Potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants

PLoS Pathog. 2017 May 18;13(5):e1006372. doi: 10.1371/journal.ppat.1006372. eCollection 2017 May.

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.

MeSH terms

  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology*
  • Coronavirus Infections / metabolism
  • Coronavirus Infections / virology*
  • Drug Evaluation, Preclinical
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Hemorrhagic Fever Virus, Crimean-Congo / drug effects*
  • Hemorrhagic Fever Virus, Crimean-Congo / enzymology
  • Hemorrhagic Fever Virus, Crimean-Congo / genetics
  • Hemorrhagic Fever, Crimean / metabolism
  • Hemorrhagic Fever, Crimean / virology*
  • Humans
  • Middle East Respiratory Syndrome Coronavirus / drug effects*
  • Middle East Respiratory Syndrome Coronavirus / enzymology
  • Middle East Respiratory Syndrome Coronavirus / genetics
  • Ubiquitin / metabolism*
  • Ubiquitination / drug effects
  • Viral Proteins / antagonists & inhibitors*
  • Viral Proteins / chemistry
  • Viral Proteins / genetics
  • Viral Proteins / metabolism

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • Ubiquitin
  • Viral Proteins

Grants and funding

MK is supported by the Division of Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW) through Excellent Chemical Research (ECHO) grant (700.59.008). BLM is a Manitoba Research Chair and is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant (RGPIN-2015-05310). BABE holds an NSERC Postgraduate Scholarship (PGS-Doctoral). WZ is supported by Mitacs Elevate program with funds from Mitacs Inc. and Centre for the Commercialization of Antibodies and Biologics (CCAB). SSS is supported by Canadian Institutes of Health Research (CIHR) grants (MOP-136956 and PJT-148510). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.