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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Virology - A Study Section (VIRA)
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Read-Connole, Elizabeth Lee
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Pennsylvania State University
Internal Medicine/Medicine
Schools of Medicine
United States
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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
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
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
Lochmann, Timothy L; Bann, Darrin V; Ryan, Eileen P et al. (2013) NC-mediated nucleolar localization of retroviral gag proteins. Virus Res 171:304-18
Nadaraia-Hoke, Shorena; Bann, Darrin V; Lochmann, Timothy L et al. (2013) Alterations in the MA and NC domains modulate phosphoinositide-dependent plasma membrane localization of the Rous sarcoma virus Gag protein. J Virol 87:3609-15
Parent, Leslie J (2011) New insights into the nuclear localization of retroviral Gag proteins. Nucleus 2:92-7
Parent, Leslie J; Gudleski, Nicole (2011) Beyond plasma membrane targeting: role of the MA domain of Gag in retroviral genome encapsidation. J Mol Biol 410:553-64
Gudleski, Nicole; Flanagan, John M; Ryan, Eileen P et al. (2010) Directionality of nucleocytoplasmic transport of the retroviral gag protein depends on sequential binding of karyopherins and viral RNA. Proc Natl Acad Sci U S A 107:9358-63
Garbitt-Hirst, Rachel; Kenney, Scott P; Parent, Leslie J (2009) Genetic evidence for a connection between Rous sarcoma virus gag nuclear trafficking and genomic RNA packaging. J Virol 83:6790-7

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