HIV-1 encodes the nuclear regulatory protein Tat, which is essential for HIV-1 replication and which primarily orchestrates HIV-1 provirus transcriptional regulation. Tat transactivation from the viral promoter (LTR), is highly dependent on complex interactions between Tat, the short leader RNA present in the 5' region of all nascent HIV-1 transcripts, TAR (Trans-activation responsive element), and a number of host cellular proteins 1234. The molecular mechanisms whereby HIV-1 gene expression is regulated by Tat occurs at distinct levels. Initially, Tat enhances transcription initiation by promoting the assembly of the RNA polII complex by interacting with various transcription factors 2. Subsequently, Tat activates elongation via two independent mechanisms: firstly, it enhances the processivity of RNA polII by interacting with elongation factors such as pTEF-b, which phosphorylates RNA polII C-terminal domain, and secondly, by recruiting histone acetyltransferase proteins which modify the chromatin template such as p300/CBP (CREB binding protein) and p300/CBP-associated factor (PCAF) and, as recently described, by interacting with BRM and BRG1, two chromatin remodellers5678910. Although the recruitment of these specific cellular factors by Tat to the HIV-1 LTR are crucial for Tat function, they only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. Furthermore, Tat can be secreted by infected cells and extracellular Tat can exert autocrine or paracrine activities via interactions with cell surface receptors including integrins, CXCR4, CD26, HSPG and LRP11.

While Tat is a small and compact protein, composed of only 86 or 101 amino acids, sequence and functional analysis reveals that Tat sequence encompasses a unique arrangement of five distinct and contiguous regions including the acidic, cysteine-rich, core, basic and glutamine-rich regions. Furthermore, Tat is subject to post-translational modifications, such as acetylation, methylation, phosphorylation and ubiquitination, thus increasing both the number and diversity of potential interfaces between Tat and cellular proteins 121314. Recently, a structural study employing nuclear magnetic resonance (NMR) spectroscopy has described Tat as a "natively unfolded" protein with fast dynamics lacking a well-structured three-dimensional fold. These characteristics would provide Tat the flexibility to interact with numerous cellular partners. Collectively these findings suggest that Tat is a potent, versatile protein suited for multiple interactions and highlights the concept that numerous protein-protein interactions underlie the molecular mechanisms of HIV-1 molecular pathogenesis 1516171819.

In this report, we have attempted to further investigate the interplay of Tat with host cell proteins. Specifically, we have designed a proteomic strategy based on affinity chromatography (AC) coupled with mass spectrometry (MS) to purify Tat interacting proteins from T-cell nuclear extracts (Figure 1). Our approach has produced the in vitro Tat nuclear interactome, which includes a total of 183 individual nuclear components, most of which have not been previously identified as Tat partners. We subsequently applied in silico analysis, to validate our dataset and develop HIV-1 Tat interaction network maps. In this report, we have focused on the description of multi-protein complexes involved in gene expression regulation, which comprised the majority of our dataset and which clearly reflects Tat primary function.

While considerable efforts have been dedicated to characterise individual proteins or specific macromolecular complexes interacting with Tat, no comprehensive characterisation of the Tat interactome has yet been reported. To place Tat into a wider context of interacting systems and pathways, we systematically analysed protein complexes interacting with Tat within the nucleus, by performing subcellular fractionation followed by AC-MS/MS. This experimental approach is prone to introducing technical artifacts or false positives which can then bias the subsequent analysis. To reduce this, we filtered the raw list of proteins, removed potential contaminants and obtained a final dataset of 183 interaction candidates. Subsequently, we employed computational tools and in silico analysis to validate our interaction dataset and to generate a Tat-interaction network representation. This has resulted in the in vitro Tat nuclear interactome in Jurkat T-cells.

Our studies have revealed that the Tat nuclear interactome exhibits unique signature(s). The motif composition analysis underlines the enrichment for domains mediating protein, RNA and DNA interactions, which collectively are highly representated in transcription, chromatin remodelling, chromosome structure and RNA processing complexes. We also noted the enrichment of three crucial motifs (HELIC, DEXDc and AAA) associated with helicase and ATPase activities essential for RNA processing, chromatin remodeling and chromosome architecture, which constitute the basis of both DNA replication and gene expression regulation. In support of this, the functional analysis demonstrated that proteins involved in chromatin remodeling, transcription regulation and RNA processing constitute the greater part of our dataset. Finally, the network reconstruction of the Tat nuclear interactome revealed the higher-order and collective behaviour of the Tat-interacting proteins, which compose large but well defined biochemical entities, involved in critical pathways mediating gene expression regulation, chromosome/chromatin structure, and nuclear architecture. Taken together, the remarkable enrichment for essential proteins together with their corresponding macromolecular complexes, and their roles in both activation and repression of gene expression indicate that the described Tat-interactome might act as a modular switch committed to control HIV-1 gene expression.

The presence of numerous, previously identified Tat-interacting partners further validates our dataset. Conversely, critical Tat cellular partners previously identified were not identified by our experimental approach. This could be the result of the level of endogenous expression of these proteins in Jurkat cells and perhaps technical limitations including the following: (i) loss of a fraction of the proteins during the sub-cellular fractionation step; (ii) proteins resistant to trypsin digestion; (iii) proteins not detected by the MS/MS step; (iv) absence of specific Tat post-translational modifications on our recombinant bait, GST-Tat, which is produced by a bacterial expression system. Indeed, Tat interaction with its cellular partner has been shown to be regulated by the acetylation state of its lysines 28, 50 and 51. Interaction of cyclinT1 with Tat requires the acetylation on lysine 28, while acetylation on lysine 50 prevents Tat interaction with Brm but is necessary for its interaction with BRG1 567256257. Nevertheless, despite absence of CyclinT1 from our interaction dataset, we were able to detect it by WB, further establishing our recombinant GST-Tat as a suitable bait.

Our experimental approach does not enable us to distinguish direct from indirect interactions. Furthermore, while we treated our nuclear extract with the Benzonase^® nuclease (included as part of the ProteoExtract^® Subcellular Proteome Extraction Kit (Calbiochem)), which has both a DNase and an RNase activities, we cannot exclude that some of the observed interactions could be mediated by residual nucleic acids.

To explore the Tat interaction network in detail, we partitioned the interacting candidates into coherent functional modules, based on their reciprocal interactions. Our results further support the recently described role of SNF proteins in Tat-dependent transcription of the provirus56722258259. Indeed, we have identified BRG1 and the various components of its related complex, SWI2/SNF2. We have also identified for the first time, WICH, an ISWI complex as a candidate Tat interactor.

Other complexes with histone-modifying activities include TRRAP/p400, a histone acetylase, and SET1, a histone methylase, both of which have critical positive effects on transcription. In addition, we isolated the histone chaperone FACT, directly involved in promoting the processivity of RNA polII through the chromatin template, which appears to be of relevance for Tat function in transcription elongation236.

Importantly, we have provided the first evidence describing a direct or indirect interaction of Tat with cellular proteins, including YY1, HDAC-1/-2, and the components of the SIN3/HDAC and NuRD complexes, previously reported to interact with the integrated HIV-1 LTR. Indeed, earlier studies have identified the presence of HDAC-1 and -2 at the HIV-1 LTR in HeLa cells containing an integrated HIV-1 LTR reporter, as well in latently HIV-1 infected cell lines (ACH2, U1, J-Lat 6.3)260261262263264265266. They mediate histone deacetylation of nuc-1, the nucleosome positioned immediately downstream of the transcription start site, and are believed to be one of the processes mediating HIV-1 provirus transcriptional silencing throughout the establishment and/or maintenance of HIV-1 latency. In addition, two recent studies have identified the presence of SIN3A and MTA1, a component of the NuRD complex, at the integrated HIV-1 LTR in Jurkat cells, respectively recruited by CBF-1 and CTIP2241243. While various studies have shown that several transcription factors (NF-κB (p50), AP-4, YY-1, c-myc, SP1, CBF-1 and LSF) can recruit HDAC-1 and -2, and SIN3A or MTA1 to the HIV-1 LTR, the molecular mechanism(s) regulating the activity and/or presence of HDAC-1/-2 and their associated complexes at the integrated HIV-1 LTR has not been fully elucidated241243260261262263264265266. The interaction of HIV-1 Tat with the SIN3/HDAC and NuRD complexes further implicates them as potential epigenetic regulators of HIV-1 post-integration latency and suggests how Tat might intersect with epigenetic pathways.

Alternatively, the enzymatic activities of the multi-protein complexes recruited by Tat, such as methylation, acetylation and deacetylation, could be directed to Tat itself and mediate post-translational modifications, as it has been shown previously with PCAF, p300/CBP, SIRT1, or SETDB1/2 1314267268.

Accumulative evidence has recently described how cellular proteins belonging to the DNA replication and the mitotic chromosome condensation machineries have been shown to carry out additional activities in gene expression regulation and/or silencing by selectively affecting the chromatin/chromosome architecture during the interphase. The identification of Tat interactions with multiple components of the cohesin, condensin, toposome, MCM and ORC complexes provide us with a new prospective on how these pathways might also influence Tat function and HIV-1 provirus expression and silencing.

In addition to its role in regulating the access of specific regulatory factors to the nucleus, the nuclear architecture can affect the genome subnuclear organisation and chromatin structure. In general, there is a strong correlation between the nuclear periphery and heterochromatin establishment and/or maintenance and accordingly gene silencing, while the NPCs have been implicated in preserving euchromatin from such a process269270. More specifically, components of the inner nuclear envelop, lamina and NPC have been described to have an important role in regulating gene expression. LBR, part of our dataset, has been shown to interact with HP1 and consequently regulates heterochromatin formation271. Other lamin-associated proteins, such as emerin and Lap2, were also identified as Tat interactors in our studies, have been shown to associate with and sequester a number of transcriptional repressors, including HDACs, BAF, NCoR and beta-catenin270272. The identification of numerous crucial elements involved the nuclear architecture, as Tat interactors, suggest that they could be involved in regulating the transcriptional state of the provirus.

The results presented here, position the viral regulatory protein at the nexus of a range of interaction networks, which play essential and diverse roles in gene expression, RNA processing, chromatin organisation, chromosome structure and nuclear architecture, and provide the first insights into the modular network properties of the Tat interactome. Ultimately, the HIV-1 Tat rewiring of cellular networks could equip the provirus with a wide repertoire of tools to orchestrate HIV-1 gene expression and confer a remarkable adaptability to a continuously changing cellular environment. Overall, this confirms that Tat transactivation function appears to be the net result of complex interactions with distinct cellular complexes highly specialised in controlling gene expression and more specifically chromosome/chromatin structure. We anticipate that the data presented here will be useful for researchers investigating HIV-1 gene regulation and further studies will delineate the biological significance of these findings.

The authors declare that they have no competing interests.

VG conceived and designed the study, planned and coordinated its execution, conducted experimental procedures, data interpretation and in silico analysis, and drafted the manuscript. LG performed GST-pull down/Western blotting experiments. NOD participated in the experimental design and performed LC MS/MS and peptide and protein identification. SP participated in the experimental design and supervised the proteomic analysis. NS participated in the interpretation of results and final editing of the manuscript. WWH supervised the study design, execution, analysis and revised the manuscript critically.