Retroviruses cause incurable malignancies and immunodeficiency diseases in animals and humans. The avian pathogen Rous sarcoma virus (RSV), the first virus linked to cancer, is the prototypic oncoretrovirus, and its use as a model system has led to significant advances in understanding the human retroviruses HIV and HTLV-I. All retroviruses must gain access to the nucleus in order to establish infection. Following reverse transcription of the RNA genome in the cytoplasm, the preintegration complex (PIC) enters the nucleus. In RSV and lentiviruses including HIV, nuclear entry appears to be an active process, as mitosis is not absolutely required. This process of nuclear import is one of the least understood aspects of the viral life cycle. In the previous funding period, we identified a nuclear import signal in the matrix (MA) domain of the RSV Gag protein, raising the possibility that MA might play a role in nuclear import of the PIC. In addition, we found that the nuclear import signal in MA mediates nuclear localization of the Gag polyprotein during the assembly phase of the replication cycle, and a CRMl-dependent nuclear export signal in the p10 sequence returns Gag to the cytosol. Interestingly, a mutant Gag protein that is defective in viral RNA packaging bypasses the nuclear compartment, suggesting a functional link between the nuclear trafficking of Gag and encapsidation of viral RNA. The major goal of the current proposal is to capitalize on these pivotal findings as a means to elucidate (1) the viral factors that control nucleocytoplasmic trafficking of Gag, (2) host cofactors that mediate nuclear entry and egress of Gag, and (3) the functional significance of transient nuclear localization of Gag. In the first Specific Aim, we propose to define the NLS in MA, the mechanism of nuclear entry, and the cellular pathways used for import.
In Aim 2, the Gag NES will be characterized and host factors that mediate export will be identified.
Specific Aim 3 focuses on testable hypotheses that address the role of MA and Gag proteins in nuclear import of the PIC and selection of genomic viral RNA for packaging into virions. Through these studies, we expect to gain insight into the essential but poorly understood mechanisms underlying viral nuclear entry and RNA encapsidation. By elucidating a fundamental pathway critical for virus replication, we hope to find targets for a new class of agents to combat retroviral diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076534-07
Application #
6744039
Study Section
Virology Study Section (VR)
Program Officer
Cole, John S
Project Start
1998-07-01
Project End
2008-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
7
Fiscal Year
2004
Total Cost
$261,975
Indirect Cost
Name
Pennsylvania State University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Bewley, Maria C; Reinhart, Lisa; Stake, Matthew S et al. (2017) A non-cleavable hexahistidine affinity tag at the carboxyl-terminus of the HIV-1 Pr55Gag polyprotein alters nucleic acid binding properties. Protein Expr Purif 130:137-145
Rye-McCurdy, Tiffiny; Olson, Erik D; Liu, Shuohui et al. (2016) Functional Equivalence of Retroviral MA Domains in Facilitating Psi RNA Binding Specificity by Gag. Viruses 8:
Kaddis Maldonado, Rebecca J; Parent, Leslie J (2016) Orchestrating the Selection and Packaging of Genomic RNA by Retroviruses: An Ensemble of Viral and Host Factors. Viruses 8:
Stake, Matthew; Singh, Deepali; Singh, Gatikrushna et al. (2015) HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins. Virology 486:307-20
Rice, Breanna L; Kaddis, Rebecca J; Stake, Matthew S et al. (2015) Interplay between the alpharetroviral Gag protein and SR proteins SF2 and SC35 in the nucleus. Front Microbiol 6:925
Bann, Darrin V; Beyer, Andrea R; Parent, Leslie J (2014) A murine retrovirus co-Opts YB-1, a translational regulator and stress granule-associated protein, to facilitate virus assembly. J Virol 88:4434-50
Rye-McCurdy, Tiffiny D; Nadaraia-Hoke, Shorena; Gudleski-O'Regan, Nicole et al. (2014) Mechanistic differences between nucleic acid chaperone activities of the Gag proteins of Rous sarcoma virus and human immunodeficiency virus type 1 are attributed to the MA domain. J Virol 88:7852-61
Stake, Matthew S; Bann, Darrin V; Kaddis, Rebecca J et al. (2013) Nuclear trafficking of retroviral RNAs and Gag proteins during late steps of replication. Viruses 5:2767-95
Webb, Joseph A; Jones, Christopher P; Parent, Leslie J et al. (2013) Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging. RNA 19:1078-88
Beyer, Andrea R; Bann, Darrin V; Rice, Breanna et al. (2013) Nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein L9. J Virol 87:1069-82

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