We are studying the interaction between retroviral envelope and receptor proteins, which mediate viral entry. Some studies involve mutating the receptor, or the envelope, to see what portions of these proteins are involved in various steps in the fusion process. Other approaches include modifying the surface charge or lipid composition of the membranes involved in fusion. We made some interesting observations related to the latter approach over the past year. In trying to make new fluorescent probes to monitor fusion events at the cell surface, we found that attaching cholesterol (a lipid) to DNA molecules caused them to bind tightly to the surface of cells. Since DNA is negatively charged, we investigated whether cell surface-bound DNA affected virus-mediated membrane fusion. We found that cholesterol-DNA probes inhibited entry by certain strains of HIV but not others. The strains that were inhibited had a high density of positive charge in a variable region of the HIV envelope protein known as the V3 loop. Previous work had shown that dextran sulfate and heparin, other negatively charged polymers, inhibited the same strains of HIV, though at much higher concentrations, presumably because these compounds do not concentrate at the cell surface. Consistent with an electrostatic interaction, the inhibitory effects of cholesterol-DNA were blocked by a positively charged polymer, DEAE-dextran. The cholesterol-DNA probes have several unique properties that make them interesting tools to study cell surface events: they are targeted to specialized membrane domains known as rafts that are involved in cell-signaling and, probably, virus-mediated cell fusion; by adjusting the length of the DNA, they might be used to probe events at different distances from the cell surface; because they hybridize to complementary DNA strands, they could be used to engineer various types of close connection between cells with complementary probes on their surfaces.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000781-07
Application #
6808839
Study Section
(LMM)
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2003
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Ou, Wu; Silver, Jonathan (2006) Stoichiometry of murine leukemia virus envelope protein-mediated fusion and its neutralization. J Virol 80:11982-90
Gilbert, Joanna; Ou, Wu; Silver, Jonathan et al. (2006) Downregulation of protein disulfide isomerase inhibits infection by the mouse polyomavirus. J Virol 80:10868-70
Ou, Wu; Silver, Jonathan (2006) Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion. Virology 350:406-17
Ou, Wu; Lu, Ning; Yu, Sloane S et al. (2006) Effect of epitope position on neutralization by anti-human immunodeficiency virus monoclonal antibody 2F5. J Virol 80:2539-47
Ou, Wu; Silver, Jonathan (2005) Efficient trapping of HIV-1 envelope protein by hetero-oligomerization with an N-helix chimera. Retrovirology 2:51
Ou, Wu; Silver, Jonathan (2005) Inhibition of murine leukemia virus envelope protein (env) processing by intracellular expression of the env N-terminal heptad repeat region. J Virol 79:4782-92
Ahn, Kwang-Soo; Ou, Wu; Silver, Jonathan (2004) Inhibition of certain strains of HIV-1 by cell surface polyanions in the form of cholesterol-labeled oligonucleotides. Virology 330:50-61
Ou, Wu; Xiong, Ying; Silver, Jonathan (2004) Quantification of virus-envelope-mediated cell fusion using a tetracycline transcriptional transactivator: fusion does not correlate with syncytium formation. Virology 324:263-72
Ou, Wu; Silver, Jonathan (2003) Role of a conserved amino-terminal sequence in the ecotropic MLV receptor mCAT1. Virology 308:101-13
Lu, Xiongbin; Xiong, Ying; Silver, Jonathan (2002) Asymmetric requirement for cholesterol in receptor-bearing but not envelope-bearing membranes for fusion mediated by ecotropic murine leukemia virus. J Virol 76:6701-9

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