The Advanced Photon Source
a U.S. Department of Energy Office of Science User Facility

Alternative Pathways for Inhibiting SARS-CoV-2

Multifunctional PLpro from SARS-CoV2 cleaves viral polyproteins and Ub and ISG15 modifications to promote viral pathogenesis. Red arrows indicate PLpro cleavage sites on SARS-CoV2 polyproteins, K48-linked polyUb, and ISG15.

When a SARS-CoV-2 virus infects a human cell, it produces a long protein that must be cut into smaller active units before the virus can proliferate. PLpro is one of two molecules that does the cutting, thereby ushering in a case of COVID-19. Interestingly, PLpro is also able to chop two human proteins: ISG15 and K48-linked polyubiquitin (polyUb). ISG15 is a critical player in our immune system’s response to viral and other infections. K48-linked polyUb tags proteins—including viral proteins—for degradation.

In human cells both ISG15 and K48-polyUb attach to viral proteins. PLpro’s ability to cleave them off affects the host’s immune response and changes the way the cell promotes cellular stability. Using the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science user facility at DOE’s Argonne National Laboratory, a team of scientists has uncovered the atomic and molecular mechanisms behind PLpro’s capacity to recognize and cleave these two host proteins. Their findings may pave the way for developing small molecule drugs that target new, alternative pathways for combating the SARS-CoV-2 virus.

Viruses replicate inside our cells by producing countless alien proteins, which our bodies try to obliterate. Cells have numerous ways of destroying unwanted proteins; one way is by attaching ubiquitin molecules to the protein, tagging it for destruction. A particularly potent “destroy me” signal is produced by K48-linked polyUb tag, a multi-unit string of ubiquitin molecules linked together in a sort of linear structure.  

ISG15 is produced in our cells when they are attacked by pathogens, including viruses. It is a central player in our innate immune defense system. Like K48-linked polyUb, it attaches to viral proteins and may play several roles in the antiviral response, including interfering with activities of newly synthesized viral proteins. Its structure is like two ubiquitins connected to each other head to tail.

When PLpro cleaves these two molecules from viral proteins, it evades the host’s ability to destroy already existing viral proteins and keep new functional viral proteins from being produced. The current study reveals how the first step in the cleaving process proceeds: PLpro recognizes and binds K48-linked Ubdimers (K48-Ub2) and ISG15 not only by a similar amino-acid sequence in their tails but also by their differing three-dimensional structures.

The team determined crystal structures of PLpro in complex with K48-Ub2 and human ISG15 substrates using X-ray diffraction at the 19-ID beamline of the Structural Biology Center at the APS. The team also employed biochemical, nuclear magnetic resonance (NMR) spectroscopy, protein engineering, and computational approaches to understand how PLpro differentiates between K48-Ub2 and human ISG15 substrates.

The scientists found that the dissimilar way in which individual units in K48-Ub2 and ISG15 connect to each other—branched or head-to-tail—makes them bind to PLpro differently. This finding introduced the team to previously unknown binding surfaces that may play a role in PLpro’s activity and could represent future controllable targets. To explore PLpro’s preference for ISG15 and K48-Ub2, the scientists used computational methods to identify substrate specificity-determining residues and validated their effects by mutating those residues and measuring changes in binding using fluorescent probes.

The discovery of the different binding surfaces  were complemented by solution NMR data that also revealed that the tail extension of both substrates has significant effect on their binding to PLpro and demonstrated preferred PLpro cleavage sites in longer K48-polyUb.  

In the future, the team is hoping to introduce mutations in PLpro to see how they  affect PLpro’s binding to ISG15 and K48-Ub2, engineering PLpro that prefers K48-UB2 over ISG15 and vice versa. Disentangling the two pathways will enable the scientists to further investigate how each pathway contributes to the virus’s virulence and disease.

The goal is to produce drugs that target PLpro’s active binding site. Ascertaining the contribution that each host protein makes to PLpro recognition and cleavage would help the team identify ways to inhibit one pathway or the other or both—a significant new avenue that will expand treatment options beyond the current focus of SARS-CoV-2 drug discovery, which has centered on changes in the virus’s spike protein or the other, C3-like protease molecule.  – Judy Myers


See: P. Wydorski1,, J. Osipuk2,3, B.T. Latham4, C. Tesar2,3, M. Endres2,3, E. Engle4, R. Jedrzejczak2,3, V. Mullapudi1, K. Michalska2,3, K. Fidelis5, D. Fushman4, A. Joachimiak2,3, L.A, Joachimiak1, “Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin,”

Nat Commun 14, 2366 (2023).

Author affiliations: 1University of Texas Southwestern Medical Center; 2University of Chicago; 3Argonne National Laboratory; 4University of Maryland; 5University of California Davis

Funding for this project was provided in part by federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract HHSN272201700060C and 75N93022C00035 (AJ). The use of SBC beamlines at the Advanced Photon Source is supported by the U.S. Department of Energy (DOE) Office of Science and operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This research was supported by NIH grant GM065334 to D.F. NMR experiments were performed at UMD on instruments supported in part by NSF grant DBI1040158, and the instrument for mass spectrometry measurements was supported by NSF grant CHE-2018860. We acknowledge Dr. Yue Li at UMD for help with Maxis-II Q-TOF measurements. EE was supported by Undergraduate Maryland Summer Scholarship. LAJ is supported by an Effie Marie Cain Scholarship in Medical Research. PMW is supported by an ODonnell Brain Institute Pilot grant.

The U.S. Department of Energy's APS at Argonne National Laboratory is one of the world’s most productive x-ray light source facilities. Each year, the APS provides high-brightness x-ray beams to a diverse community of more than 5,000 researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. Researchers using the APS produce over 2,000 publications each year detailing impactful discoveries and solve more vital biological protein structures than users of any other x-ray light source research facility. APS x-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being.

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