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.

Nucleolar Relocalization of RBM14 by Influenza A Virus NS1 Protein

“…We sought to characterize a hit from our results to better understand its role in viral replication. RNA binding motif protein 14 (RBM14) has not been previously characterized in the context of respiratory virus infection; however, it has been shown to interact with human immunodeficiency virus type 1 (HIV-1) Rev ( 23 ) and Tat ( 24 ) proteins and to modulate the expression of p24 ( 41 ). For influenza virus, affinity-purification mass spectrometry (AP-MS) studies have linked RBM14 to the components of the IAV polymerase complex, NEP, and NS1 ( 42 ), but no validation has been performed.…”

“…RBM14 is typically nuclear ( 21 ) and has been identified as a component of nuclear paraspeckles ( 22 ). Its known interactions with human viruses are limited to human immunodeficiency virus type 1 (HIV-1) ( 23 , 24 ) and Epstein-Barr virus (EBV) ( 25 ). RBM14 has been implicated in innate immunity through its interaction with SMURF2 ( 26 ) and TRAF3 ( 27 ) and through its role in DNA sensing by cGAS ( 22 ).…”

Semen exosomes

“…Using SE to repress HIV-1 transcription allowed us to identify transcription elongation as another target of SE in provirus transcription. To our knowledge, this is the first report to suggest remodeling [164], mRNA splicing [165], Rev function [274], microRNA biogenesis [275], and reverse transcriptase (RT) function [166][167][168][169][170]. Interestingly, we previously found that SE regulate the ratio of HIV-1 RT subunits [63].…”