@article{magliocca_vancini_hernandez_brown_2016, title={Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly}, volume={90}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00948-16}, abstractNote={ABSTRACT
          
            A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface.
            35
            S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer.
          
          
            IMPORTANCE
            Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.
          }, number={18}, journal={JOURNAL OF VIROLOGY}, author={Magliocca, Joseph and Vancini, Ricardo and Hernandez, Raquel and Brown, Dennis T.}, year={2016}, month={Sep}, pages={8372–8380} }
 @article{schuchman_vancini_piper_breuer_ribeiro_farreira_magliocca_emmerich_hernandez_brown_2016, title={The role of the vacuolar ATPase in alphavirus replication.}, volume={27}, journal={Molecular Biology of the Cell}, author={Schuchman, R. and Vancini, R. and Piper, A. and Breuer, D. and Ribeiro, M. and Farreira, D. and Magliocca, J. and Emmerich, V. and Hernandez, R. and Brown, D.}, year={2016} }
 @misc{vancini_hernandez_brown_2015, title={Alphavirus Entry into Host Cells}, volume={129}, journal={Molecular basis of viral infection}, author={Vancini, R. and Hernandez, R. and Brown, D.}, year={2015}, pages={33–62} }
 @article{vancini_wang_ferreira_hernandez_brown_2013, title={Alphavirus Genome Delivery Occurs Directly at the Plasma Membrane in a Time- and Temperature-Dependent Process}, volume={87}, ISSN={["0022-538X"]}, DOI={10.1128/jvi.03412-12}, abstractNote={ABSTRACT
          It is widely held that arboviruses such as the alphavirus Sindbis virus gain entry into cells by a process of receptor-mediated endocytosis followed by membrane fusion in the acid environment of the endosome. We have used an approach of direct observation of Sindbis virus entry into cells by electron microscopy and immunolabeling of virus proteins with antibodies conjugated to gold beads. We found that upon attaching to the cell surface, intact RNA-containing viruses became empty shells that could be identified only by antibody labeling. We found that the rate at which full particles were converted to empty particles increased with time and temperature. We found that this entry event takes place at temperatures that inhibit both endosome formation and membrane fusion. We conclude that entry of alphaviruses is by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins.}, number={8}, journal={JOURNAL OF VIROLOGY}, author={Vancini, Ricardo and Wang, Gongbo and Ferreira, Davis and Hernandez, Raquel and Brown, Dennis T.}, year={2013}, month={Apr}, pages={4352–4359} }
 @article{vancini_kramer_ribeiro_hernandez_brown_2013, title={Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane}, volume={435}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2012.10.013}, abstractNote={Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.}, number={2}, journal={VIROLOGY}, author={Vancini, Ricardo and Kramer, Laura D. and Ribeiro, Mariana and Hernandez, Raquel and Brown, Dennis}, year={2013}, month={Jan}, pages={406–414} }
 @article{kononchik_vancini_brown_2011, title={Alphavirus adsorption to mosquito cells as viewed by freeze fracture immunolabeling}, volume={415}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2011.04.011}, abstractNote={Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction of the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor.}, number={2}, journal={VIROLOGY}, author={Kononchik, Joseph P. and Vancini, Ricardo and Brown, Dennis T.}, year={2011}, month={Jul}, pages={132–140} }
 @article{smith_nanda_spears_ribeiro_vancini_piper_thomas_thomas_brown_hernandez_2011, title={Structural mutants of dengue virus 2 transmembrane domains exhibit host-range phenotype}, volume={8}, ISSN={["1743-422X"]}, DOI={10.1186/1743-422x-8-289}, abstractNote={There are over 700 known arboviruses and at least 80 immunologically distinct types that cause disease in humans. Arboviruses are transmitted among vertebrates by biting insects, chiefly mosquitoes and ticks. These viruses are widely distributed throughout the world, depending on the presence of appropriate hosts (birds, horses, domestic animals, humans) and vectors. Mosquito-borne arboviruses present some of the most important examples of emerging and resurgent diseases of global significance. A strategy has been developed by which host-range mutants of Dengue virus can be constructed by generating deletions in the transmembrane domain (TMD) of the E glycoprotein. The host-range mutants produced and selected favored growth in the insect hosts. Mouse trials were conducted to determine if these mutants could initiate an immune response in an in vivo system. The DV2 E protein TMD defined as amino acids 452SWTMKILIGVIITWIG467 was found to contain specific residues which were required for the production of this host-range phenotype. Deletion mutants were found to be stable in vitro for 4 sequential passages in both host cell lines. The host-range mutants elicited neutralizing antibody above that seen for wild-type virus in mice and warrant further testing in primates as potential vaccine candidates. Novel host-range mutants of DV2 were created that have preferential growth in insect cells and impaired infectivity in mammalian cells. This method for creating live, attenuated viral mutants that generate safe and effective immunity may be applied to many other insect-borne viral diseases for which no current effective therapies exist.}, journal={VIROLOGY JOURNAL}, author={Smith, Katherine M. and Nanda, Kavita and Spears, Carla J. and Ribeiro, Mariana and Vancini, Ricardo and Piper, Amanda and Thomas, Gwynneth S. and Thomas, Malcolm E. and Brown, Dennis T. and Hernandez, Raquel}, year={2011}, month={Jun} }
 @article{nanda_vancini_ribeiro_brown_hernandez_2009, title={A high capacity Alphavirus heterologous gene delivery system}, volume={390}, ISSN={["0042-6822"]}, DOI={10.1016/j.virol.2009.05.026}, abstractNote={A novel replication competent Sindbis virus based gene delivery vector has been developed for the introduction of genetic cargo into cell lines in vitro and potentially, animal models in vivo. This delivery system expands the previous uses of Sindbis virus as a gene delivery system in that no replicons are required and the resulting cargo containing virus particles are infectious. The heterologous vector is based on a morphological mutant in C, Ser180/Gly183 which produces larger than the normal size T=4 virus particles of 70 nm in size. This mutant produced particles up to 205 nm in size equal to a triangulation number of 36. It was postulated that because the Ser180/Gly183 mutant was capable of assembling such large particles, that increasing the size of the RNA genome incorporated into this mutant capsid protein would favor the assembly of larger than T=4 wild type sized virions. The first generation prototype larger vehicle, described here, carries a approximately 18 kb cDNA insert, however it is conceivable that RNA as large as 32 kb could be transcribed and packaged. The large variant produces a high virus titer of approximately 10(9) pfu/ml from either mammalian or insect cells in culture. Multiple passages of the virus show no loss of the inserted genetic material.}, number={2}, journal={VIROLOGY}, author={Nanda, Kavita and Vancini, Ricardo and Ribeiro, Mariana and Brown, Dennis T. and Hernandez, Raquel}, year={2009}, month={Aug}, pages={368–373} }