HIV infection of target cells requires at least three different molecules, the HIV receptor DC4, a coreceptor such as CXCR4 or CCR5, and the viral envelope protein. Much is known about the structure, biochemistry, and molecular biology of these essential proteins. Unfortunately, our understanding of the cell biology of these molecules and HIV entry limits our ability to understand how these proteins interact over time to mediate fusion between the virial and cellular membranes. To gain insights into this process, we will utilize a system recently developed in my laboratory, which allows individual virions of HIV to be imaged using fluorescent microscopy. The studies outlined here will combine our ability to visualize HIV interaction with living cells with the methods of modern cell biology to gain new insights into the process of HIV entry. Our preliminary studies reveal that the HIV receptor and coreceptor(s) are specifically localized on the cell surface to regions of active membrane activity. This observation suggests that the localization of these molecules is regulated. Using fluorescent protein fusions to CD4 and coreceptor(s) we will determine how the presence of HIV effects the cell biology of these cell surface proteins during infection. Using methods which allow the fusion of viral and cellular membranes to be detected visually we will determine the spatial interactions between the viral envelope protein and receptor/coreceptor(s) required for virion entry. Finally, we will utilize fluorescent HIV to characterize the role of DC-SIGN. The studies proposed here are significant for completing our understanding of HIV entry into cells because they will provide an important'context for the wealth of details which are currently known about HIV entry. This understanding can in turn be useful for the current push to bring compounds which specifically target, and disrupt, HIV entry to the clinic.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052051-08
Application #
7758256
Study Section
Special Emphasis Panel (ZRG1-AARR-A (07))
Program Officer
Sharma, Opendra K
Project Start
2002-04-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
8
Fiscal Year
2010
Total Cost
$383,127
Indirect Cost
Name
Northwestern University at Chicago
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Flegler, Ayanna J; Cianci, Gianguido C; Hope, Thomas J (2014) CCR5 conformations are dynamic and modulated by localization, trafficking and G protein association. PLoS One 9:e89056
Bhattacharyya, Suchita; Hope, Thomas J (2013) Cellular factors implicated in filovirus entry. Adv Virol 2013:487585
Berro, Reem; Klasse, Per Johan; Lascano, Danny et al. (2011) Multiple CCR5 conformations on the cell surface are used differentially by human immunodeficiency viruses resistant or sensitive to CCR5 inhibitors. J Virol 85:8227-40
Bhattacharyya, Suchita; Hope, Thomas J; Young, John A T (2011) Differential requirements for clathrin endocytic pathway components in cellular entry by Ebola and Marburg glycoprotein pseudovirions. Virology 419:1-9
Bhattacharyya, Suchita; Warfield, Kelly L; Ruthel, Gordon et al. (2010) Ebola virus uses clathrin-mediated endocytosis as an entry pathway. Virology 401:18-28
Platt, Emily J; Kozak, Susan L; Durnin, James P et al. (2010) Rapid dissociation of HIV-1 from cultured cells severely limits infectivity assays, causes the inactivation ascribed to entry inhibitors, and masks the inherently high level of infectivity of virions. J Virol 84:3106-10
Fahrbach, K M; Barry, S M; Anderson, M R et al. (2010) Enhanced cellular responses and environmental sampling within inner foreskin explants: implications for the foreskin's role in HIV transmission. Mucosal Immunol 3:410-8
Melar, Marta; Ott, David E; Hope, Thomas J (2007) Physiological levels of virion-associated human immunodeficiency virus type 1 envelope induce coreceptor-dependent calcium flux. J Virol 81:1773-85
Campbell, Edward M; Perez, Omar; Melar, Marta et al. (2007) Labeling HIV-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell. Virology 360:286-93
Fahrbach, Kelly M; Barry, Sheila M; Ayehunie, Seyoum et al. (2007) Activated CD34-derived Langerhans cells mediate transinfection with human immunodeficiency virus. J Virol 81:6858-68

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