RNA with Azvudine incorporated one nucleotide upstream of 3’-end resists cleavage by SARS-CoV-2 proofreading exonuclease

Abstract The cytidine analog Azvudine (Azv) showed promising therapeutic effects against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections, leading to its conditional authorization as a COVID-19 treatment in China. Once Azvudine enters infected cells, it is converted to the active form, Azvudine-triphosphate, which has been shown to inhibit polymerases of several viruses. Here we show that when Azvudine is incorporated into RNA by the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), it hinders incorporation of the subsequent nucleotide, but this hindrance can be partially overcome in the presence of natural nucleotides. Moreover, the 3’-Azv-terminated RNA does not show any resistance to the SARS-CoV-2 proofreading exonuclease (ExoN). However, we found that the incorporation of Azvudine at the penultimate position of the RNA prevents RNA cleavage by the ExoN. Because Azvudine partially terminates RNA synthesis and 3’-Azv-terminated RNA does not show any ExoN resistance, the penultimate ExoN-resistant RNA configuration is a less frequent event. Although these finding do not establish a direct RdRp-mediated inhibition as the primary reason for reported Azvudine’s therapeutic effect, the position-dependent ExoN resistance observed here provide mechanistic insight for the development of novel nucleotide analogs that potentially inhibit RdRp and resist the viral proofreading mechanism of coronaviruses.