Alphaviruses and flaviviruses are small enveloped plus-sense RNA viruses with structurally and functionally-related membrane fusion proteins. These viruses include important human pathogens such as the encephalitic alphaviruses and chikungunya virus, and the flaviviruses dengue, yellow fever, and West Nile virus. Many of these viruses are classified as category A, B or C priority pathogens, yet in spite of their medica importance there are no available antiviral therapies. Although alphaviruses and flaviviruses are well-characterized structurally, relatively little is known about host factors involved in the viru lifecycle. Alphaviruses and flaviviruses infect cells by clathrin-mediated entry and a membrane fusion reaction triggered by the low pH in the endocytic pathway. New alphavirus particles bud through the host cell plasma membrane, while nascent flaviviruses bud into the endoplasmic reticulum. The long-term goal of this grant is to define the molecular mechanisms of alphavirus and flavivirus entry and exit and the role of host proteins in these processes. Our studies will focus on the highly-developed alphavirus experimental systems Semliki Forest virus and sindbis virus, and the emerging alphavirus pathogen chikungunya virus. Three integrated and complementary approaches will be used to address key features of alphavirus entry and exit: 1. We will define the roles of cellular proteins identified by our genome-wide siRNA screen for novel host factors involved in the alphavirus lifecycle. Hits were prioritized by bioinformatics analysis, confirmed with several alphaviruses, and differentially screened for key steps in virus infection. The mechanisms by which these host proteins promote or inhibit alphavirus entry, replication and exit will now be determined. 2. The small viral membrane protein 6K promotes alphavirus budding by an unknown mechanism that may involve direct effects and/or effects on cellular proteins. Our preliminary results show that the cellular membrane protein BST2/tetherin inhibits alphavirus release. We will define the mechanism of tetherin inhibition and determine if tetherin is antagonized by 6K or other viral proteins. We will determine the activity of 6K in directly modulating membrane curvature at the bud site. 3. Biochemical and imaging approaches will be used to study the cell biology of alphavirus exit, focusing in particular on the transport mechanism and assembly site of the viral nucleocapsid core, and on the properties of the final envelopment step and the mechanistic roles of host and viral proteins. Results will further our understanding the molecular mechanisms of alphavirus entry and exit, provide critical information on viral disease mechanisms and novel strategies for anti-viral therapy, and suggest fruitful new avenues for research on the flaviviruses.
This project will study the mechanisms used by membrane-enveloped alphaviruses to enter cells and to assemble and exit from cells. Alphaviruses include important human pathogens that cause serious diseases and are potential biodefense threats, and for which there are no antiviral therapies. The alphaviruses are highly developed experimental systems that also teach us about the general functions of membrane-enveloped viruses and cellular membrane processes.
|Martinez, Maria Guadalupe; Snapp, Erik-Lee; Perumal, Geoffrey S et al. (2014) Imaging the alphavirus exit pathway. J Virol 88:6922-33|
|Zheng, Yan; Kielian, Margaret (2013) Imaging of the alphavirus capsid protein during virus replication. J Virol 87:9579-89|
|Pierson, Theodore C; Kielian, Margaret (2013) Flaviviruses: braking the entering. Curr Opin Virol 3:3-12|
|Zheng, Aihua; Umashankar, Mahadevaiah; Kielian, Margaret (2010) In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions. PLoS Pathog 6:e1001157|
|Sanchez-San Martin, Claudia; Liu, Catherine Y; Kielian, Margaret (2009) Dealing with low pH: entry and exit of alphaviruses and flaviviruses. Trends Microbiol 17:514-21|
|Umashankar, M; Sanchez-San Martin, Claudia; Liao, Maofu et al. (2008) Differential cholesterol binding by class II fusion proteins determines membrane fusion properties. J Virol 82:9245-53|
|Markosyan, Ruben M; Kielian, Margaret; Cohen, Fredric S (2007) Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell. J Virol 81:11218-25|
|Taylor, Gwen M; Hanson, Phyllis I; Kielian, Margaret (2007) Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding. J Virol 81:13631-9|
|Chanel-Vos, Chantal; Kielian, Margaret (2006) Second-site revertants of a Semliki Forest virus fusion-block mutation reveal the dynamics of a class II membrane fusion protein. J Virol 80:6115-22|
|Kielian, Margaret (2006) Class II virus membrane fusion proteins. Virology 344:38-47|
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