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Dual Upstream Open Reading Frames Control Translation of a Herpesviral Polycistronic mRNA


Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of multicentric Castleman's disease, primary effusion lymphoma and Kaposi's sarcoma. KSHV expresses a number of transcripts with the potential to generate multiple proteins, yet relies on the cellular translation machinery that is primed to synthesize only one protein per mRNA. Here we report that the viral transcript encompassing ORF35-37 is able to direct synthesis of both ORF35 and ORF36, while the 3'-proximal ORF37 is translated from a monocistronic transcript under separate transcriptional regulation. While ORF36 encodes the KSHV protein kinase and functions to phosphorylate and activate the anti-viral prodrug ganciclovir, ORF37 expresses SOX (shutoff and exonuclease), a viral protein responsible for promoting widespread degradation of host mRNAs. Surprisingly, ORF36 is robustly translated as a downstream cistron from the ORF35-37 polycistronic transcript in a cap-dependent manner. Two short, upstream open reading frames (uORFs) were identified within the 5' UTR of the polycistronic mRNA. While both uORFs function as negative regulators of ORF35, unexpectedly, translation of the downstream ORF36 gene occurs by a termination-reinitiation mechanism after translation of both uORF1 and uORF2. The sequence of the viral uORFs appears unimportant, and they instead function to position the translation machinery to favor translation of the downstream major ORF. Furthermore, the newly developed KSHV genetic system was used to introduce a mutation into the viral genome to disrupt uORF2 and this single nucleotide change leads to a dramatic drop in infectious viral titer. Positional conservation of uORFs within a number of related viruses suggests that this may be a common γ-herpesviral adaptation of a host translational regulatory mechanism. Thus, KSHV uses a host strategy generally reserved to repress translation to instead allow for the expression of an internal gene.

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