All enveloped viruses enter cells by a membrane fusion reaction and produce progeny virus by budding through a cellular membrane. Semliki Forest virus (SFV) is a well-characterized alphavirus that infects cells by low pH-triggered membrane fusion and exits by budding through the cell plasma membrane. Membrane fusion is mediated by the transmembrane E1 protein, a """"""""class II virus membrane fusion protein"""""""" that is structurally related to the fusion proteins of flaviviruses. At low pH E1 inserts its fusion peptide loop into cholesterol-containing target membranes and refolds to a hairpin-like trimer conformation to drive the fusion reaction. During virus budding, E1 forms an icosahedral lattice that organizes the structure of the new virus particle. The overall goal of this grant is to define the molecular features of the entry and exit of viruses having class II fusion proteins. Our studies focus on the highly-developed SFV system and on the flavivirus dengue virus (DV), a class A pathogen and an important human health problem world-wide. Our previous studies demonstrated important roles of the ij loop adjacent to the fusion loop at the tip of E1. The ij loop is involved in alphavirus cholesterol dependence, and mutation of a highly conserved histidine in the ij loop blocked SFV at a late stage in fusion. We will define the functions of the SFV and DV ij loop and conserved histidine using mutagenesis and fusion analysis. Membrane insertion of SFV and DV fusion proteins will be characterized and compared using site-specific fluorescent probes to follow the membrane interactions of the fusion and ij loops. SFV budding produces a highly organized particle and requires a critical number of envelope proteins on the plasma membrane. The regulation of cell surface envelope protein levels by cellular and viral processes will be determined and their role in budding will be defined. The cellular ESCRT machinery is important in the budding of many RNA viruses, and we will determine its functions in the alphavirus lifecycle. We will also screen to identify novel cellular proteins involved in virus assembly and budding. Understanding the molecular mechanisms of virus fusion and budding will provide critical insights into virus disease mechanisms, potential anti-viral therapies, and the membrane fusion and budding reactions of eukaryotic cells.
| 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 |
| 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 |
| Ooi, Yaw Shin; Dubé, Mathieu; Kielian, Margaret (2015) BST2/tetherin inhibition of alphavirus exit. Viruses 7:2147-67 |
| 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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