Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

Abstract

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.

Stable Occupancy of the Crimean-Congo Hemorrhagic Fever Virus-Encoded Deubiquitinase Blocks Viral Infection

“…A synthetic Ub variant (UbV) that binds the CCHFV OTU with high levels of affinity and specificity in vitro was recently described (UbV-CC4) (28). UbV-CC4 resembles cellular Ub, but the consensus LRLRGG motif required for cleavage and conjugation of Ub to proteins is mutated ( Fig.…”

“…The nairoviral OTU domain displays a high degree of sequence diversity, potentially affecting the binding affinity of UbV-CC4. For example, UbV-CC4's Tyr 68 improves hydrophobic packing with OTU residues Thr 10 , Val 12 , and Val 18 (28). Thr 10 is conserved across the four tested CCHFV OTUs, whereas the other tested nairovirus species predominantly encode a glutamic acid at that position.…”

“…TR-FRET. UbV-CC4 and the unmodified UbV-AA control have been described previously (28). The genes were synthesized and inserted into a pcDNA3 vector.…”

Structure and Function of Viral Deubiquitinating Enzymes

“…Recently, UbVs have been identified that potently and selectively inhibit the DUB, deISGylating, and polyprotein processing activities of MERS-CoV PL pro . Furthermore, expression of the UbVs in MERS-CoVinfected cells resulted in a 4-log reduction in infectious viral progeny [171], demonstrating the promising potential for virus-specific therapies developed using Ub phage display methods.…”

Peptides meet ubiquitin: Simple interactions regulating complex cell signaling

“…Large phage‐displayed libraries of UbVs, designed by us and others through computational and structural analysis of ubiquitin interactions, yielded specific inhibitors of USPs (ubiquitin‐specific proteases) . Similarly, by randomizing solvent‐exposed residues, we generated potent and selective UbV inhibitors of the SCF (Skp1‐Cullin‐F‐box) E3 ligase family and DUBs from pathogenic viruses, and we developed both inhibitors and activators of the HECT (homologous to the E6AP carboxyl terminus) E3 ligase family …”