Viral pathogens possessing nonsegmented, negative-sense RNA genomes are the cause of devastating diseases worldwide (e.g. Ebola, measles, and rabies viruses). Virion assembly and release (budding) are important late events in the replication cycles of these viruses; however, the molecular mechanisms employed by negative-sense RNA viruses to bud and separate from cells remain largely undefined. The experiments described in this proposal are significant for the following reasons: i) They will provide fundamental information on the mechanisms of virus budding (Sp.
Aim 1). ii) They not only will provide information about the virus, but also may provide information concerning the role of the host cell in virus budding (Sp.
Aim 2). iii) They may be applicable for the development of future DNA-based vaccines (Sp.
Aim 3). A highly conserved proline-rich motif (PY motif) within the M protein of vesicular stomatitis virus (VSV) has been implicated in mediating both release of M protein from mammalian cells and interactions with specific domains (WW-domains) of cellular proteins. The biological significance of budding domains within the VSV M protein will be tested using reverse-genetics techniques and electron microscopic analysis. Virus-host interactions mediated by the viral PY motif and the cellular WW-domain will be characterized both in vitro and in vivo. The biological relevance of these protein-protein interactions will be assessed in both yeast and mammalian cells. Lastly, the ability of the PY motif from the VSV M protein to direct the budding of an otherwise nonbudding, heterologous protein will be tested in a functional budding assay. Overall, the experiments described in this proposal not only will provide important insights into the molecular aspects of budding of rhabdoviruses, but also will likely provide important insights into the budding mechanisms of other negative-sense RNA viruses that possess conserved PY or PY-like motifs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI046499-01A2
Application #
6430794
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Meegan, James M
Project Start
2001-12-01
Project End
2006-11-30
Budget Start
2001-12-01
Budget End
2002-11-30
Support Year
1
Fiscal Year
2002
Total Cost
$277,375
Indirect Cost
Name
University of Pennsylvania
Department
Pathology
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Irie, Takashi; Carnero, Elena; GarcĂ­a-Sastre, Adolfo et al. (2012) In Vivo Replication and Pathogenesis of Vesicular Stomatitis Virus Recombinant M40 Containing Ebola Virus L-Domain Sequences. Infect Dis (Auckl) 5:59-64
Okumura, Atsushi; Harty, Ronald N (2011) Rabies virus assembly and budding. Adv Virus Res 79:23-32
Harty, Ronald N (2009) No exit: targeting the budding process to inhibit filovirus replication. Antiviral Res 81:189-97
Wirblich, Christoph; Tan, Gene S; Papaneri, Amy et al. (2008) PPEY motif within the rabies virus (RV) matrix protein is essential for efficient virion release and RV pathogenicity. J Virol 82:9730-8
Okumura, Atsushi; Pitha, Paula M; Harty, Ronald N (2008) ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc Natl Acad Sci U S A 105:3974-9
Han, Ziying; Licata, Jillian M; Paragas, Jason et al. (2007) Permeabilization of the plasma membrane by Ebola virus GP2. Virus Genes 34:273-81
Irie, Takashi; Carnero, Elena; Okumura, Atsushi et al. (2007) Modifications of the PSAP region of the matrix protein lead to attenuation of vesicular stomatitis virus in vitro and in vivo. J Gen Virol 88:2559-67
Johnson, Reed F; Bell, Peter; Harty, Ronald N (2006) Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology. Virol J 3:31
Han, Ziying; Harty, Ronald N (2005) Packaging of actin into Ebola virus VLPs. Virol J 2:92

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