis Virus Glycoprotein E1 is Divided into Two Discrete Domains at Amino Acid 129 by Disufide Bridge Connections. J. Virol. 74: 9313-9316 [abstract]
Hernandez, R., T. Luo1 and D.T. Brown (2001) Exposure to low pH is not required for the penetration of mosquito cells by Sindbis virus. J. Virol. 75:2010-2013 [abstract]
Pletnev, S., W. Zhang, S. Mukhopadhyay, B. Fisher, R. Hernandez, D. T. Brown, T. S. Baker, M. G. Rossmann and R. J. Kuhn (2001). Location of Carbohydrate Sites on Sindbis Virus Glycoprpteins Shows that E1 Forms an Icosahedreal Scaffold. Cell: Vol 105. 127-136 [ abstract]
Davis Ferreira, Raquel Hernandez, Michelle Horton and Dennis T. Brown (2003)Morphological variants of Sindbis virus produced by a mutation in the capsid protein. Virology 307: 54-6695.
Hernandez, R., Nelson, S., Salm, J. R., Brown, D. T., and Alpert, A. J. (2004). Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column. J Virol Methods 120(2), 141-9.
Hernandez, R., Sinodis, C., Horton, M., Ferreira, D., Yang, C., and Brown, D. T. (2003). Deletions in the transmembrane domain of a sindbis virus glycoprotein alter virus infectivity, stability, and host range. J Virol 77(23), 12710-9.
Paredes, A. M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., and Brown, D. T. (2004). Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-86.
Steevenson Nelson, Raquel Hernandez, Davis Ferreira, and Dennis T. Brown. (2005) In Vivo processing and isolation of furin protease sensitive alphavirus glycoproteins: A new technique for producing mutations in virus assembly, Virology 332: 629-639
Hernandez, R., Ferreira, D., Sinodis, C., Litton, K. & Brown, D. T. (2005). Single amino acid insertions at the junction of the sindbis virus E2 transmembrane domain and endodomain disrupt virus envelopment and alter infectivity. J Virol 79, 7682-97.
Nelson, S., Hernandez, R., Ferreira, D. & Brown, D. T. (2005). In vivo processing and isolation of furin protease-sensitive alphavirus glycoproteins: a new technique for producing mutations in virus assembly. Virology 332, 629-39.
Sharp, J. S., S. Nelson, D. Brown, and K. B. Tomer. (2006). Structural characterization of the E2 glycoprotein from Sindbis by lysine biotinylation and LC-MS/MS. Virology. 348:216-223
West, J., and D. T. Brown. (2006). The role of a conserved tripeptide in the endodomain of Sindbis virus glycoprotein E2 in virus assembly and function. J. Gen Virol 87:657-654.
Caryn L. Heldt,a Raquel Hernandez,b Usharani Mudiganti,b Patrick V. Gurgel,c Dennis T. Brown,b Ruben G. Carbonell,a*( 2006) A colorimetric assay for viral agents which produce cytopathic effects , J Virol Meth. 135: 56-65
John West, Raquel Hernandez, Davis Ferreira and Dennis T. Brown* (2006) Mutations in the endodomain of Sindbis virus Glycoprotein E2 define sequences critical for virus assembly. J. Virol. 80:4458-4468
Raquel Hernandez*, Christine Sinodis and Dennis T. Brown (2006) Sindbis Virus: Propagation, Quantitation, and Storage. Current Protocols in Microbiology
Whitehurst, C. B., Willis, J. H., Sinodis, C. N., Hernandez, R., and Brown, D. T. (2006). Single and multiple deletions in the transmembrane domain of the Sindbis virus E2 glycoprotein identify a region critical for normal virus growth. Virology 347(1), 199-207.
Mudiganti, U., R. Hernandez, D. Ferreira, and D. T. Brown. 2006. Sindbis virus infection of two model insect cell systems--a comparative study. Virus Res 122:28-34.
Wang, G., R. Hernandez, K. Weninger, and D. T. Brown. 2007. Infection of cells by Sindbis virus at low temperature. Virology 362:461-7.
Whitehurst, C. B., E. J. Soderblom, M. L. West, R. Hernandez, M. B. Goshe, and D. T. Brown. 2007. Location and role of free cysteinyl residues in the sindbis virus e1 and e2 glycoproteins. J Virol 81:6231-40.
Updated May 11, 2007
