Abstract
BackgroundOne facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.ResultsOur approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.ConclusionWe have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.
Highlights
One facet of the complexity underlying the biology of HIV-1 resides in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly
Peptide mixtures were separated by liquid chromatography (LC) prior to tandem mass spectrometry analysis (MS/MS)
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
Summary
One facet of the complexity underlying the biology of HIV-1 resides in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. They can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. 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 [1-4]. 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. 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 LRP[11]
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