Progress in understanding virus entry has been dominated by pH-dependent viruses, principally because entry can be synchronized by lowering pH and quantitative assays exist to measure this en masse fusion event. Much less attention has been paid to pH-independent viruses such as retroviruses. One of the most important unresolved issues in this field is where retroviruses penetrate the cell membrane and if cellular cues, other than receptor interaction, are required to trigger entry. To identify factors important for Murine leukemia virus entry we designed and screened a custom siRNA library targeting genes important for endocytosis and receptor trafficking. We compared infection of Friend murine leukemia virus (Fr-MLV) to Vesicular stomatitis virus, Venezuelan equine encephalitis virus and Ebola using an envelope pseudotyping system. Genes corresponding to the Rac1-PAK1-LIMK1 pathway of actin regulation were identified as key for infection by Fr-MLV but not the other viruses. Also, Dynamin, EEA1 and Eps15R were identified as important. The latter 3 genes play important roles in endocytosis. Since the only difference between the pseudotypes is the source of envelope protein used, it is likely that the differences seen were due to differences in entry pathway. In this proposal we will test the role of the actin and endocytosis for Murine leukemia virus entry. The role of actin may be for trafficking of receptor across the cell surface or endocytosis itself. We have developed a new virus entry assay that measures virus entry kinetics in real time. The assay provides the highest possible detail for entry measurements. This assay allows us to define the role of each gene for entry and permits us to distinguish roles in trafficking or endocytosis. This study will provide new insight into the entry process of retroviruses including HIV and enveloped viruses in general. Knowledge of the entry pathway will in turn aid in development of drugs that will block this first step in infection and prevent cell to cell spread of virus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063513-05
Application #
7812123
Study Section
Virology - B Study Section (VIRB)
Program Officer
Park, Eun-Chung
Project Start
2006-05-15
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
5
Fiscal Year
2010
Total Cost
$249,194
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
Wu, Yu; Pons, Valérie; Goudet, Amélie et al. (2017) ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments. Sci Rep 7:15567
Sakurai, Yasuteru; Kolokoltsov, Andrey A; Chen, Cheng-Chang et al. (2015) Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment. Science 347:995-8
Moller-Tank, Sven; Kondratowicz, Andrew S; Davey, Robert A et al. (2013) Role of the phosphatidylserine receptor TIM-1 in enveloped-virus entry. J Virol 87:8327-41
Kondratowicz, Andrew S; Hunt, Catherine L; Davey, Robert A et al. (2013) AMP-activated protein kinase is required for the macropinocytic internalization of ebolavirus. J Virol 87:746-55
Miller, Mary E; Adhikary, Shramika; Kolokoltsov, Andrey A et al. (2012) Ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection. J Virol 86:7473-83
Chen, Zeming; Kolokoltsov, Andrey A; Wang, Jia et al. (2012) GRB2 interaction with the ecotropic murine leukemia virus receptor, mCAT-1, controls virus entry and is stimulated by virus binding. J Virol 86:1421-32
Kondratowicz, Andrew S; Lennemann, Nicholas J; Sinn, Patrick L et al. (2011) T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proc Natl Acad Sci U S A 108:8426-31
Saeed, Mohammad F; Kolokoltsov, Andrey A; Albrecht, Thomas et al. (2010) Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog 6:e1001110
Gavicherla, Balramakrishna; Ritchey, Lisa; Gianfelice, Antonella et al. (2010) Critical role for the host GTPase-activating protein ARAP2 in InlB-mediated entry of Listeria monocytogenes. Infect Immun 78:4532-41
Kolokoltsov, Andrey A; Saeed, Mohammad F; Freiberg, Alexander N et al. (2009) Identification of novel cellular targets for therapeutic intervention against Ebola virus infection by siRNA screening. Drug Dev Res 70:255-265

Showing the most recent 10 out of 16 publications