HIV RNA splicing regulation by PKR

The Laboratory of Experimental Virology at the AMC (LEV) is engaged in studies of RNA-mediated control of HIV replication and it is here that collaboration with Kaempfer, an expert in RNA structure and function, is highly beneficial. Critical for the replication of human immunodeficiency virus-1 (HIV) is the expression of virus-encoded gene products that regulate HIV transcription (tat) and mRNA splicing (rev). These and additional HIV proteins are translated on spliced mRNA, rendering the regulation of HIV mRNA splicing a critical step in the infectious life cycle . Understanding of this control is the topic of our proposed collaboration. Such insight is potentially a key to the design of novel HIV antagonists. Kaempfer?s hypothesis, supported by early results, is that regulation of HIV-1 mRNA splicing follows in the footsteps of the control used by the human gene encoding tumor necrosis factor (TNF-α), a cytokine pivotal for protective immunity that is expressed promptly during inflammatory responses. Kaempfer?s lab in Israel discovered that splicing of TNF- mRNA is rendered highly efficient by an intrinsic 104-nucleotide cis-acting RNA element that activates the RNA-dependent stress kinase, PKR . While the classical role of activated PKR is to inhibit translation through phosphorylation of translation initiation factor eIF2α, splicing of TNF- pre-mRNA depends positively on its ability to activate PKR . Kaempfer has shown that PKR activation is used solely to enhance TNF-α mRNA splicing, not to repress its translation3. Yet, upregulation of splicing upon PKR activation requires phosphorylation of eIF2α substrate3. This remarkable finding demonstrates a dual function for the same fundamental regulatory mechanism, eIF2α phosphorylation resulting from RNA-mediated activation of PKR, in the control of mRNA translation and splicing, but with opposite outcomes. Considering that the host cell for HIV infection is the T lymphocyte that expresses TNF-, and that all species of HIV-1 mRNA contain a potent PKR activator element (TAR), Kaempfer postulated that splicing of HIV mRNA might also depend on PKR activation and indeed, on phosphorylation of eIF2α, a novel and ground-breaking concept. To this end, Kaempfer initiated collaboration with LEV in 2010, taking advantage of its strong RNA orientation and HIV replication capability. In the course of 2010-2011, work between the two laboratories, detailed below, yielded promising preliminary data suggesting that this hypothesis may indeed be correct. The aims of this proposal are to provide cogent proof for that concept. Given keen interest in HIV worldwide, such proof necessarily must be robust. Kaempfer?s work on RNA activators of PKR in human cytokine mRNA was published in top-flight journals including Cell , Genes & Development2 and Nature Chemical Biology . Thus, the participants in this collaboration have a strong record in their respective fields and preliminary data that show feasibility and justify an intensive collaboration, with a high likelihood of success.