Date Published: May 18, 2017
Publisher: Public Library of Science
Author(s): Wei Zhang, Ben A. Bailey-Elkin, Robert C. M. Knaap, Baldeep Khare, Tim J. Dalebout, Garrett G. Johnson, Puck B. van Kasteren, Nigel J. McLeish, Jun Gu, Wenguang He, Marjolein Kikkert, Brian L. Mark, Sachdev S. Sidhu, Sean P.J. Whelan.
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.
Ubiquitination is a post-translational modification mediated by an enzyme cascade that results in the conjugation of ubiquitin (Ub) to cellular proteins [1, 2]. This process is regulated in part through activity of cellular deubiquitinating enzymes (DUBs), which remove Ub from cellular proteins [1, 2]. Given the essential role of the Ub system in regulating a large number of critical cellular processes, it is not surprising that viruses have acquired the means to modulate this system in order to promote infection and replication in host cells . In particular, virus-encoded DUBs reverse the ubiquitination process to alter host signaling pathways critical to the induction of cellular antiviral and pro-inflammatory innate immune responses . In addition to removing Ub molecules from host proteins, many viral DUBs (vDUBs) also remove the Ub-like protein interferon-stimulated gene 15 (ISG15) to further suppress antiviral responses [4, 5]. Importantly, a number of vDUBs also play an essential role in viral replication [4–6]. Together, the replicative and/or deubiquitinating activities of viral proteases contribute directly to pathogenesis during viral infection in vivo , making them ideal antiviral drug targets.
The continued introduction of viruses from zoonotic sources into human populations poses a serious and constant threat to human health . In the case of coronavirus infection, therapeutic options are limited, and vaccine development remains in progress [8, 29]. Here, we describe a unique protein engineering platform that can be used to rapidly generate UbVs to selectively block the activity of vDUBs from a range of evolutionarily distinct viral lineages. Indeed, UbVs generated against MERS-CoV almost completely abolished the replication of this virus in host cells. Meanwhile, none of the UbVs generated in this study likely cross-reacted with human DUBs as no toxicity was observed upon expression of UbVs, demonstrating their potential therapeutic safety.