Rotaviruses are a major cause of acute gastroenteritis in children and livestock globally. While the translation of rotavirus RNAs (RV-mRNAs) requires host translational machinery, RV-mRNAs do not fully resemble eukaryotic mRNAs; most notably, instead of a 3’ poly(A) tail RV mRNAs have a conserved 3’ GUGACC motif. The viral protein NSP3 facilitates RV-mRNA translation by binding this 3’ motif and associating with the cellular translation initiation complex, supporting RV-mRNA circularisation required for translation initiation. Using in-house developed bioinformatic pipelines, we have identified a conserved upstream, in-frame start codon (uAUG) in the NSP3 segment, 3 codons upstream of the canonical start codon (AUG0) that is present in 48% of circulating strains. The distribution of the uAUG is clade-specific, but not host-specific. We found that both uAUG and AUG0, individually but not together, are dispensable for successful rescue of rotavirus strain SA11. While removing the uAUG did not significantly change replication kinetics, the mutant without the canonical AUG0 is attenuated. Removing either start codon resulted in different rates of cell death during infection, where removing the uAUG accelerated cell death and removing the AUG0 slowed down cell death. The NSP3 mutant panel elicited significant, cell type-independent differences in the production of the viral protein VP6. We are currently performing mass spectrometry to investigate the cell pathways contributing to the observed differences in cell death rates, and to characterise any potential effects of the uAUG on global viral protein synthesis.