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. 2020 May 18;94(11):e01734-19.
doi: 10.1128/JVI.01734-19. Print 2020 May 18.

Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

Xufang Deng  1 , Yafang Chen  2 , Anna M Mielech  1 , Matthew Hackbart  1 , Kristina R Kesely  3 , Robert C Mettelman  1 , Amornrat O'Brien  1 , Mackenzie E Chapman  2 , Andrew D Mesecar  2   3 , Susan C Baker  4
Affiliations

Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

Xufang Deng et al. J Virol. .

Abstract

Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous in vitro studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction (P < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis.IMPORTANCE Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.

Keywords: DUB activity; IFN antagonist; PLP2; PLP2 structure; coronavirus; papain-like protease.

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Figures

FIG 1
FIG 1
X-ray structure of the MHV PLP2-ubiquitin complex and residues involved in ubiquitin binding. (A) Overall structure of MHV PLP2-C1716S-Ub complex. Domains are color coded as follows: yellow, Ub; purple, Ubl2 domain of PLP2; orange, thumb domain; cyan, palm domain; green, fingers domain. Residue D1772 of PLP2 is located outside the active site, which is circled in black. (B) Hydrogen bond interactions between MHV PLP2 R1803 and the backbone of A46 of ubiquitin. (C) Binding interactions between MHV PLP2 D1772 and two arginine residues (R42 and R72) of ubiquitin. (D) Hydrophobic interactions between F1812 of MHV PLP2 and ubiquitin residues I44 and V70. The 2Fo-Fc maps (blue) surrounding the residues are contoured at 1σ in each panel. The PDB coordinates for the MHV PLP2-C1716S-Ub complex are available under PDB code 5WFI. (E) Sequence alignment (MultAlign) of coronavirus papain-like protease/deubiquitinating domains from MHV (amino acids [aa] 1606 to 1911, GenBank accession number AAX23975), SARS (aa 1541 to 1854, accession number ACZ72209), SARS-CoV-2 (aa 1564 to 1878, accession number QHO60603), and MERS (aa 1480 to 1803, accession number AHY21467). Amino acids are colored according to similarity using the RISER coloring scheme. Numbering shown is based on MHV sequence. Amino acids mutated in this study are indicated with a black asterisk, the catalytic cysteine is indicated by a blue asterisk, and those amino acids that bind ubiquitin and were mutated in this study are boxed in green. The active site substrate binding loop also involved in binding inhibitors of SARS is shown highlighted in yellow. The sequence alignment was created using ESPript3.
FIG 2
FIG 2
Structure-guided mutagenesis of MHV PLP2 reveals that D1772A disrupts ubiquitin binding and reduces DUB activity. (A) Relative kinetic activities of three mutant MHV PLP2 enzymes toward three substrates: Z-RLRGG-AMC (green), Ub-AMC (blue), and ISG15-AMC (yellow) compared to the wild-type enzyme. (B) Steady-state kinetic parameters for wild-type and D1772A mutant enzymes. (C) Sequence alignment of Ub and ISG15 from human and mouse generated by Clustal Omega. The two arginine residues of Ub (R42 and R72) that interact with D1772 are indicated by arrows. R72 is conserved between Ub and ISG15, whereas R42 (shaded in yellow) is only present in Ub. Accession numbers are as follows: PDB code 1UBQ (Human_Ub); Uniprot accession number P62984 (Mouse_Ub); GenBank accession number AAH09507 (Human_ISG15); and GenBank accession number AAI09347 (Mouse_ISG15). The sequence alignment was created using ESPript.
FIG 3
FIG 3
D1772A substitution in the coronavirus papain-like protease Ub-binding site reduces DUB activity and interferon antagonism without reducing protease activity. (A) Western blot assessing the DUB activity of PLP2. (B) IFN antagonism of PLP2 was determined using an IFN-luciferase (Luc) reporter stimulated by N-RIG-I expression. The reporter activity of vector control was set to 100% (indicated by a dash line). Values are presented as means ± standard deviation (SD) and were statistically analyzed using an unpaired t test. **, P < 0.01; ***, P < 0.001. (C and D) Protease activity was evaluated using (C) a trans-cleavage assay that detects the cleaved products by Western blot and (D) a pGlo biosensor assay that is activated by PLP2-mediated cleavage of the substrate. Values are presented as means ± SD and were statistically analyzed using an unpaired t test at each time point. “n.s.” indicates that the values at the tested time points are not significantly different. Data are representative of at least two independent experiments.
FIG 4
FIG 4
Evaluating the replication kinetics of, and level of interferon activation by, WT MHV and DUBmut in cell culture. (A) Replication kinetics of WT and DUBmut virus in DBT cells. (B) IFN-α11 mRNA levels in WT- and DUBmut-infected BMDMs were assessed at indicated time points by reverse transcription-quantitative PCR (qRT-PCR). (C) IFN-α protein levels in the supernatants of infected BMDMs were evaluated at the times indicated. (D) Comparison of IFN-α11 mRNA levels in B6 versus MDA5−/− BMDMs at 12 h postinfection (h p.i.). (E) Assessment of levels of viral nucleocapsid (N) mRNA by qRT-PCR. (F) Replication kinetics of WT and DUBmut virus in BMDM cells. Data are representative of at least two independent experiments and are presented as means ± SD. Data in panels B and C were statistically analyzed using unpaired t tests. *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
Evaluating replication and pathogenesis of MHV-DUBmut in mice. Four- (A) or 6-week-old (B) mice were infected with the indicated doses of MHV. Viral titer in livers and spleens isolated from WT- or DUBmut virus-infected mice was determined by plaque assay. The number of mice in each group is shown in parentheses. Data were statistically analyzed using unpaired t tests and are presented as means ± standard error of the mean (SEM). (C) H&E staining of liver sections from infected mice at 3 and 5 days postinfection (d p.i.). Representative MHV-associated liver lesions are indicated by arrows.
FIG 6
FIG 6
Alignment of the SARS-CoV-2 PLpro domain with the X-ray structure of the closely related SARS-CoV PLpro domain in complex with ubiquitin. (A) X-ray structure of SARS-CoV PLpro-ubiquitin-aldehyde complex (blue) (PDB code 4MM3) with each of its domains labeled as finger, palm, thumb, and Ubl2. Ubiquitin-aldehyde is colored yellow. The SARS-CoV-2 PLpro structure (cyan) was modeled by first mutating the residues of SARS PLpro in the X-ray structure to those of SARS-CoV-2 PLpro based upon the sequence alignment in Fig. 1E. The SARS-CoV-2 PLpro-ubiquitin-aldehyde complex was then refined using the structure-factor amplitudes and initial phases of the SARS PLP-ubiquitin-aldehyde complex (PDB code 4MM3). The residues that are different between SARS-CoV PLpro and SARS-CoV-2 PLpro are highlighted as sticks. (B) Potential interactions between L1803 of SARS-CoV PLpro and SARS-CoV-2 PLpro with residue A46 of ubiquitin. (C) Predicted interaction between E1772 of SARS-CoV PLpro and SARS-CoV-2 PLpro and residues R42 and R72 of ubiquitin. (D) Potential interactions between residues I44, V70, and R42 of ubiquitin with residues M1812 of SARS-CoV PLpro and SARS-CoV-2 PLpro.
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