ABSTRACT
Viruses use IRES sequences within their RNA to hijack translation machinery and thereby rapidly replicate in host cells. While this process has been extensively studied in bulk assays, the dynamics of hijacking at the single-molecule level remain unexplored in living cells. To achieve this, we developed a bicistronic biosensor encoding complementary repeat epitopes in two ORFs, one translated in a Cap-dependent manner and the other translated in an IRES-mediated manner. Using a pair of complementary probes that bind the epitopes co-translationally, our biosensor lights up in different colors depending on which ORF is being translated. In combination with single-molecule tracking and computational modeling, we measured the relative kinetics of Cap versus IRES translation and show: (1) Two non-overlapping ORFs can be simultaneously translated within a single mRNA; (2) EMCV IRES-mediated translation sites recruit ribosomes less efficiently than Cap-dependent translation sites but are otherwise nearly indistinguishable, having similar mobilities, sizes, spatial distributions, and ribosomal initiation and elongation rates; (3) Both Cap-dependent and IRES-mediated ribosomes tend to stretch out translation sites; (4) Although the IRES recruits two to three times fewer ribosomes than the Cap in normal conditions, the balance shifts dramatically in favor of the IRES during oxidative and ER stresses that mimic viral infection; and (5) Translation of the IRES is enhanced by translation of the Cap, demonstrating upstream translation can positively impact the downstream translation of a non-overlapping ORF. With the ability to simultaneously quantify two distinct translation mechanisms in physiologically relevant live-cell environments, we anticipate bicistronic biosensors like the one we developed here will become powerful new tools to dissect both canonical and non-canonical translation dynamics with single-molecule precision.