Alphaviruses are small enveloped plus-sense RNA viruses with highly organized structures. They include important human pathogens such as Chikungunya virus and the encephalitic alphaviruses, which are classified as category B or C priority pathogens. In spite of the medical importance of the alphaviruses there are currently no licensed antiviral therapies or vaccines. Alphaviruses infect cells through endocytosis and low pH-triggered fusion, replicate in the cytoplasm, and bud through the plasma membrane. Budding requires the specific 1:1 interaction of the E2 viral envelope protein with the capsid protein in the nucleocapsid core. This grant will address key questions on alphavirus assembly and budding 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 pathogen Chikungunya virus. We propose three integrated aims to define the alphavirus exit pathway: 1. Alphavirus remodeling of the cytoskeleton. Alphaviruses remodel the host cell cytoskeleton to generate long intercellular extensions that contain actin and stable microtubules and can transmit virus particles to new cells. Induction of extensions requires the capsid-E2 interaction and can be recapitulated by expression of the viral structural proteins in the absence of virus infection. We will determine the properties of the extensions, the viral protein requirements for their generation, and the cellular signaling pathways that the virus coopts to cause this dramatic remodeling. 2. Tetherin inhibition of alphavirus release. BST2/Tetherin is an interferon-induced host membrane protein that acts to inhibit the release of a number of enveloped viruses from their host cells. Tetherin has two isoforms, with L-tetherin containing a 12 residue extension of the amino terminal cytoplasmic domain compared to S-tetherin. Our evidence indicates that alphavirus restriction by tetherin has a novel isoform specificity: only L-tetherin causes efficient restriction, without a role for an alphavirus-encoded tetherin antagonist. We will determine the mechanism of this unique restriction profile by comparison of the activities of L- vs. S-tetherin in the alphavirus exit pathway. 3. Pathway of capsid protein incorporation into virus. We are developing a targeted BirA method to tag capsid protein with biotin at specific cellular locations on the cytoplasmic face of the endoplasmic reticulum, Golgi, or plasma membranes, or in the cytoplasm. We will exploit this novel method to track capsid protein using pulse-chase biotinylation together with biochemical and morphological assays. Results will define the precursor to the alphavirus nucleocapsid and its traffic to the plasma membrane. Together these aims will reveal critical mechanisms in the alphavirus exit pathway and provide important functional information on host cell proteins during virus infection.
This project will study the mechanisms used by membrane-enveloped alphaviruses to assemble and exit from cells and infect new host 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.
| Brown, Rebecca S; Wan, Judy J; Kielian, Margaret (2018) The Alphavirus Exit Pathway: What We Know and What We Wish We Knew. Viruses 10: |
| Martinez, Maria Guadalupe; Kielian, Margaret (2016) Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission. PLoS Pathog 12:e1006061 |
| Stiles, Katie M; Kielian, Margaret (2016) Role of TSPAN9 in Alphavirus Entry and Early Endosomes. J Virol 90:4289-97 |
| Ooi, Yaw Shin; Dubé, Mathieu; Kielian, Margaret (2015) BST2/tetherin inhibition of alphavirus exit. Viruses 7:2147-67 |
| Kleinfelter, Lara M; Jangra, Rohit K; Jae, Lucas T et al. (2015) Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion. MBio 6:e00801 |
| Zheng, Yan; Kielian, Margaret (2015) An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly. Virology 484:412-20 |
| Martinez, Maria Guadalupe; Snapp, Erik-Lee; Perumal, Geoffrey S et al. (2014) Imaging the alphavirus exit pathway. J Virol 88:6922-33 |
| Kielian, Margaret (2014) Mechanisms of Virus Membrane Fusion Proteins. Annu Rev Virol 1:171-89 |
| Ooi, Yaw Shin; Stiles, Katie M; Liu, Catherine Y et al. (2013) Genome-wide RNAi screen identifies novel host proteins required for alphavirus entry. PLoS Pathog 9:e1003835 |
| Zheng, Yan; Kielian, Margaret (2013) Imaging of the alphavirus capsid protein during virus replication. J Virol 87:9579-89 |
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