Membrane fusion provides a conceptually simple mechanism for enveloped viruses to deliver their genomes into the cytoplasm of target cells. In contrast, the mechanisms used by nonenveloped viruses to penetrate cellular membranes and release their genomes into the cell remain poorly understood. This application proposes a program of structural studies of poliovirus cell entry intermediates as a simple model for understanding how nonenveloped viruses gain entry into the cell. The problem is framed in the context of several specific questions: 1) What is the nature of the virus-receptor interaction and how is receptor binding coupled to the induction of conformational changes? 2) What is the sequence of conformational changes that ultimately leads to cell entry and the release of the viral RNA? 3) What is the nature of the interaction of the virus with the cell membrane that leads to the internalization of the RNA? 4) How is the RNA released from the particle and into the cell? From a structural perspective, these questions cover over six orders of magnitude in scale from the atomic level (tenths of nanometers) to the cellular level (tens of microns). This range is well beyond the scope of any one structural method. The project seeks to develop a hybrid approach combining x-ray crystallography, cryoelectron microscopy, cyroelectron tomography and optical microscopy to develop a series of structural snapshots of cell entry intermediates of poliovirus;and to use these structures to address questions posed by, and pose questions for, supporting biochemical and genetic studies.
The specific aims i nclude extending structural studies of soluble and membrane-associated poliovirus cell entry intermediates in vitro and initiating studies using a combination of fluorescence microscopy and electron tomography to probe the location, environment and roles of entry intermediates in cells. The goal will be to combine the high-resolution structures derived from the in vitro studies with information about the location of intracellular intermediates to define structural changes associated with cell entry and delivery of the genome into the cytoplasm in the context of the intact cell at an unprecedented level of detail. The information derived from these studies will be directly relevant to understanding the detailed cell entry mechanism of a group of viruses (the enteroviruses and rhinoviruses) that includes a number of significant human pathogens, and will provide important insights into the more general question of how nonenveloped viruses enter cells. The structural methods developed in these studies will also be generally useful to a wide range of questions in cell biology.

Public Health Relevance

The project seeks to understand how poliovirus and related viruses such as rhinoviruses, coxsackieviruses, and enteroviruses (all of which are significant human pathogens) enter cells. The findings are also expected to be more generally relevant to many other viruses which lack a lipid envelop. Understanding of the viral entry pathways could lead to the development of novel antivirals.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Park, Eun-Chung
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Harvard University
Schools of Medicine
United States
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Zhao, Zhao; Zhang, Meng; Hogle, James M et al. (2018) DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry. J Am Chem Soc 140:10639-10643
Nasr, Mahmoud L; Baptista, Diego; Strauss, Mike et al. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49-52
Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen et al. (2017) Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes. PLoS Pathog 13:e1006197
Strauss, Mike; Schotte, Lise; Karunatilaka, Krishanthi S et al. (2017) Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State. J Virol 91:
Strauss, Mike; Schotte, Lise; Thys, Bert et al. (2016) Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site. J Virol 90:3496-505
Schotte, Lise; Thys, Bert; Strauss, Mike et al. (2015) Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs). Antimicrob Agents Chemother 59:4695-706
Strauss, Mike; Filman, David J; Belnap, David M et al. (2015) Nectin-like interactions between poliovirus and its receptor trigger conformational changes associated with cell entry. J Virol 89:4143-57
Panjwani, Anusha; Strauss, Mike; Gold, Sarah et al. (2014) Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore. PLoS Pathog 10:e1004294
Ma, Hsin-Chieh; Liu, Ying; Wang, Chunling et al. (2014) An interaction between glutathione and the capsid is required for the morphogenesis of C-cluster enteroviruses. PLoS Pathog 10:e1004052
Moustafa, Ibrahim M; Korboukh, Victoria K; Arnold, Jamie J et al. (2014) Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase. J Biol Chem 289:36229-48

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