Paragraph: The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques. Emerging viruses pose a tremendous challenge to human health. While vaccine-based approaches are desirable in terms of infection prevention in the longer term, alternative antiviral strategies are needed, especially when providing treatment options for infected patients during acute outbreaks. Here we applied protein engineering technology to target virus-encoded deubiquitinating enzymes of two viruses with significant impact on human health: Middle East respiratory syndrome coronavirus (MERS-CoV) and Crimean-Congo hemorrhagic fever virus (CCHFV). This resulted in the rapid identification of ubiquitin variant (UbV) inhibitors that bound with high affinity and specificity to the viral deubiquitinating enzymes. These proteins inhibited the catalytic activities of the deubiquitinating enzymes and almost completely blocked MERS-CoV infection. This work provides proof-of-principle that structurally diverse, virus-specific deubiquitinating enzymes can be selectively targeted through rational protein design technology, and therefore opens new avenues for quickly developed molecularly tailored therapy across a broad spectrum of viral pathogens that infect humans, livestock and plants. Rapid development of antiviral agents PLOS Pathogens | https://doi. 
 Title: Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants Author summary
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