Retroviruses co-opt cellular pathways to facilitate the assembly of virus particles that spread infection. Identifying the host factors usurped by retroviruses for virus assembly will enhance our understanding of retrovirus-cell interactions and may uncover new targets for antiviral therapy. Although host factors engaged in late steps of retrovirus assembly and release have been identified, the early stages of assembly remain poorly understood because the molecular events that initiate assembly have been difficult to study. However, our discovery that the Rous sarcoma virus (RSV) Gag protein, which orchestrates the assembly process, undergoes nuclear trafficking has allowed us to study early events in the assembly pathway that occur in the nucleus. Using this system, we showed that nuclear trafficking of RSV Gag is crucial for the formation of infectious virus particles, and mutants that bypass the nucleus incorporate reduced amounts of viral genomic RNA. Furthermore, our structural studies demonstrate that Gag binding to nuclear export machinery is facilitated by Gag-RNA interactions, providing a mechanism for the regulated transport of viral ribonucleoprotein complexes. New data from our laboratory suggest that RSV Gag may traffic to sites of active transcription to recognize and select viral genomic RNA in the nucleus. Therefore, our research may define a novel paradigm for how retroviruses select and package their genomes. In this application, we propose two Specific Aims to test the novel hypothesis that Gag nuclear trafficking facilitates recognition, selection, and packaging of the genomic RNA. These mechanistic experiments take advantage of our well-characterized toolbox of Gag mutants with altered nuclear trafficking and RNA packaging properties. We will utilize our unique set of reagents to investigate whether Gag is tethered near sites of RNA synthesis to mediate co-transcriptional packaging of viral genomic RNA. In addition, we will use mass spectrometry to identify host factors that interact with RSV Gag and deep sequencing to analyze cellular RNA targets of Gag. Proteomic analysis of Gag-interacting factors from diverse retroviruses that undergo nuclear localization will broaden the impact of this application and provide insight into potential common functions of retroviral Gag proteins in the nucleus. Through this well-crafted, focused experimental plan, we will shed light on fundamental aspects of retrovirus assembly, genomic RNA packaging, and nuclear events to reveal new information about the interplay between retroviral pathogens and their host cells.

Public Health Relevance

Retroviruses cause cancer and immunodeficiency syndromes in people and animals. This proposal focuses on understanding molecular mechanisms that direct intracellular trafficking of the Gag protein and genomic RNA during virus assembly. Results of this research may enhance our understanding of how retroviruses produce new virus particles, potentially leading to new antiviral targets and enhanced retroviral vectors for gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA076534-16A1
Application #
8760613
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1998-07-01
Project End
2019-06-30
Budget Start
2014-08-11
Budget End
2015-06-30
Support Year
16
Fiscal Year
2014
Total Cost
$279,752
Indirect Cost
$99,752
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 Pr55(Gag) 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
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
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

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