title: A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1.
abstract: BACKGROUND
  Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex.
  
  
  RESULTS
  To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells.
  
  
  CONCLUSIONS
  Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev's activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function.
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. min-hsuan.lin@qimrberghofer.edu.au.
authors.forename: Min-Hsuan
authors.initials: MH
authors.lastname: Lin
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. haran.sivakumaran@qimrberghofer.edu.au.
authors.forename: Haran
authors.initials: H
authors.lastname: Sivakumaran
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia. a.jones@imb.uq.edu.au.
authors.forename: Alun
authors.initials: A
authors.lastname: Jones
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. dongsheng.lin@qimrberghofer.edu.au.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Li
authors.affiliation: Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, Australia. c.harper@uq.edu.au.
authors.forename: Callista
authors.initials: C
authors.lastname: Harper
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. ting.wei@qimrberghofer.edu.au.
authors.forename: Ting
authors.initials: T
authors.lastname: Wei
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. hongping.jin@qimrberghofer.edu.au.
authors.forename: Hongping
authors.initials: H
authors.lastname: Jin
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. lina.rustanti@qimrberghofer.edu.au.
authors.forename: Lina
authors.initials: L
authors.lastname: Rustanti
authors.affiliation: Australian Infectious Disease Research Centre, The University of Queensland, St. Lucia, Queensland, Australia. f.meunier@uq.edu.au.|Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, Australia. f.meunier@uq.edu.au.
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Meunier
authors.affiliation: Australian Infectious Disease Research Centre, The University of Queensland, St. Lucia, Queensland, Australia. k.spann@uq.edu.au.|School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, Australia. k.spann@uq.edu.au.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Spann
authors.affiliation: QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia. david.harrich@qimrberghofer.edu.au.|Australian Infectious Disease Research Centre, The University of Queensland, St. Lucia, Queensland, Australia. david.harrich@qimrberghofer.edu.au.
authors.forename: David
authors.initials: D
authors.lastname: Harrich
mesh_terms: D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D040901:Proteomics; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000037:antagonists & inhibitors*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases