The mechanism of retroviral entry into cells involves binding of viral envelope (ENV) proteins to cellular receptors followed by a set of conformational changes in ENV giving rise to virus-cell membrane fusion. However, these events are not well understood. This proposal is aimed at better understanding this critical first step of viral replication using the avian leukosis virus (ALV)-receptor model system. The first specific aim is to determine the molecular basis for the existence of two functionally-distinct forms of the TVBS1 receptor for subgroups B, D, and E ALV. These studies will provide crucial new insights into the mechanism of entry used by these viruses and into the non-reciprocal receptor-interference pattern observed between these viruses. Furthermore, the results of these studies should also have important implications for understanding the biological significance of non-reciprocal receptor interference seen with other types of retroviruses. The second specific aim is to characterize conformational changes in ALV ENV glycoproteins following receptor-binding and low pH activation, and to test the hypothesis that these events are sufficient to stimulate ALV ENV-driven membrane fusion. These studies build upon our novel and unexpected finding that ALV ENV-driven membrane fusion requires receptor-binding followed by a low pH-activated step and will test the sufficiency of these events for viral entry. The third specific aim is to identify viral entry determinants of ALV ENV and receptor proteins, studies that will help us to build rational models for ALV entry into cells and that will be invaluable for validating the biological significance of crystal structures of ALV ENV-receptor complexes once they become available. The fourth specific aim addresses whether ALV use a clathrin-dependent endocytic pathway for entry into cells. Together, these studies will significantly advance our understanding of how retroviruses enter cells. Understanding this process in detail should lead to better antiviral therapeutic strategies and aid with viral targeting approaches for gene therapy protocols.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA070810-06
Application #
6127933
Study Section
Virology Study Section (VR)
Program Officer
Cole, John S
Project Start
1996-06-25
Project End
2005-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
6
Fiscal Year
2000
Total Cost
$205,667
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Babel, Angeline Rose; Bruce, James; Young, John A T (2007) The hr1 and fusion peptide regions of the subgroup B avian sarcoma and leukosis virus envelope glycoprotein influence low pH-dependent membrane fusion. PLoS One 2:e171
Wickersham, Ian R; Lyon, David C; Barnard, Richard J O et al. (2007) Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53:639-47
Bruce, James W; Bradley, Kenneth A; Ahlquist, Paul et al. (2005) Isolation of cell lines that show novel, murine leukemia virus-specific blocks to early steps of retroviral replication. J Virol 79:12969-78
Lim, Kwang-Il; Narayan, Shakti; Young, John A T et al. (2004) Effects of lipid rafts on dynamics of retroviral entry and trafficking: Quantitative analysis. Biotechnol Bioeng 86:650-60
Melikyan, G B; Barnard, R J O; Markosyan, R M et al. (2004) Low pH is required for avian sarcoma and leukosis virus Env-induced hemifusion and fusion pore formation but not for pore growth. J Virol 78:3753-62
Narayan, Shakti; Young, John A T (2004) Reconstitution of retroviral fusion and uncoating in a cell-free system. Proc Natl Acad Sci U S A 101:7721-6
Barnard, Richard J O; Narayan, Shakti; Dornadula, Geethanjali et al. (2004) Low pH is required for avian sarcoma and leukosis virus Env-dependent viral penetration into the cytosol and not for viral uncoating. J Virol 78:10433-41
Narayan, Shakti; Barnard, Richard J O; Young, John A T (2003) Two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity. J Virol 77:1977-83
Yu, Xuemei; Wang, Qing-Yin; Guo, Ying et al. (2003) Kinetic analysis of binding interaction between the subgroup A Rous sarcoma virus glycoprotein SU and its cognate receptor Tva: calcium is not required for ligand binding. J Virol 77:7517-26
Barnard, R J O; Young, J A T (2003) Alpharetrovirus envelope-receptor interactions. Curr Top Microbiol Immunol 281:107-36

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