Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors. Author summary N-myristoylation is a lipidic co- or post-translational modification of proteins that is catalyzed by the enzyme N-myristoyltransferase (NMT). In this study, we use IMP-1088, an ultrapotent inhibitor of human NMTs, to query whether targeting the N-myristolation of viral proteins can serve as an effective antiviral strategy against VACV. Through quantitative chemical proteomics, we demonstrated that IMP-1088 blocks the N-myristoylation of L1, a VACV protein essential for viral entry. Progeny virus particles generated in the presence of IMP-1088 lacked N-myrisoylated L1, which considerably reduced their infectivity. Other steps in the viral life cycle, including replication, morphogenesis and viral egress were not directly impacted by IMP-1088-mediated NMT inhibition. While the N-myristoylation of two other EFC proteins, namely A16 and G9, was also abrogated by IMP-1088 treatment, mutational studies revealed they were dispensable for viral entry in the presence of a functional L1 protein. Taken together, these data illustrate the mechanism of action of IMP-1088 as a potential poxviral inhibitor.

Automated summary

N-myristoylation plays a critical role in VACV infection, and the NMT inhibitor IMP-1088 can effectively inhibit viral replication and infectivity.