A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

Retrovirology

David Harrich¹  Kirsten Spann³  Frederic A Meunier⁵  Lina Rustanti²  Hongping Jin²  Ting Wei²  Callista Harper⁵  Dongsheng Li²  Alun Jones⁴  Haran Sivakumaran²  Min-Hsuan Lin² 

[1]Australian Infectious Disease Research Centre, The University of Queensland, St. Lucia, Queensland, Australia

[2]QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia

[3]School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland, Australia

[4]Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia

[5]Queensland Brain Institute, The University of Queensland, St. Lucia, Queensland, Australia

关键词: RNA export;    DDX1;    Helicase;    Nullbasic;    Tat;    Rev;    HIV-1;   

Others  :  1151493
DOI  :  10.1186/s12977-014-0121-9

 received in 2014-09-29, accepted in 2014-12-03,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Lin et al.; licensee BioMed Central Ltd.

【 图 表 】

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