Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant public health burden with limited treatment options. Many ß-coronaviruses, including SARS-CoV-2, gain entry to host cells through the interaction of SARS-CoV-2 spike protein with membrane-bound ACE2 (angiotensin-converting enzyme 2). Given its necessity for SARS-CoV-2 infection, ACE2 represents a potential therapeutic target in COVID-19. However, early attempts focusing on ACE2 in COVID-19 have not validated it as a druggable target nor identified other ACE2-related novel proteins for therapeutic intervention. Here, we identify a mechanism for ACE2 protein modulation by the deubiquitinase (DUB) enzyme UCHL1 (ubiquitin carboxyl-terminal hydrolase isozyme L1). ACE2 is constitutively ubiquitinated and degraded by the proteasome in lung epithelia. SARS-CoV-2 spike protein cellular internalization increased ACE2 protein abundance by decreasing its degradation. Using an siRNA library targeting 96 human DUBs, we identified UCHL1 as a putative regulator of ACE2 function as a viral receptor. Overexpressed UCHL1 preserved ACE2 protein abundance, whereas silencing of the DUB in cells destabilized ACE2 through increased polyubiquitination. A commercially available small molecule inhibitor of UCHL1 DUB activity decreased ACE2 protein concentrations coupled with inhibition of SARS-CoV-2 infection in epithelial cells. These findings describe a unique pathway of ACE2 regulation uncovering UCHL1 as a potential therapeutic target to modulate COVID-19 viral entry as a platform for future small molecule design and testing.

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15).

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

Ubiquitin specific peptidase 25 alleviates acute lung injury and suppresses the inflammatory response in lung epithelial cells.

As COVID-19 spreads over the world, the treatment of acute lung injury (ALI) has attracted much attention. Considering ubiquitin-specific protease (USP) 25 has been relevant to inflammation, this article focused on its role in ALI and its regulatory mechanism. Lipopolysaccharide (LPS) was applied to separately stimulate mice and human lung epithelial cells to establish in vivo and in vitro ALI models. To discover the effects of USP25 overexpression on mouse, lung pathology, inflammatory factor levels, edema, number of inflammatory cells, and downstream protein levels were evaluated. USP25 overexpression in mice could alleviate LPS-induced lung tissue lesions and edema, and reduce inflammatory factors and inflammatory cells. It also inhibited the levels of downstream TRAF6, MAPK pathway-related proteins, and Fos Proto-Oncogene (FOS) in vivo. Furthermore, BEAS-2B cells were transfected with TNF receptor-associated factor 6 (TRAF6) plasmids to study the role of TRAF6 in the regulatory mechanism of USP25. TRAF6 overexpression was found to reverse the functions of USP25 overexpression on cells. In conclusion, USP25 reduced ALI and inhibited inflammation in lung epithelial cells via regulating TRAF6/MAPK/FOS signaling.

Discovery of host-directed modulators of virus infection by probing the SARS-CoV-2-host protein-protein interaction network.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin-specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that the host proteins HDAC2, BRD4 and USP10 have antiviral functions. We observed marked differences in antiviral effects across cell lines, which may have consequences for identification of selective modulators of viral infection or potential antiviral therapeutics. While network-based approaches enable systematic identification of host targets and selective compounds that may modulate the SARS-CoV-2 interactome, further developments are warranted to increase their accuracy and cell-context specificity.

USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING.

Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFNs (IFN-λ) exhibit cell-type specific and long-lasting functions in auto-inflammation, tumorigenesis, and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2'3'-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication, and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.

RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa.

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.

USP10 regulates B cell response to SARS-CoV-2 or HIV-1 nanoparticle vaccines through deubiquitinating AID.

Activation-induced cytidine deaminase (AID) initiates class-switch recombination and somatic hypermutation (SHM) in antibody genes. Protein expression and activity are tightly controlled by various mechanisms. However, it remains unknown whether a signal from the extracellular environment directly affects the AID activity in the nucleus where it works. Here, we demonstrated that a deubiquitinase USP10, which specifically stabilizes nuclear AID protein, can translocate into the nucleus after AKT-mediated phosphorylation at its T674 within the NLS domain. Interestingly, the signals from BCR and TLR1/2 synergistically promoted this phosphorylation. The deficiency of USP10 in B cells significantly decreased AID protein levels, subsequently reducing neutralizing antibody production after immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or human immunodeficiency virus type 1 (HIV-1) nanoparticle vaccines. Collectively, we demonstrated that USP10 functions as an integrator for both BCR and TLR signals and directly regulates nuclear AID activity. Its manipulation could be used for the development of vaccines and adjuvants.

