Viral budding (membrane deformation away from the host cell) is the final required step in the replication of enveloped viruses such as HIV and Ebola virus (EBOV). To bud, HIV and EBOV hijack cellular trafficking proteins normally involved in cell surface receptor downregulation through the multivesicular body (MVB) pathway, including Nedd4-family ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs). By co-opting ubiquitin ligases and ESCRTs, viral infections can interfere with the sorting of endogenous proteins, such as epidermal growth factor receptor and the CXCR4 chemokine receptor and may impact the sorting of other receptors, such as the delta opioid receptor and beta2- adrenergic receptors, with wide ranging ramifications. For instance, disruption of delta opioid receptor trafficking by viral infection could have implications for T-cell activation and HIV replication, particularly in a setting of opioid abuse. Ubiquitin ligases and the ESCRTs are highly conserved in S. cerevisiae, making it a good model system for understanding these interactions. In MVB sorting ubiquitin ligases have both enzymatic (ubiquitination) and non-enzymatic (adaptor) roles, but it is less clear which ligase activities are required for viral budding. We will systematically address the role of ubiquitin ligase interactions on the budding ofthe viral structural proteins HIV Gag and EBOV VP40, using the yeast system. We will begin by mapping the interactions of Gag with the ubiquitin ligase Rsp5 (a Nedd4 homolog) and then examine the functional impact of defects in ligase binding and ubiquitination on the budding of Gag and VP40 viral-like particles (VLPs). Then we will investigate the impact of viral proteins on the trafficking of endogenous MVB cargos using pulse-chase kinetic analysis of cargo trafficking in the setting of Gag and VP40 expression. Finally, we will begin the development of viral budding inhibitors using a rational design approach that combines In silico inhibitor screening with laboratory based experimental testing and validation. The goal of this research is to understand the molecular mechanisms of HIV and Ebola virus budding, the final step in replication. This work will contribute to the development of novel therapeutics to treat viral infections. Additionally, this knowledge may help us to understand potentially wide-ranging consequences of viral infection, including oncogenesis, cadiomyopathy, addiction, and neurodegeneration. The insights gained from this research will have broad application to the study of other enveloped viruses, including Epstein- Barr virus. Human T-cell Lymophotropic virus. Influenza virus, and others.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30DA026762-03
Application #
8286405
Study Section
Special Emphasis Panel (ZRG1-AARR-H (22))
Program Officer
Avila, Albert
Project Start
2010-07-01
Project End
2013-05-17
Budget Start
2012-07-01
Budget End
2013-05-17
Support Year
3
Fiscal Year
2012
Total Cost
$30,761
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Norgan, Andrew P; Davies, Brian A; Azmi, Ishara F et al. (2013) Relief of autoinhibition enhances Vta1 activation of Vps4 via the Vps4 stimulatory element. J Biol Chem 288:26147-56
Norgan, Andrew P; Arguello, Heather E; Sloan, Lynne M et al. (2013) A method for reducing the sloughing of thick blood films for malaria diagnosis. Malar J 12:231