Enveloped viruses release their nucleocapsids into the cytoplasm by merging their membrane with the cell membrane. The majority of these viruses is internalized by cells and fuses with endosomes. The elucidation of the molecular mechanisms of virus-endosome fusion and its regulation has been hampered by the highly dynamic nature of endosomes and the lack of access to these compartments. We have developed non- invasive imaging techniques that permit the time-resolved visualization of the critical steps of virus entry, from hemifusion (lipid transfer), to small pore formation (release o small content markers) and pore enlargement (release of the capsid). The recently implemented imaging assay enables (i) measurements of the pH in virus- carrying endosomes and (ii) detection of the resulting fusion events. We propose to apply these imaging and other approaches to define the mechanism of entry of the Avian Sarcoma and Leukosis Virus (ASLV), which is an excellent model for elucidating the entry pathways used by disparate viruses. The two-step triggering of ASLV-endosome fusion - binding to a cognate receptor and exposure to low pH - permit an unprecedented control over the virus entry process. Our pilot data suggest that, strikingly, the ASLV fusion with early acidic endosomes is restricted in some cell types and occurs in late endosomal compartments. These findings imply that ASLV fusion is regulated by cellular factors and that this virus may undergo hemifusion or form a small fusion pore in early endosomes, while relying on host factors to drive the energetically unfavorable step of pore enlargement. To test this hypothesis, we will: (1) investigate the spatio-temporal regulation of ASLV fusion;(2) examine the determinants of ASLV pore enlargement;and (3) define the role of endosomal lipids in ASLV fusion. These studies will provide critical insights into the ASLV fusion and its regulation by host factors. Knowledge of the mechanisms of ASLV fusion will offer a conceptual framework for studies of human viruses, such as HIV-1, Hepatitis C virus and Ebola virus, which enter host cells via the endocytic pathway and rely on a multitude of host factors.

Public Health Relevance

Enveloped viruses deposit their genome into a host cell by fusing their membrane with a cell membrane. We will track the entry of single retroviral particles into a host cell, from uptake to a local merger of the viral and cellular membranes and the release of the viral genome. Pinpointing the sites of viral fusion in different cell types will she light on the host factors facilitating this process and guide future antiviral strategies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Park, Eun-Chung
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Emory University
Schools of Medicine
United States
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Sood, Chetan; Francis, Ashwanth C; Desai, Tanay M et al. (2017) An improved labeling strategy enables automated detection of single-virus fusion and assessment of HIV-1 protease activity in single virions. J Biol Chem 292:20196-20207
Desai, Tanay M; Marin, Mariana; Mason, Caleb et al. (2017) pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion. J Biol Chem 292:7817-7827
Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong et al. (2017) Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12:150-167
Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana et al. (2016) Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion. J Virol Methods 233:62-71
Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min et al. (2016) Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger. PLoS Pathog 12:e1005373
Padilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki et al. (2014) Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging. Retrovirology 11:47
Matos, Pedro M; Marin, Mariana; Ahn, Byungwook et al. (2013) Anionic lipids are required for vesicular stomatitis virus G protein-mediated single particle fusion with supported lipid bilayers. J Biol Chem 288:12416-25
Padilla-Parra, Sergi; Marin, Mariana; Gahlaut, Nivriti et al. (2013) Fusion of mature HIV-1 particles leads to complete release of a gag-GFP-based content marker and raises the intraviral pH. PLoS One 8:e71002
Padilla-Parra, Sergi; Matos, Pedro M; Kondo, Naoyuki et al. (2012) Quantitative imaging of endosome acidification and single retrovirus fusion with distinct pools of early endosomes. Proc Natl Acad Sci U S A 109:17627-32
Padilla-Parra, Sergi; Marin, Mariana; Kondo, Naoyuki et al. (2012) Synchronized retrovirus fusion in cells expressing alternative receptor isoforms releases the viral core into distinct sub-cellular compartments. PLoS Pathog 8:e1002694

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