Host Cell Interactome of HIV-1 Rev Includes RNA Helicases Involved in Multiple Facets of Virus Production

Abstract

The HIV-1 Rev protein plays a key role in the late phase of virus replication. It binds to the Rev Response Element found in underspliced HIV mRNAs, and drives their nuclear export by the CRM1 receptor pathway. Moreover, mounting evidence suggests that Rev has additional functions in viral replication. Here we employed proteomics and statistical analysis to identify candidate host cell factors that interact with Rev. For this we studied Rev complexes assembled in vitro with nuclear or cytosolic extracts under conditions emulating various intracellular environments of Rev. We ranked the protein-protein interactions by combining several statistical features derived from pairwise comparison of conditions in which the abundance of the binding partners changed. As a validation set, we selected the eight DEAD/H box proteins of the RNA helicase family from the top-ranking 5% of the proteins. These proteins all associate with ectopically expressed Rev in immunoprecipitates of cultured cells. From gene knockdown approaches, our work in combination with previous studies indicates that six of the eight DEAD/H proteins are linked to HIV production in our cell model. In a more detailed analysis of infected cells where either DDX3X, DDX5, DDX17, or DDX21 was silenced, we observed distinctive phenotypes for multiple replication features, variously involving virus particle release, the levels of unspliced and spliced HIV mRNAs, and the nuclear and cytoplasmic concentrations of these transcripts. Altogether the work indicates that our top-scoring data set is enriched in Rev-interacting proteins relevant to HIV replication. Our more detailed analysis of several Revinteracting DEAD proteins suggests a complex set of functions for the helicases in regulation of HIV mRNAs. HIV-1 utilizes many host cell factors for its replication (1-3), similar to other viruses. There is strong interest in identifying and understanding these components to shed light on the molecular mechanisms of virus replication. Moreover, this can provide the potential for developing new therapeutics. The HIV Rev protein is a key regulator of viral replication that is critical for the late stages of virus replication (4, 5). The bestcharacterized function of Rev involves its potent stimulation of the nuclear export of unspliced and singly spliced ("underspliced") HIV transcripts that encode the viral structural proteins and accessory factors (5). In the absence of Rev, these transcripts are retained in the nucleus because of their incomplete splicing. At the molecular level, Rev binds and oligomerizes along the 351-nt Rev Response Element (RRE) 1 (6) in the env gene that is present in all underspliced HIV transcripts. Rev contains a classical leucine-rich nuclear export sequence that recruits CRM1, a transport receptor of the karyopherin family (7,8). CRM1 commonly is used for nuclear export of cellular proteins, and only infrequently is involved in cellular mRNA export (9, 10). Upon binding to the RRE together with the GTP-bound form of Ran, CRM1 forms the core ...

Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA

“…31 Our results support such a model in the control of vRNA encapsidation that involves subtle changes to the composition and trafficking of the HIV-1 RNP. hnRNP A2/B1 is a common component of several relevant HIV-1 complexes that have been recently characterized at the proteomics level 9,33,65 and these hnRNPs likely exert functions during the transit of HIV-1 complexes from the nucleus to and within the cytoplasm, perhaps acting in concert with Rev at multiple levels.…”

“…[6][7][8] Several host proteins have been shown to influence Rev's activity by direct interaction or by influencing the ribonucleoprotein (RNP) context of the RNA. [9][10][11][12] Rev also has effects on splicing regulation 13,14 as well as on downstream gene expression events at the translation level. [15][16][17] Efforts to understand Rev's functions during viral replication cycle have provided several key leads in the identification of potential therapeutic strategies and targets 18,19 and have provided critical information on the processes governing nuclear RNA export in general.…”

Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4+T Cells

“…We initially identified NonO interacting with PICs in HIV-1-infected CD4 + T cell lines, 9 and subsequent studies have also identified NonO to be an HIV-1 interactant. [10][11][12] However, the significance of these interactions and whether NonO plays a crucial role in HIV-1 infection have not been answered. We addressed these questions by investigating the effect of NonO KD on HIV-1 infection and characterized what stages of the replication cycle were affected.…”

“…11 Furthermore, NonO was identified in an analysis of the Rev interactome in HeLa cells, and the association between NonO and Rev was enhanced by the presence of the Rev response element. 12 These studies suggest that NonO may affect multiple steps of the HIV-1 lifecycle including integration. However, the role of NonO in HIV-1 infection has not been clearly characterized.…”

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

“…Extensive research has identified cellular proteins by direct and indirect interactions with Rev that facilitate transport of RRE-containing viral mRNA out of the nucleus [34,[42][43][44]. We had shown that a mutant Tat protein, we call Nullbasic, has an intriguing ability to inhibit Rev function resulting from its mislocalization from the nucleolus to the nucleoplasm and cytoplasm.…”