title: Cooperativity among Rev-associated nuclear export signals regulates HIV-1 gene expression and is a determinant of virus species tropism.
abstract: Murine cells exhibit a profound block to HIV-1 virion production that was recently mapped to a species-specific structural attribute of the murine version of the chromosomal region maintenance 1 (mCRM1) nuclear export receptor and rescued by the expression of human CRM1 (hCRM1). In human cells, the HIV-1 Rev protein recruits hCRM1 to intron-containing viral mRNAs encoding the Rev response element (RRE), thereby facilitating viral late gene expression. Here we exploited murine 3T3 fibroblasts as a gain-of-function system to study hCRM1's species-specific role in regulating Rev's effector functions. We show that Rev is rapidly exported from the nucleus by mCRM1 despite only weak contributions to HIV-1's posttranscriptional stages. Indeed, Rev preferentially accumulates in the cytoplasm of murine 3T3 cells with or without hCRM1 expression, in contrast to human HeLa cells, where Rev exhibits striking en masse transitions between the nuclear and cytoplasmic compartments. Efforts to bias Rev's trafficking either into or out of the nucleus revealed that Rev encoding a second CRM1 binding domain (Rev-2xNES) or Rev-dependent viral gag-pol mRNAs bearing tandem RREs (GP-2xRRE), rescue virus particle production in murine cells even in the absence of hCRM1. Combined, these results suggest a model wherein Rev-associated nuclear export signals cooperate to regulate the number or quality of CRM1's interactions with viral Rev/RRE ribonucleoprotein complexes in the nucleus. This mechanism regulates CRM1-dependent viral gene expression and is a determinant of HIV-1's capacity to produce virions in nonhuman cell types.
  
  
  OBJECTIVE
  Cells derived from mice and other nonhuman species exhibit profound blocks to HIV-1 replication. Here we elucidate a block to HIV-1 gene expression attributable to the murine version of the CRM1 (mCRM1) nuclear export receptor. In human cells, hCRM1 regulates the nuclear export of viral intron-containing mRNAs through the activity of the viral Rev adapter protein that forms a multimeric complex on these mRNAs prior to recruiting hCRM1. We demonstrate that Rev-dependent gene expression is poor in murine cells despite the finding that, surprisingly, the bulk of Rev interacts efficiently with mCRM1 and is rapidly exported from the nucleus. Instead, we map the mCRM1 defect to the apparent inability of this factor to engage Rev multimers in the context of large viral Rev/RNA ribonucleoprotein complexes. These findings shed new light on HIV-1 gene regulation and could inform the development of novel antiviral strategies that target viral gene expression.
authors.affiliation: McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Mounavya
authors.initials: M
authors.lastname: Aligeti
authors.affiliation: McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Behrens
authors.affiliation: McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA Morgridge Institute for Research, Madison, Wisconsin, USA.
authors.forename: Ginger M
authors.initials: GM
authors.lastname: Pocock
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Johannes
authors.initials: J
authors.lastname: Schindelin
authors.affiliation: Department of Computer and Information Science, University of Constance, Constance, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Dietz
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin, USA Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Eliceiri
authors.affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.
authors.forename: Chad M
authors.initials: CM
authors.lastname: Swanson
authors.affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Malim
authors.affiliation: McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA Morgridge Institute for Research, Madison, Wisconsin, USA Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Ahlquist
authors.affiliation: McArdle Laboratory for Cancer Research and Institute for Molecular Virology, University of Wisconsin-Madison, Madison, Wisconsin, USA nsherer@wisc.edu.
authors.forename: Nathan M
authors.initials: NM
authors.lastname: Sherer
mesh_terms: D000818:Animals; D002460:Cell Line; D005347:Fibroblasts / Q000821:virology; D015967:Gene Expression Regulation, Viral; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D051379:Mice; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D056189:Viral Tropism; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism*; D000097606:Exportin 1 Protein
chemical_list: D028884:Karyopherins; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097606:Exportin 1 Protein; C514860:rev protein, Human Immunodeficiency Virus-1