Dennis T. Brown
BA Chemistry/Biology, University of Pennsylvania,
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]
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 Virol79, 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.
Wang, G., Hernandez, R., Weninger, K., and Brown, D. T. (2007). Infection of cells by Sindbis virus at low temperature. Virology 362(2), 461-7.
Hernandez, R., Paredes, A., and Brown, D. T. (2008). Sindbis virus conformational changes induced by a neutralizing anti-E1 monoclonal antibody. J Virol 82(12), 5750-60. Featured on journal cover
Kononchik, J. P., Jr., Nelson, S., Hernandez, R., and Brown, D. T. (2009). Helical virus particles formed from morphological subunits of a membrane containing icosahedral virus. Virology 385(2), 285-93.
Nanda, K., R. Vancini, et al. (2009). "A high capacity Alphavirus heterologous gene delivery system." Virology 390(2): 368-373.
Hafer, A., Whittlesey, et.al. (2009). “ Differential incorporation of cholesterol by Sindbis virus grown in mammalian or insect cells” J Virol 83 (18): 9113-21.
Brown, D.T. and Raquel Hernandez. (2010). Host Range Virus Mutants as Vaccines for Arthropod Vectored Viruses. Nova Acta Leopoldina NF 98, Nr. 361, 201-213.
Mudiganti, U., Hernandez, R., and Brown, D. T. (2010). Insect response to alphavirus infection--establishment of alphavirus persistence in insect cells involves inhibition of viral polyprotein cleavage. Virus Res 150(1-2), 73-84.
He, L., Piper, A., Meilleur, F., Myles, D. A., Hernandez, R., Brown, D. T., and Heller, W. T. (2010). The structure of Sindbis virus produced from vertebrate and invertebrate hosts as determined by small-angle neutron scattering. J Virol 84(10), 5270-6.
Hernandez R, Brown DT.Growth and maintenance of baby hamster kidney (BHK) cells. Curr Protoc Microbiol. 2010 May;Chapter 4:Appendix 4H.
Hernandez R, Sinodis C, Brown DT.Sindbis virus: propagation, quantification, and storage. Curr Protoc Microbiol. 2010 Feb;Chapter 15:Unit15B.1.
Hunt, S. R., Hernandez, R., and Brown, D. T. (2011). Role of the Vacuolar-ATPase in Sindbis Virus Infection. J Virol 85(3), 1257-66.
Kononchik, J. P., R. Vancini, et al. (2011). "Alphavirus adsorption to mosquito cells as viewed by freeze fracture immunolabeling." Virology 415(2): 132-140
Smith, K.M., Nanda, K., Spears, C.J., Ribeiro, M.,
Vancini, R., Piper, A., Thomas, G.S., Thomas, M.E., Brown, D.T., Hernandez, R.,
2011. Structural mutants of dengue virus 2 transmembrane domains exhibit
host-range phenotype. Virol J 8, 289.
Lilin He, Amanda Piper, Flora Meilleur, Raquel Hernandez, William T. Heller, Dennis T. Brown. 2011 Conformational changes in Sindbis virus induced by decreased pH revealed by small-angle neutron scattering J Virol 84(10): 5270-5276.
Featured on journal cover
Ricardo Vancini; Angel Paredes; Mariana Ribeiro; Kevin Blackburn; Davis Ferreira; Joseph P. Kononchik Jr. Raquel Hernandez and Dennis Brown, 2012. Espirito Santo Virus: A new Birnavirus that replicates in insect cells Journal of virology 86(5): 2390-2399
Featured on journal cover
Brown, D. T. and R. Hernandez (2012). "Infection of cells by alphaviruses." Advances in experimental medicine and biology 726: 181-199.
Vancini, R., L. D. Kramer, M. Ribiero, R. Hernandez and D.T. Brown. (2013). "Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane." Virology 435(2): 406-414
Vancini, R., G. Wang, D. Ferreira, R. Hernandez and D.T. Brown (2013). "Alphavirus genome delivery occurs directly at the plasma membrane in a time- and temperature-dependent process." J Virol 87(8): 4352-4359.
Featured on Journal Cover
Updated April 1, 2013