The extent of neuroradiological findings in COVID-19 shows correlation with blood biomarkers, Glasgow coma scale score and days in intensive care.

BACKGROUND AND PURPOSE: A wide range of neuroradiological findings has been reported in patients with coronavirus disease 2019 (COVID-19), ranging from subcortical white matter changes to infarcts, haemorrhages and focal contrast media enhancement. These have been descriptively but inconsistently reported and correlations with clinical findings and biomarkers have been difficult to extract from the literature. The purpose of this study was to quantify the extents of neuroradiological findings in a cohort of patients with COVID-19 and neurological symptoms, and to investigate correlations with clinical findings, duration of intensive care and biomarkers in blood. MATERIAL AND METHODS: Patients with positive SARS-CoV-2 and at least one new-onset neurological symptom were included from April until July 2020. Nineteen patients were examined regarding clinical symptoms, biomarkers in blood and MRI of the brain. In order to quantify the MRI findings, a semi-quantitative neuroradiological severity scale was constructed a priori, and applied to the MR images by two specialists in neuroradiology. RESULTS AND CONCLUSIONS: The score from the severity scale correlated significantly with blood biomarkers of CNS injury (glial fibrillary acidic protein, total-tau, ubiquitin carboxyl-terminal hydrolase L1) and inflammation (C-reactive protein), Glasgow Coma Scale score, and the number of days spent in intensive care. The underlying radiological assessments had inter-rater agreements of 90.5%/86% (for assessments with 2/3 alternatives). Total intraclass correlation was 0.80. Previously reported neuroradiological findings in COVID-19 have been diverse and heterogenous. In this study, the extent of findings in MRI examination of the brain, quantified using a structured report, shows correlation with relevant biomarkers.

SARS-CoV-2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets.

The current work investigated SARS-CoV-2 Nucleocapsid (NCAP or N protein) interactors in A549 human lung cancer cells using a SILAC-based mass spectrometry approach. NCAP interactors included proteins of the stress granule (SG) machinery and immunoregulators. NCAP showed specific interaction with the SG proteins G3BP1, G3BP2, YTHDF3, USP10 and PKR, and translocated to SGs following oxidative stress and heat shock. Treatment of recombinant NCAP with RNA isolated from A549 cells exposed to oxidative stress-stimulated NCAP to undergo liquid-liquid phase separation (LLPS). RNA degradation using RNase A treatment completely blocked the LLPS property of NCAP as well as its SG association. The RNA intercalator mitoxantrone also disrupted NCAP assembly in vitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP.

Biomarkers and Immune Repertoire Metrics Identified by Peripheral Blood Transcriptomic Sequencing Reveal the Pathogenesis of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global crisis; however, our current understanding of the host immune response to SARS-CoV-2 infection remains limited. Herein, we performed RNA sequencing using peripheral blood from acute and convalescent patients and interrogated the dynamic changes of adaptive immune response to SARS-CoV-2 infection over time. Our results revealed numerous alterations in these cohorts in terms of gene expression profiles and the features of immune repertoire. Moreover, a machine learning method was developed and resulted in the identification of five independent biomarkers and a collection of biomarkers that could accurately differentiate and predict the development of COVID-19. Interestingly, the increased expression of one of these biomarkers, UCHL1, a molecule related to nervous system damage, was associated with the clustering of severe symptoms. Importantly, analyses on immune repertoire metrics revealed the distinct kinetics of T-cell and B-cell responses to SARS-CoV-2 infection, with B-cell response plateaued in the acute phase and declined thereafter, whereas T-cell response can be maintained for up to 6 months post-infection onset and T-cell clonality was positively correlated with the serum level of anti-SARS-CoV-2 IgG. Together, the significantly altered genes or biomarkers, as well as the abnormally high levels of B-cell response in acute infection, may contribute to the pathogenesis of COVID-19 through mediating inflammation and immune responses, whereas prolonged T-cell response in the convalescents might help these patients in preventing reinfection. Thus, our findings could provide insight into the underlying molecular mechanism of host immune response to COVID-19 and facilitate the development of novel therapeutic strategies and effective vaccines.

Qing-Fei-Pai-Du decoction and wogonoside exert anti-inflammatory action through down-regulating USP14 to promote the degradation of activating transcription factor 2.

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.

