Date Published: January 31, 2013
Publisher: Public Library of Science
Author(s): Lisa M. Kronstad, Kevin F. Brulois, Jae U. Jung, Britt A. Glaunsinger, Dirk P. Dittmer.
The Kaposi’s sarcoma-associated herpesvirus (KSHV) protein kinase, encoded by ORF36, functions to phosphorylate cellular and viral targets important in the KSHV lifecycle and to activate the anti-viral prodrug ganciclovir. Unlike the vast majority of mapped KSHV genes, no viral transcript has been identified with ORF36 positioned as the 5′-proximal gene. Here we report that ORF36 is robustly translated as a downstream cistron from the ORF35–37 polycistronic transcript in a cap-dependent manner. We identified two short, upstream open reading frames (uORFs) within the 5′ UTR of the polycistronic mRNA. While both uORFs function as negative regulators of ORF35, unexpectedly, the second allows for the translation of the downstream ORF36 gene by a termination-reinitiation mechanism. 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.
Translation initiation of eukaryotic mRNAs is dependent on the 5′ mRNA cap and proceeds by ribosomal scanning until recognition of an AUG codon in a favorable context , . As a consequence of the translation machinery not engaging start codons at internal positions within the mRNA, eukaryotic transcripts generally encode only one functional protein. For the majority of mRNAs the most 5′-proximal AUG is selected, however strategies exist to bypass upstream start codons to enable downstream initiation. For example, leaky scanning can occur if the nucleotides flanking the 5′-proximal AUG are not in the Kozak consensus sequence (ccRccAUGG), allowing the 40S ribosomal subunit to instead engage a downstream methionine codon , . Alternatively, when an upstream AUG is followed shortly thereafter by an in-frame termination codon, ribosomes can reinitiate translation, albeit with reduced efficiency, at a downstream AUG. These upstream open reading frames (uORFs) presumably permit translation of a downstream gene because factors necessary for initiation have not yet dissociated during the short elongation period. Notably, uORFs are common regulatory elements in eukaryotic transcripts, and generally function to reduce translation of the major ORF , . Additional, although rare, examples of internal ORF translation also exist, for example after ribosome shunting over a highly structured upstream sequence –, or upon direct 40S recruitment via internal ribosome entry sites (IRESs) –.
In this study, we describe a novel functionally bicistronic viral mRNA that is translated via a unique adaption of ribosomal reinitiation. In other characterized examples of viral translation via a reinitiation mechanism, expression of the downstream gene is significantly tempered as a consequence of ribosomal engagement at an upstream start codon , –. Aside from being bicistronic, translation from the KSHV ORF35–37 transcript is unusual in that the protein product of ORF36 is at least as robustly expressed as the 5′ ORF35 despite the fact that the ORF35 start codon is in a favorable sequence context. We reveal that a key mechanism underlying this phenotype involves the position of a short uORF overlapping the start codon of ORF35, which enables translation of ORF36 (Figure 8). These findings provide the first example of cap-dependent non-canonical translation in KSHV and illustrate a novel strategy to translate polycistronic mRNA.