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

Lin, Min-Hsuan, Sivakumaran, Haran, Jones, Alun, Li, Dongsheng, Harper, Callista, Wei, Ting, Jin, Hongping, Rustanti, Lina, Meunier, Frederic A., Spann, Kirsten and Harrich, David (2014) A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1. Retrovirology, 11 1: 121.1-121.16. doi:10.1186/s12977-014-0121-9


Author Lin, Min-Hsuan
Sivakumaran, Haran
Jones, Alun
Li, Dongsheng
Harper, Callista
Wei, Ting
Jin, Hongping
Rustanti, Lina
Meunier, Frederic A.
Spann, Kirsten
Harrich, David
Title A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1
Journal name Retrovirology   Check publisher's open access policy
ISSN 1742-4690
Publication date 2014-12-01
Sub-type Article (original research)
DOI 10.1186/s12977-014-0121-9
Open Access Status DOI
Volume 11
Issue 1
Start page 121.1
End page 121.16
Total pages 16
Place of publication London, United Kingdom
Publisher BioMed Central
Language eng
Formatted 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.
Keyword HIV-1
Rev
Tat
Nullbasic
Helicase
DDX1
RNA export
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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Created: Fri, 23 Jan 2015, 20:22:14 EST by Susan Allen on behalf of Institute for Molecular Bioscience