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

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 Ͻ 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. 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 do...

Coronavirus Endoribonuclease and Deubiquitinating Interferon Antagonists Differentially Modulate the Host Response during Replication in Macrophages

“…Loss of EndoU activity significantly alters gene expression and cytokine production in macrophages, which results in the attenuation of EndoUmut viruses in vivo (9). In contrast, we report mild attenuation of the DUBmut virus in mice (18) and show here that DUBmut and MHV-A59 generate similar patterns of transcriptional activation in macrophages. While macrophages have been documented to be important for controlling CoV infections, it is also important to appreciate that CoVs infect multiple cell types, including as epithelial cells in the lung and intestines.…”

“…Instead, these results suggest that the timing and the magnitude of the host antiviral response are critical for determining the outcome of infection in macrophages ( Fig. 6) and for pathogenesis in infected animal (18). Our observation that the EndoUmut virus induces an earlier and more profoundly elevated type I interferon response than even the DUBmut virus implies that there is a threshold of IFN expression that must be breached before the cell mounts an effective antiviral response.…”

“…Although we observed an enhanced IFN response during DUBmut infection of BMDMs, this phenotype did not lead to dramatic differences in pathogenesis during infection of mice (18). To further investigate the differences between the host responses to DUBmut versus wild-type virus infection, we reduced the cutoff stringency to at least 2-fold differential expression with a significant q value (q Ͻ 0.05) and a normalized read count of at least 50.…”

“…Wild-type MHV strain A59 (GenBank accession number AY910861) and EndoUmut (H262A) were previously generated by reverse genetics and confirmed by deep sequencing. DUBmut (D1772A) was generated as described in the companion paper (18).…”

“…Previous studies demonstrated that CoV PLP2s/PLpros are multifunctional, with catalytic residues that mediate protease, deubiquitinase (DUB), and deISGylating (deconjugating interferon-stimulated gene 15 [ISG15] molecule from modified substrates) activities (13)(14)(15)(16)(17). In our companion manuscript (18), we describe the X-ray structure-guided identification of a residue in MHV PLP2 that is required for DUB activity, but not protease activity. Using reverse genetics, we generated an MHV that expressed the DUB mutant enzyme, and we found that DUBmut virus is mildly attenuated in mice.…”