Molecular insight into the specific interactions of the SARS-Coronavirus-2 nucleocapsid with RNA and host protein.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin-A, Pin1, and 14-3-3τ. Regarding RNA interactions, the nucleocapsid protein N-terminal folded domain preferentially interacts with smaller RNA fragments relative to the C-terminal region, suggesting an initial RNA engagement is largely dictated by this N-terminal region followed by weaker interactions to the C-terminal region. The nucleocapsid protein forms 10 nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine-arginine-rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin-A and host 14-3-3τ. Considering these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine-arginine-rich region. Our studies identify many of the diverse interactions that may be important for SARS-CoV-2 pathology during infection.

SARS-CoV-2 Pattern Provides a New Scoring System and Predicts the Prognosis and Immune Therapeutic Response in Glioma.

OBJECTIVE: Glioma is the most common primary malignancy of the adult central nervous system (CNS), with a poor prognosis and no effective prognostic signature. Since late 2019, the world has been affected by the rapid spread of SARS-CoV-2 infection. Research on SARS-CoV-2 is flourishing; however, its potential mechanistic association with glioma has rarely been reported. The aim of this study was to investigate the potential correlation of SARS-CoV-2-related genes with the occurrence, progression, prognosis, and immunotherapy of gliomas. METHODS: SARS-CoV-2-related genes were obtained from the human protein atlas (HPA), while transcriptional data and clinicopathological data were obtained from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Glioma samples were collected from surgeries with the knowledge of patients. Differentially expressed genes were then identified and screened, and seven SARS-CoV-2 related genes were generated by LASSO regression analysis and uni/multi-variate COX analysis. A prognostic SARS-CoV-2-related gene signature (SCRGS) was then constructed based on these seven genes and validated in the TCGA validation cohort and CGGA cohort. Next, a nomogram was established by combining critical clinicopathological data. The correlation between SCRGS and glioma related biological processes was clarified by Gene set enrichment analysis (GSEA). In addition, immune infiltration and immune score, as well as immune checkpoint expression and immune escape, were further analyzed to assess the role of SCRGS in glioma-associated immune landscape and the responsiveness of immunotherapy. Finally, the reliability of SCRGS was verified by quantitative real-time polymerase chain reaction (qRT-PCR) on glioma samples. RESULTS: The prognostic SCRGS contained seven genes, REEP6, CEP112, LARP4B, CWC27, GOLGA2, ATP6AP1, and ERO1B. Patients were divided into high- and low-risk groups according to the median SARS-CoV-2 Index. Overall survival was significantly worse in the high-risk group than in the low-risk group. COX analysis and receiver operating characteristic (ROC) curves demonstrated excellent predictive power for SCRGS for glioma prognosis. In addition, GSEA, immune infiltration, and immune scores indicated that SCRGS could potentially predict the tumor microenvironment, immune infiltration, and immune response in glioma patients. CONCLUSIONS: The SCRGS established here can effectively predict the prognosis of glioma patients and provide a potential direction for immunotherapy.

The role of cyclophilins in viral infection and the immune response.

Cyclophilins (Cyps) are a subgroup of peptidyl-prolyl cis-trans isomerases (PPIases) that contain a highly conserved domain of PPIases. Sixteen Cyps have been identified in humans, among which the functions of five classical Cyp subtypes (CypA, B, C, D, and 40) have been studied in more detail. Cyps are widely expressed in almost all human tissues and are involved in several intracellular signaling pathways such as oxidative stress, mitochondrial dysfunction, cell migration, and apoptosis. Several studies have also demonstrated that Cyps play an important role in the development of cardiovascular diseases, neurodegeneration, cancer, and other diseases. However, as regulators of intercellular communication, Cyps have increasingly attracted attention as a result of their implications in viral infection. The specific motifs of Cyps can be targeted by viral proteins and thus promote either a viral infection or an antiviral response. This review highlights the present understanding of Cyps in viral infection and immune response. These effects will facilitate revealing the molecular mechanisms of several diseases induced by viruses and may provide novel insight into the development of corresponding drug-based treatment methods.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

The Cyclophilin-Dependent Calcineurin Inhibitor Voclosporin Inhibits SARS-CoV-2 Replication in Cell Culture.

Kidney transplant recipients (KTRs) are at increased risk for a more severe course of COVID-19, due to their pre-existing comorbidity and immunosuppression. Consensus protocols recommend lowering immunosuppression in KTRs with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but the optimal combination remains unclear. Calcineurin inhibitors (CNIs) are cornerstone immunosuppressants used in KTRs and some have been reported to possess antiviral activity against RNA viruses, including coronaviruses. Here, we evaluated the effect of the CNIs tacrolimus, cyclosporin A, and voclosporin (VCS), as well as other immunosuppressants, on SARS-CoV-2 replication in cell-based assays. Unexpected, loss of compound due to plastic binding and interference of excipients in pharmaceutical formulations (false-positive results) complicated the determination of EC50 values of cyclophilin-dependent CNI's in our antiviral assays. Some issues could be circumvented by using exclusively glass lab ware with pure compounds. In these experiments, VCS reduced viral progeny yields in human Calu-3 cells at low micromolar concentrations and did so more effectively than cyclosporin A, tacrolimus or other immunosuppressants. Although, we cannot recommend a particular immunosuppressive regimen in KTRs with COVID-19, our data suggest a potential benefit of cyclophilin-dependent CNIs, in particular VCS in reducing viral progeny, which warrants further clinical evaluation in SARS-CoV-2-infected KTRs.

Cathepsin B is a potential therapeutic target for coronavirus disease 2019 patients with lung adenocarcinoma.

Coronavirus disease 2019 (COVID-19) was declared a serious global public health emergency. Hospitalization and mortality rates of lung cancer patients diagnosed with COVID-19 are higher than those of patients presenting with other cancers. However, the reasons for the outcomes being disproportionately severe in lung adenocarcinoma (LUAD) patients with COVID-19 remain elusive. The present study aimed to identify the possible causes for disproportionately severe COVID-19 outcomes in LUAD patients and determine a therapeutic target for COVID-19 patients with LUAD. We used publicly available data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and various bioinformatics tools to identify and analyze the genes implicated in SARS-CoV-2 infection in LUAD patients. Upregulation of the SARS-CoV-2 infection-related molecules dipeptidyl peptidase 4, basigin, cathepsin B (CTSB), methylenetetrahydrofolate dehydrogenase, and peptidylprolyl isomerase B rather than angiotensin-converting enzyme 2 may explain the relatively high susceptibility of LUAD patients to SARS-CoV-2 infection. CTSB was highly expressed in the LUAD tissues after SARS-CoV-2 infection, and its expression was positively correlated with immune cell infiltration and proinflammatory cytokine expression. These findings suggest that CTSB plays a vital role in the hyperinflammatory response in COVID-19 patients with LUAD and is a promising target for the development of a novel drug therapy for COVID-19 patients.

N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization.

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirus via interferon-λ in vitro and in mice.

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.

Advances and challenges in the prevention and treatment of COVID-19.

Since the end of 2019, a new type of coronavirus pneumonia (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been spreading rapidly throughout the world. Previously, there were two outbreaks of severe coronavirus caused by different coronaviruses worldwide, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). This article introduced the origin, virological characteristics and epidemiological overview of SARS-CoV-2, reviewed the currently known drugs that may prevent and treat coronavirus, explained the characteristics of the new coronavirus and provided novel information for the prevention and treatment of COVID-19.

Inhibition of SARS-CoV-2 Infection by the Cyclophilin Inhibitor Alisporivir (Debio 025).

Cyclophilins play a key role in the life cycle of coronaviruses. Alisporivir (Debio 025) is a nonimmunosuppressive analogue of cyclosporine with potent cyclophilin inhibition properties. Alisporivir reduced SARS-CoV-2 RNA production in a dose-dependent manner in Vero E6 cells, with a 50% effective concentration (EC50) of 0.46 ± 0.04 µM. Alisporivir inhibited a postentry step of the SARS-CoV-2 life cycle. These results justify rapidly conducting a proof-of-concept phase 2 trial with alisporivir in patients with SARS-CoV-2 infection.