RNA features governing zinc finger antiviral protein sensitivity enable attenuated virus design

Synonymous recoding of viral genome can attenuate their replication, but can have pleiotropic effects, with multiple mechanisms contributing to attenuation. We set out to design recoded viral genomes whose attenuation was specific and conditional. The zinc finger antiviral protein (ZAP) recognizes CpG dinucleotides and targets CpG-rich RNAs for depletion, but RNA features such as CpG numbers, spacing and surrounding nucleotide composition that enable specific modulation by ZAP are undescribed. Using synonymously mutated HIV-1 genomes, we define several sequence features that govern ZAP sensitivity and stable attenuation. Using features defined using HIV-1, we then designed a mutant enterovirus A71 genome whose attenuation was also stable and strictly ZAP-dependent, both in cell culture and in mice. This conditionally attenuated enterovirus A71 elicited neutralizing antibodies that were protective against wild-type enterovirus 71 infection and disease. Elucidation of the determinants of ZAP sensitivity can thus enable the rational design of conditionally attenuated viral vaccines. Overall design: The goal of the experiment was to analyze RNA sequences of the HIV-1 genome bound the Zinc Finger Antiviral Protein (ZAP). ZAP:RNA complexes were extracted and purified from 293T cells, reversed transcribed and analysed by NGS. Two repeated experiments are included; HIV-1 mutants include (1) one with 15 CpGs located ~11nt from each other; (2) one with 15 CpGs located ~32nt from each other and (3) one mutant with 0 CpGs.