Inhibition of the Cellular Deubiquitinase UCHL1 Suppresses SARS-CoV-2 Replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease (COVID-19), a global pandemic with a significant mortality rate worldwide (1). Despite rapid advancements in our understanding of SARS-CoV-2 biology, our understanding of how host factors regulate viral infection and their role in driving COVID-19 disease severity in patients with preexisting medical conditions (e.g., chronic lung disease) or comorbidities (e.g., cigarette smoking) remains limited (2, 3). UCHL1 (ubiquitin C-terminal hydrolase 1) is a deubiquitinase (DUB) and cellular orthologue of PLpro (coronavirus papain-like protease) (4, 5). Prior studies have demonstrated levels of UCHL1 are altered during SARS-CoV-2 infection (in vitro and in vivo), in response to overexpression of SARS-CoV-2 proteins (e.g., ORF3, ORF9b, and E) in vitro, up-regulated in the airway epithelium of cigarette smokers in vivo and associated with idiopathic pulmonary fibrosis and COVID-19 prognosis (6–9). However, the role of UCHL1 in regulating SARS-CoV-2 infection is largely unknown. To investigate the role of UCHL1 in regulating SARS-CoV-2 infection, we used an in vitromodel of the humanmucociliary airway epithelium using immortalized (BCi-NS1.1) and primary human bronchial epithelial cells (HBECs) differentiated on air–liquid interface (ALI) culture (10–12). All methods related to ALI culture, SARS-CoV-2 propagation and infection, analysis of SARS-CoV-2 replication, propagation of recombinant lentivirus vectors, and generation of stable cell lines with drug selection have been previously described (11–13). Information on individual reagents is listed in Table E1 in the data supplement. Differentiated ALI cultures of BCiNS1.1 cells are a suitable model in which to study SARS-CoV-2 (10), with replication kinetics (i.e., increased expression of SARS-CoV-2 nucleocapsid and production of infectious virus as a function of time) comparable to those observed in ALI cultures of primary HBECs (Figures 1A and 1B) (11). Therefore, using the BCi-NS1.1 ALI model, we next tested the impact of inhibiting UCHL1 enzymatic activity during SARS-CoV-2Washington strain (isolate USA-WA1/2020) infection using nontoxic concentrations (<10μM) of the small molecule LDN-57444 as quantified by lactate dehydrogenase activity (Figures 1C and 1D). Our results demonstrate that LDN-57444 treatment (1μMand 10μM) suppresses SARS-CoV-2 replication in a dose-dependent manner, with 10μMLDN-57444 resulting in a significant decrease in expression of virus nucleocapsid at 24 (82.3%), 48 (92.5%), and 72 (97.1%) hours postinfection (hpi) and 1μM treatment significantly suppressing expression at 48 (65.5%) and 72 (54.6%) hpi (Figure 1E). Furthermore, we observed a significant decrease in virus production at 72 hpi with 1μM (89.5%) and 10μM (99.4%) LDN-57444 (Figure 1F), along with a corresponding reduction in nucleocapsid-positive cells (Figure 1G). These findings were further validated in ALI cultures of primary HBECs, in which 1μMLDN-57444 significantly decreased (53.7%) nucleocapsid expression at 72 hpi (Figure 1H). In addition toWashington strain replication, LDN-57444 treatment suppresses replication of multiple SARS-CoV-2 variants, including Alpha (isolate USA/CA_CDC_5574/ 2020), Beta (isolate hCoV-19/USA/MD-HP01542/2021, Lineage B.1.351), Delta (isolate hCoV-19/USA/MD-HP05647/2021, Lineage B.1.617), and Omicron (isolate hCoV-19/USA/MD-HP20874/2021, Lineage B.1.1.529), by a range of 70.7–99.2% (Figure 1I). Importantly, using in vitro enzyme activity assays for the SARS-CoV-2 proteases PLpro and 3CL, we demonstrated that LDN-57444 has no inhibitory effects on the activity of either protease, suggesting that the antiviral effects observed with LDN-57444 are due to inhibition of UCHL1 activity rather than direct effects on virus protease activity (Figure 1J). To assess the impact of UCHL1 overexpression on SARS-CoV-2 replication, we generated a stable BCi-NS1.1 cell line that overexpresses UCHL1 using lentivirus vector delivery and G418 selection. Importantly, overexpression of UCHL1 has no deleterious effects on airway epithelial cell differentiation, as demonstrated by hematoxylin and eosin staining (Figure 1K) and quantitative PCR analysis for mRNA expression of airway epithelial cell markers (Figure 1L). Furthermore, we observed no changes in mRNA expression of SARS-CoV-2 entry factors (ACE2, TMPRSS2, CTSL, and FURIN) in response to UCHL1 overexpression (Figure 1L). Based on the knowledge that UCHL1 regulates protein levels posttranscriptionally via its DUB activity, rather than directly regulating gene transcription (4, 14), we investigated ACE2 and TMPRSS2 protein levels byWestern blotting. Consistent with the gene-expression patterns of ACE2 and TMPRSS2 (Figure 1L), UCHL1 overexpression led to no consistent change in ACE2 or TMPRSS2 protein levels compared with “empty” control cells (Figures 1M and 1N). Relative to ALI-differentiated empty cells, infection of UCHL1-overexpressing cultures with SARS-CoV-2 (Washington strain) led to a significant increase (5.87 fold) in the expression of virus nucleocapsid at 72 hpi, suggesting that increased protein levels of UCHL1 can promote virus replication (Figure 1O). A recent study by Bednash and colleagues (14) using submerged two-dimensional cultures of transformed airway epithelial cells and primary HBECs demonstrated that UCHL1modulates ACE2 protein stability and that inhibition of UCHL1 DUB activity leads to a decrease in ACE2 protein levels and a reduction in SARS-CoV-2 entry. Our study further supports these findings and demonstrates that UCHL1 enzyme activity is 1) critical for SARS-CoV-2 replication in differentiated mucociliary airway epithelium and 2) a potentially This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern.