HIV co-opts a cellular antiviral mechanism, activation of stress kinase PKR by its RNA, to enable splicing of rev/tat mRNA

BackgroundActivation of RNA-dependent stress kinase PKR, especially by viral double-stranded RNA, induces eukaryotic initiation factor 2 alpha-chain (eIF2 alpha) phosphorylation, attenuating thereby translation. We report that this RNA-mediated negative control mechanism, considered a cornerstone of the cell's antiviral response, positively regulates splicing of a viral mRNA.ResultsExcision of the large human immunodeficiency virus (HIV) rev/tat intron depends strictly on activation of PKR by the viral RNA and on eIF2 alpha phosphorylation. Rev/tat mRNA splicing was blocked by viral PKR antagonists Vaccinia E3L and Ebola VP35, as well as by a trans-dominant negative mutant of PKR, yet enhanced by overexpressing PKR. Expression of non-phosphorylatable mutant eIF2 alpha S51A, but not of wild type eIF2 alpha, abrogated efficient splicing of rev/tat mRNA. By contrast, expression of eIF2 alpha S51D, a phosphomimetic mutant of eIF2 alpha, left rev/tat mRNA splicing intact. Unlike eIF2 alpha S51A, eIF2 alpha S51D does not inhibit eIF2 alpha phosphorylation by activated PKR. All HIV mRNA species contain terminal trans-activation response (TAR) stem-loop sequences that potentially could activate PKR, yet even upon TAR deletion, HIV mRNA production remained sensitive to inhibitors of PKR activation. Bioinformatic and mutational analyses revealed a compact RNA pseudoknot upstream of 3 '-terminal TAR that promotes splicing by activating PKR. Supporting its essential role in control of splicing, this pseudoknot is conserved among diverse HIV and nonhuman primate SIVcpz isolates. The pseudoknot and 3 '-terminal TAR collaborate in mediating PKR-regulated splicing of rev/tat intron, the pseudoknot being dominant.ConclusionsOur results on HIV provide the first example of a virus co-opting activation of PKR by its RNA, a cellular antiviral mechanism, to promote splicing. They raise the question whether other viruses may use local activation of host kinase PKR through RNA elements within their genome to achieve efficient splicing of their mRNA. Our experiments reveal an indispensable role for eIF2 alpha phosphorylation in HIV rev/tat mRNA splicing that accounts for the need for PKR activation.