Alphaviruses and flaviviruses 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, but in spite of their medical importance there are no available antiviral therapies. Alphaviruses and flaviviruses infect cells by a membrane fusion reaction that is triggered by low pH during virus endocytic uptake and mediated by structurally similar membrane fusion proteins termed class II. These fusion proteins are synthesized together with a companion protein that dimerizes with the fusion protein and protects it from low pH during transit to the plasma membrane. Late in the secretory pathway the companion proteins are proteolytically processed by cellular furin, thereby priming the dimer to dissociate a low pH during virus entry. The fusion proteins then drive virus membrane fusion by inserting a hydrophobic fusion loop into the target membrane and refolding to a hairpin-like homotrimer. This proposal will address the following critical questions in the mechanism and biogenesis of class II fusion proteins: 1. The roles of E3 and E2 interactions during alphavirus entry and biogenesis. Furin processing converts the alphavirus p62 companion protein to mature E2 and an E3 peptide. We will define the critical dimer dissociation steps that mediate the initial uncovering of the fusion loop on E1 and the subsequent complete dissociation of the dimer to permit E1 homotrimer formation. We will determine the function of E3 in protecting E1 from low pH during virus biogenesis, and the role of a conserved E2 CXXC motif in promoting E1 disulfide bond formation. 2. Molecular mechanisms of class II protein-membrane interaction. We will use innovative fluorescence methods to study the mechanism of membrane insertion of the alphavirus fusion protein, and we will determine the specific protein loops involved, the properties of insertion and the role of cholesterol in this process. We will develop a novel cross-linking assay to define the cooperative lateral interactions between E1 trimers in the target membrane and we will determine their role in virus-membrane fusion. 3. Novel class II pH protection mechanisms. Recent studies demonstrate that the rubella virus (RuV) fusion protein E1 has a typical class II structure, but its companion protein E2 is not proteolytically processed. RuV E1 is thus an important paradigm for a growing group of low pH-triggered viral class II proteins in which fusion is not regulated by companion protein processing. We will define the novel mechanisms that protect the RuV fusion protein from low pH inactivation during virus biogenesis.

Public Health Relevance

This project will study the membrane fusion mechanism that mediates infection by the alphaviruses and flaviviruses, which include important human pathogens that cause serious diseases and are potential biodefense threats. The virus fusion reaction is tightly controlled to occur at the correct place and time during virus entry, and is silenced during the exit of the virus from the host cell. Understanding these fusion and regulatory mechanisms will ultimately allow the design of targeted antiviral therapies.

National Institute of Health (NIH)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Repik, Patricia M
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Albert Einstein College of Medicine
Anatomy/Cell Biology
Schools of Medicine
United States
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Zheng, Aihua; Yuan, Fei; Kleinfelter, Lara M et al. (2014) A toggle switch controls the low pH-triggered rearrangement and maturation of the dengue virus envelope proteins. Nat Commun 5:3877
Uchime, Onyinyechukwu; Fields, Whitney; Kielian, Margaret (2013) The role of E3 in pH protection during alphavirus assembly and exit. J Virol 87:10255-62
Vigant, Frederic; Lee, Jihye; Hollmann, Axel et al. (2013) A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion. PLoS Pathog 9:e1003297
Fields, Whitney; Kielian, Margaret (2013) A key interaction between the alphavirus envelope proteins responsible for initial dimer dissociation during fusion. J Virol 87:3774-81
Sánchez-San Martín, Claudia; Nanda, Soumya; Zheng, Yan et al. (2013) Cross-inhibition of chikungunya virus fusion and infection by alphavirus E1 domain III proteins. J Virol 87:7680-7
Pierson, Theodore C; Kielian, Margaret (2013) Flaviviruses: braking the entering. Curr Opin Virol 3:3-12
Liu, Catherine Y; Kielian, Margaret (2012) Identification of a specific region in the e1 fusion protein involved in zinc inhibition of semliki forest virus fusion. J Virol 86:3588-94
Zheng, Yan; Sánchez-San Martín, Claudia; Qin, Zhao-ling et al. (2011) The domain I-domain III linker plays an important role in the fusogenic conformational change of the alphavirus membrane fusion protein. J Virol 85:6334-42
Roman-Sosa, Gleyder; Kielian, Margaret (2011) The interaction of alphavirus E1 protein with exogenous domain III defines stages in virus-membrane fusion. J Virol 85:12271-9
Liu, Catherine Y; Besanceney, Christen; Song, Yifan et al. (2010) Pseudorevertants of a Semliki forest virus fusion-blocking mutation reveal a critical interchain interaction in the core trimer. J Virol 84:11624-33

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