Understanding the pathway(s) that a virus follows during infectious penetration will greatly facilitate the development of useful entry inhibitors. We outline plans to use contemporary tools of live-cell and single molecule imaging to provide quantitative descriptions ofthe invasion of mammalian cells by viral pathogens, including analysis of molecular mechanisms of membrane fusion. We will use an advanced live-cell, spinning-disk confocal fluorescence imaging configuration, with very sensitive optics and optimized illumination, for long time-course studies of viral entry and penetration, and we will develop software for 3D multi-tracking to enable analysis of all particles within the field of view in a given experiment. We will apply these technologies in close collaboration with Projects 1, 2 and 3 to analyse the pathways of entry of pseudotyped VSV particles bearing the glycoproteins from representatives of a broad range of enveloped RNA viruses and to determine how candidate entry inhibitors block progression along the normal entry route. We will use genome-edited cells expressing at endogenous markers for various compartments of endocytic pathways (clathrin, Rab proteins, etc.) to avoid the perturbations often associated with ectopic overexpression, and we will generate such cell lines for a number of additional proteins identified as potential targets in Project 3. Our long-term goals are to contribute a 3D time course in vivo to the characterization of candidate inhibitors and in so doing to achieve a substantially more detailed molecular description of endosomal pathways and of the proteins along those pathways that regulate or subvert viral entry.

Public Health Relevance

Direct visualization of viruses as they enter living cells, as provided by advanced imaging technologies and modes of image analysis, will facilitate the development of entry inhibitors and uncover unexpected features of the endosomal uptake of viral pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
1U19AI109740-01
Application #
8655311
Study Section
Special Emphasis Panel (ZAI1-LR-M (J1))
Project Start
Project End
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
$962,853
Indirect Cost
$234,157
Name
Harvard University
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Pitts, Jared D; Li, Pi-Chun; de Wispelaere, Melissanne et al. (2017) Antiviral activity of N-(4-hydroxyphenyl) retinamide (4-HPR) against Zika virus. Antiviral Res 147:124-130
Raaben, Matthijs; Jae, Lucas T; Herbert, Andrew S et al. (2017) NRP2 and CD63 Are Host Factors for Lujo Virus Cell Entry. Cell Host Microbe 22:688-696.e5
Salgado, Eric N; Upadhyayula, Srigokul; Harrison, Stephen C (2017) Single-particle detection of transcription following rotavirus entry. J Virol :
Wang, May K; Lim, Sun-Young; Lee, Soo Mi et al. (2017) Biochemical Basis for Increased Activity of Ebola Glycoprotein in the 2013-16 Epidemic. Cell Host Microbe 21:367-375
Filippakis, Harilaos; Alesi, Nicola; Ogorek, Barbara et al. (2017) Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R. Oncotarget 8:38099-38112
Clark, Margaret J; Miduturu, Chandra; Schmidt, Aaron G et al. (2016) GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein. Cell Chem Biol 23:443-52
Chou, Yi-ying; Cuevas, Christian; Carocci, Margot et al. (2016) Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor. J Virol 90:4494-510
Piccinotti, Silvia; Whelan, Sean P J (2016) Rabies Internalizes into Primary Peripheral Neurons via Clathrin Coated Pits and Requires Fusion at the Cell Body. PLoS Pathog 12:e1005753
Chou, Yi-Ying; Krupp, Annabel; Kaynor, Campbell et al. (2016) Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9. Sci Rep 6:36921
Taylor, Travis J; Diaz, Fernando; Colgrove, Robert C et al. (2016) Production of immunogenic West Nile virus-like particles using a herpes simplex virus 1 recombinant vector. Virology 496:186-193

Showing the most recent 10 out of 21 publications