title: Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.
abstract: HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.
  
  
  OBJECTIVE
  HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Behrens
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Mounavya
authors.initials: M
authors.lastname: Aligeti
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Morgridge Institute for Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Ginger M
authors.initials: GM
authors.lastname: Pocock
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Higgins
authors.affiliation: McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA nsherer@wisc.edu.
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Sherer
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002460:Cell Line; D002478:Cells, Cultured; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008954:Models, Biological; D019913:Nuclear Localization Signals / Q000737:chemistry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D034443:RNA Transport; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1
keywords: CRM1; Gag; RNA trafficking; Rev; exportin-1; human immunodeficiency virus; nuclear export signal; nuclear pore complex; nucleolus; retroviruses