The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication on patients treated with HAART. It has been suggested that successful depletion of such latent reservoirs will require a combination of therapeutic agents that can specifically and efficiently act on cells harboring latent HIV-1 provirus. Using Jurkat-LAT-GFP cells, a tractable model of HIV-1 latency, we have found that bryostatin -1 reactivates HIV-1 through a classical PKC-dependent pathway. Bryostatin-1 also activates MAPKs and NF-κB pathways and synergizes with HDAC inhibitors to reactivate HIV-1 from latency. Bryostatin-1 downregulates the expression of the HIV-1 co-receptors CD4 and CXCR4 and prevented de novo HIV-1 infection in susceptible cells. We applied proteomic methods to investigate major changes in protein expression in Jurkat-LAT-GFP under latency and reactivation conditions. We identified up-regulation of proteins that may be involved in the innate anti-HIV-1 response (NKEF-A and MHD2) and in different cell functions (i.e. cofilin-1 and transgelin-2) of the host cells. PKC agonists may represent a valuable pharmacological approach to purge latent HIV from cellular reservoirs and at the moment, the only clinically available PKC agonist is bryostatin-1. This drug has been tested in numerous clinical trials and its pharmacokinetics and toxicity in humans is well known. Moreover, bryostatin-1 potently synergizes with other HDAC inhibitors commonly used in the medical practice such as valproic acid. Therefore, bryostatin-1, alone or in combination with HDAC inhibitors, could be used in HAART treated patients to validate the hypothesis that reactivating HIV-1 from latency could purge HIV-1 reservoirs.

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The ubiquitin E3 ligase SIAH2 is an important regulator of the hypoxic response as it leads to the ubiquitin/proteasomal degradation of prolyl hydroxylases such as PHD3, which in turn increases the stability of hypoxia-inducible factor (HIF)-1α. In the present study, we identify the serine/threonine kinase DYRK2 as SIAH2 interaction partner that phosphorylates SIAH2 at five residues (Ser16, Thr26, Ser28, Ser68, and Thr119). Phosphomimetic and phospho-mutant forms of SIAH2 exhibit different subcellular localizations and consequently change in PHD3 degrading activity. Accordingly, phosphorylated SIAH2 is more active than the wild-type E3 ligase and shows an increased ability to trigger the HIF-1α-mediated transcriptional response and angiogenesis. We also found that SIAH2 knockdown increases DYRK2 stability, whereas SIAH2 expression facilitates DYRK2 polyubiquitination and degradation. Hypoxic conditions cause a SIAH2-dependent DYRK2 polyubiquitination and degradation which ultimately also results in an impaired SIAH2 phosphorylation. Similarly, DYRK2-mediated phosphorylation of p53 at Ser46 is impaired under hypoxic conditions, suggesting a molecular mechanism underlying chemotherapy resistance in solid tumors.

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