Inhibitory effects of molnupiravir on Crimean–Congo hemorrhagic fever virus polymerase

The order <i>Bunyavirales</i> encompasses a diverse group of segmented negative-sense RNA viruses, many of which can cause severe human disease. Their global distribution paired with multiple routes of transmission by arthropods and rodents cause risks to public health. However, effective antiviral drugs are not yet approved. Molnupiravir is an orally available prodrug of β-D-N⁴-hydroxycytidine (NHC) and shows a broad spectrum of antiviral activity. This includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and also several members of the <i>Bunyavirales</i>, such as Crimean-Congo hemorrhagic fever virus (CCHFV). For SARS-CoV-2, the active triphosphate form of NHC (NHC-TP) targets the viral RNA-dependent RNA polymerase (RdRp) and causes lethal mutagenesis; however, the mechanism of inhibition of CCHFV RdRp remains elusive. To address this problem, we employed a combination of biochemical studies, structural modeling, and cell-based, antiviral assays. The results of this study support a unifying mechanism consistent with lethal mutagenesis. Binding and/or incorporation of NHC-TP by CCHFV RdRp is likely facilitated by a conserved glutamine in close proximity to the active site. Next-generation sequencing revealed that NHC induced predominantly G-to-A and C-to-U transitions in CCHFV. Collectively, these results provide mechanistic evidence to consider the development of mutagenic nucleotides as possible treatments for CCHFV infection.