Translational Regulation in the 5′UTR of Enteroviruses
The genus Enterovirus consists of a large group of positive-sense RNA viruses, with over 100 serotypes known to be human pathogens. Disease phenotype in humans ranges from sub- clinical to acute flaccid paralysis and treatment options for infection with these viruses are limited, with only few antivirals and vaccienes available. Recent work has shown that many enteroviruses encode their proteins within two open reading frames (ORFs), the polyprotein ORF (ppORF) and the upstream ORF (uORF). The uORF encodes a small protein, UP. In addition to the canonical uORF start codon (uAUG), situated on the domain six (dVI) stem loop structure within the viral internal ribosome entry site (IRES), some viruses have additional AUGs both up and downstream of this structure. However, whether these represent alternative sites of translation initiation has not yet been determined. Since many enteroviruses have additional AUGs in the region of the dVI AUG, we evaluated its effects on translation in three frames in coxsackievirus A13 (CVA-13). CVA-13 naturally has an additional AUG (uuAUG) 10 nucleotides upstream of the dVI AUG (uAUG), where UP is encoded. Analysis of the translational landscape of infected cells, through profiling of initiating ribosomes, demonstrated that both the uAUG and uuAUG codons can be used as sites of translation initiation. Using luciferase reporters, we determined the efficiency of Enterovirus alphacoxsackie and coxsackiepol IRES-mediated translation in three frames, further confirming that alternative initiation sites exist in natural isolates and that both upstream AUGs can be utilised for translation initiation. In some cases, UP translation initiates at the uuAUG rather than the canonical uAUG. Thus, uAUG-proximal start codons must also be considered when defining the coding capacity of enteroviruses. Although alternative start codons within enteroviruses provide additional sites for translation initiation, in CVA-13, the presence of the uuAUG is not necessary for translation initiation events and is not implicated in viral fitness in any condition tested thus far. However, the uuAUG may prove to be functional within an undefined context, such as cellular stress or within a more complex system such as a mouse model. We illustrated the differences in translation initiation patterns between enteroviruses with distinct coding strategies through ribosome profiling, going on to further to look at how translation is regulated in echovirus 7 (E7). Here we show the effects of the induction of cellular stress and translation inhibition on E7 uORF expression, revealing the preferential increase of uORF expression over ppORF in these conditions. Furthermore, we modulated the usage of the E7 uORF by alteration of the dVI stem loop RNA secondary structure to adopt a ‘stabilised’ and ‘destabilised’ conformation. Taken together, these data demonstrate the remarkable plasticity of Enterovirus genomes, providing a broader understanding of translation initiation events and how they are regulated. Given the complex systems which Enteroviruses infect such as the gastrointestinal (GI) tracts and the brain in some cases, the search for the most relevant model system is always at the forefront of enterovirus research. We showed the successful use of human intestinal organoids to evaluate the growth and susceptibility of infection with a panel of enteroviruses as a model system for the GI tract. We also show that human induced pluripotent stem cell derived neurons can also be used for the evaluation of viral infection with neurotropic enteroviruses. This body of work provides insights into translational regulation in enteroviruses. We show that translation initiation events in several enteroviruses can occur upstream of the ppORF start codon. Moreover, we demonstrate that both sequence and structure of the dVI stem loop is tightly regulated by several enteroviruses with key features necessary for efficient virus translation.
Enterovirus, Translation initiation, uORF, Intestinal organoids
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