Genome-wide screening of SARS-CoV-2 infection-related genes based on the blood leukocytes sequencing data set of patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is a global epidemic disease caused by a novel virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing serious adverse effects on human health. In this study, we obtained a blood leukocytes sequencing data set of COVID-19 patients from the GEO database and obtained differentially expressed genes (DEGs). We further analyzed these DEGs by protein-protein interaction analysis and Gene Ontology enrichment analysis and identified the DEGs closely related to SARS-CoV-2 infection. Then, we constructed a six-gene model (comprising IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1) by logistic regression analysis and calculated the area under the ROC curve (AUC) for the diagnosis of COVID-19. The AUC values of the training group, testing group, and entire group were 0.930, 0.914, and 0.921, respectively. The six genes were highly expressed in patients with COVID-19 and positively correlated with the expression of SARS-CoV-2 invasion-related genes (ACE2, TMPRSS2, CTSB, and CTSL). The risk score calculated by this model was also positively correlated with the expression of TMPRSS2, CTSB, and CTSL, indicating that the six genes were closely related to SARS-CoV-2 infection. In conclusion, we comprehensively analyzed the functions of DEGs in the blood leukocytes of patients with COVID-19 and constructed a six-gene model that may contribute to the development of new diagnostic and therapeutic ideas for COVID-19. Moreover, these six genes may be therapeutic targets for COVID-19.

Blood neurofilament light chain and total tau levels at admission predict death in COVID-19 patients.

BACKGROUND AND AIMS: Patients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU). METHODS: We collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels. RESULTS: We found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan-Meier curves indicated that total tau levels at admission accurately predict mortality. CONCLUSIONS: Blood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome.

SARS-CoV-2 Spike Targets USP33-IRF9 Axis via Exosomal miR-148a to Activate Human Microglia.

SARS-CoV-2, the novel coronavirus infection has consistently shown an association with neurological anomalies in patients, in addition to its usual respiratory distress syndrome. Multi-organ dysfunctions including neurological sequelae during COVID-19 persist even after declining viral load. We propose that SARS-CoV-2 gene product, Spike, is able to modify the host exosomal cargo, which gets transported to distant uninfected tissues and organs and can initiate a catastrophic immune cascade within Central Nervous System (CNS). SARS-CoV-2 Spike transfected cells release a significant amount of exosomes loaded with microRNAs such as miR-148a and miR-590. microRNAs gets internalized by human microglia and suppress target gene expression of USP33 (Ubiquitin Specific peptidase 33) and downstream IRF9 levels. Cellular levels of USP33 regulate the turnover time of IRF9 via deubiquitylation. Our results also demonstrate that absorption of modified exosomes effectively regulate the major pro-inflammatory gene expression profile of TNFα, NF-κB and IFN-ß. These results uncover a bystander pathway of SARS-CoV-2 mediated CNS damage through hyperactivation of human microglia. Our results also attempt to explain the extra-pulmonary dysfunctions observed in COVID-19 cases when active replication of virus is not supported. Since Spike gene and mRNAs have been extensively picked up for vaccine development; the knowledge of host immune response against spike gene and protein holds a great significance. Our study therefore provides novel and relevant insights regarding the impact of Spike gene on shuttling of host microRNAs via exosomes to trigger the neuroinflammation.

Identification of novel human USP2 inhibitor and its putative role in treatment of COVID-19 by inhibiting SARS-CoV-2 papain-like (PLpro) protease.

Human ubiquitin carboxyl-terminal hydrolase-2 (USP2) inhibitors, such as thiopurine analogs, have been reported to inhibit SARS-CoV papain-like proteases (PLpro). The PLpro have significant functional implications in the innate immune response during SARS-CoV-2 infection and considered an important antiviral target. Both proteases share strikingly similar USP fold with right-handed thumb-palm-fingers structural scaffold and conserved catalytic triad Cys-His-Asp/Asn. In this urgency situation of COVID-19 outbreak, there is a lack of in-vitro facilities readily available to test SARS-CoV-2 inhibitors in whole-cell assays. Therefore, we adopted an alternate route to identify potential USP2 inhibitor through integrated in-silico efforts. After an extensive virtual screening protocol, the best compounds were selected and tested. The compound Z93 showed significant IC50 value against Jurkat (9.67 µM) and MOTL-4 cells (11.8 µM). The binding mode of Z93 was extensively analyzed through molecular docking, followed by MD simulations, and molecular interactions were compared with SARS-CoV-2. The relative binding poses of Z93 fitted well in the binding site of both proteases and showed consensus π-π stacking and H-bond interactions with histidine and aspartate/asparagine residues of the catalytic triad. These results led us to speculate that compound Z93 might be the first potential chemical lead against SARS-CoV-2 PLpro, which warrants in-vitro evaluations.