The long-term goal of this project is to elucidate the molecular mechanisms and cellular pathways of influenza infection. To accomplish this goal, we will develop state-of-the-art instrumentation for real-time fluorescence imaging of single viruses and single viral genes. These single-particle approaches will allow us to directly visualize the cellular entry process of influenza viruses in real time, to dissect individual stages of the entry pathway(s) that may be difficult if not impossible to detect by ensemble methods, and to obtain a better understanding of the cellular entry mechanisms of influenza. Influenza, representative of many medically important viruses, is a paradigm for understanding viral entry processes. Influenza viruses enter cells via several steps, including (1) receptor-mediated endocytosis, (2) endocytic trafficking of the viruses to late endosomes where viral fusion with endosomes leads to the release viral genes into the cytoplasm, and (3) nuclear import of these viral genes. Three sets of experiments are designed to investigate these viral entry steps.
Specific Aim 1 : Single-particle tracking will be used to investigate the endocytosis mechanisms used by the influenza viruses. We will address important questions including: (i) which cellular endocytic pathways are exploited by influenza to enter cells; (ii) what are the molecular characteristics of these pathways; (iii) How viruses are targeted to endocytic machinery; and/or (iv) how endocytic machinery assembles around viruses? Specific Aim 2: Single-particle tracking will be used to elucidate the endocytic trafficking of influenza viruses inside cells. We will address: (i) how viruses are transported inside cells; (ii) how viruses are trafficked between different endocytic compartments; and (iii) how viruses are sorted.
Specific Aim 3 : Single-particle tracking will be used to characterize the ? regulated nuclear import of influenzal genes, in the form of viral ribonucleoproteins (vRNPs). We will address: (i) the transport mechanisms of vRNPs in cells and (ii) the molecular mechanisms that regulate the nuclear import of vRNPs? The above experiments promise to provide new insights into the cellular entry process of influenza viruses. The new methodology developed here can be extended to study other families of viruses and may in the future lead to new designs of anti-viral drugs and therapeutics. These techniques can also be applied to other molecular and cellular biological systems. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068518-04
Application #
7286279
Study Section
Virology Study Section (VR)
Program Officer
Lewis, Catherine D
Project Start
2004-09-22
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2007
Total Cost
$272,128
Indirect Cost
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Kim, Doory; Deerinck, Thomas J; Sigal, Yaron M et al. (2015) Correlative stochastic optical reconstruction microscopy and electron microscopy. PLoS One 10:e0124581
Jia, Shu; Vaughan, Joshua C; Zhuang, Xiaowei (2014) Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function. Nat Photonics 8:302-306
Wang, Siyuan; Moffitt, Jeffrey R; Dempsey, Graham T et al. (2014) Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging. Proc Natl Acad Sci U S A 111:8452-7
Chung, Jean-Ju; Shim, Sang-Hee; Everley, Robert A et al. (2014) Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility. Cell 157:808-22
Sun, Eileen; He, Jiang; Zhuang, Xiaowei (2013) Dissecting the role of COPI complexes in influenza virus infection. J Virol 87:2673-85
He, Jiang; Sun, Eileen; Bujny, Miriam V et al. (2013) Dual function of CD81 in influenza virus uncoating and budding. PLoS Pathog 9:e1003701
Sun, Eileen; He, Jiang; Zhuang, Xiaowei (2013) Live cell imaging of viral entry. Curr Opin Virol 3:34-43
Vaughan, Joshua C; Dempsey, Graham T; Sun, Eileen et al. (2013) Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy. J Am Chem Soc 135:1197-200
Doksani, Ylli; Wu, John Y; de Lange, Titia et al. (2013) Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell 155:345-356
Kim, Ingyu; Pan, Weijun; Jones, Sara A et al. (2013) Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes. J Cell Biol 200:419-28

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