mRNA technology has widely incorporated modified nucleotides to temper innate immune activation by foreign RNA and achieve high levels of heterologous protein expression. However, the incorporation of modified nucleotides has had limited application to self-amplifying (sa) RNAs, as it impeded heterologous protein expression. Here, we used a reporter replicon of the attenuated TC-83 strain of Venezuelan equine encephalitis virus (VEEV) to investigate the impact of modified nucleotide incorporation on the replication capacity of the saRNA in transfected cells. ψ and m1ψ-modified molecules exhibited a profound defect in RNA synthesis compared to unmodified saRNA. Interestingly, the RNA synthesis levels of m5C-modified RNAs were similar to unmodified molecules, positioning m5C as a promising candidate for saRNA modification. Moreover, we found that saRNAs carrying mutations associated with resistance to nucleotide analog polymerase inhibitors partially overcame the negative impact of m1ψ-modified nucleotide incorporation on RNA synthesis. Overall, we uncovered a previously unappreciated link between polymerase fidelity and saRNA amplification.

RNA VACCINES

MODIFIED NUCLEOTIDES

ALPHAVIRUS

POLYMERASE FIDELITY

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