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1.
Infectious Medicine ; 2022.
Article in English | ScienceDirect | ID: covidwho-1699253

ABSTRACT

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. Results In this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. Conclusion In conclusion, our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.

2.
J Immunol ; 208(3): 753-761, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1614089

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.


Subject(s)
Autophagy , COVID-19/immunology , Coronavirus RNA-Dependent RNA Polymerase/physiology , Interferon Type I/biosynthesis , Methyltransferases/physiology , Protein Serine-Threonine Kinases/metabolism , RNA Helicases/physiology , SARS-CoV-2/physiology , Sequestosome-1 Protein/metabolism , Viral Nonstructural Proteins/physiology , Beclin-1/antagonists & inhibitors , Cell Line , Down-Regulation , Humans , Immune Evasion , Immunity, Innate , Immunoprecipitation , Interferon Type I/genetics , Multiprotein Complexes , Protein Aggregates , Protein Interaction Mapping
3.
Cell Res ; 31(12): 1230-1243, 2021 12.
Article in English | MEDLINE | ID: covidwho-1475291

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.


Subject(s)
COVID-19/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/drug therapy , COVID-19/mortality , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cytokines/genetics , Cytokines/metabolism , Down-Regulation/drug effects , ErbB Receptors/metabolism , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Indoles/pharmacology , Indoles/therapeutic use , Lung/pathology , Lung/virology , Mice , Mice, Transgenic , Mutation , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Survival Rate , Transcriptome/drug effects , Viral Load/drug effects , Virus Internalization
4.
Front Med (Lausanne) ; 8: 595773, 2021.
Article in English | MEDLINE | ID: covidwho-1158348

ABSTRACT

Asymptomatic carriers contribute to the spread of Coronavirus Disease 2019 (COVID-19), but their clinical characteristics, viral kinetics, and antibody responses remain unclear. A total of 56 COVID-19 patients without symptoms at admission and 19 age-matched symptomatic patients were enrolled. RNA of SARS-CoV-2 was tested using transcriptase quantitative PCR, and the total antibodies (Ab), IgG, IgA, and IgM against the SARS-CoV-2 were tested using Chemiluminescence Microparticle Immuno Assay. Among 56 patients without symptoms at admission, 33 cases displayed symptoms and 23 remained asymptomatic throughout the follow-up period. 43.8% of the asymptomatic carriers were children and none of the asymptomatic cases had recognizable changes in C-reactive protein or interleukin-6, except one 64-year-old patient. The initial threshold cycle value of nasopharyngeal SARS-CoV-2 in asymptomatic carriers was similar to that in pre-symptomatic and symptomatic patients, but the positive viral nucleic acid detection period of asymptomatic carriers (9.63 days) was shorter than pre-symptomatic patients (13.6 days). There were no obvious differences in the seropositive conversion rate of total Ab, IgG, and IgA among the three groups, though the rates of IgM varied largely. The average peak IgG and IgM COI of asymptomatic cases was 3.5 and 0.8, respectively, which is also lower than those in symptomatic patients with peaked IgG and IgM COI of 4.5 and 2.4 (p < 0.05). Young COVID-19 patients seem to be asymptomatic cases with early clearance of SARS-CoV-2 and low levels of IgM generation but high total Ab, IgG, and IgA. Our findings provide empirical information for viral clearance and antibody kinetics of asymptomatic COVID-19 patients.

5.
Front Med (Lausanne) ; 7: 579406, 2020.
Article in English | MEDLINE | ID: covidwho-937452

ABSTRACT

Objective: To investigate the clinical characteristics of children with coronavirus disease 2019 (COVID-19) and identify the occurrence of viral shedding of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) during follow-up. Methods: We retrospectively retrieved data from pediatric patients with COVID-19 from the Shenzhen Third People's Hospital in China. The dynamics of SARS-CoV-2 and antibodies against SARS-CoV-2 were analyzed during hospitalization and after discharge. Results: From January 23 to March 15, 2020, a total of 28 pediatric patients were diagnosed with COVID-19 and were followed for at least 1 month. The median age was 7 years (IQR 3.5-10) and none of the children progressed to severe COVID-19 during hospitalization. Ten patients tested positive for SARS-CoV-2 1 month after discharge while four patients tested positive during the 2nd month after discharge. Only three of 12 children showed detectable immunoglobulin-M (IgM) on day 5, 18, and 21 after illness onset, respectively. Conclusions: COVID-19 disease was relatively mild among children while a number did test positive after discharge from the hospital. Public health initiatives should thus adapt control measures targeted toward children.

6.
Ann Transl Med ; 8(17): 1084, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-842908

ABSTRACT

BACKGROUND: The characteristics, significance and potential cause of positive SARS-CoV-2 diagnoses in recovered coronavirus disease 2019 (COVID-19) patients post discharge (re-detectable positive, RP) remained elusive. METHODS: A total of 262 COVID-19 patients discharged from January 23 to February 25, 2020 were enrolled into this study. RP and non-RP (NRP) patients were grouped according to disease severity, and the characterization at re-admission was analyzed. SARS-CoV-2 RNA and plasma antibody levels were measured, and all patients were followed up for at least 14 days, with a cutoff date of March 10, 2020. RESULTS: A total of 14.5% of RP patients were detected. These patients were characterized as young and displayed mild and moderate conditions compared to NRP patients while no severe patients were RP. RP patients displayed fewer symptoms but similar plasma antibody levels during their hospitalization compared to NRP patients. Upon hospital readmission, these patients showed no obvious symptoms or disease progression. All 21 close contacts of RP patients were tested negative for viral RNA and showed no suspicious symptoms. Eighteen out of 24 of RNA-negative samples detected by the commercial kit were tested positive for viral RNA using a hyper-sensitive method, suggesting that these patients were potential carriers of the virus after recovery from COVID-19. CONCLUSIONS: Our results indicated that young patients, with a mild diagnosis of COVID-19 are more likely to display RP status after discharge. These patients show no obvious symptoms or disease progression upon re-admission. More sensitive RNA detection methods are required to monitor these patients. Our findings provide information and evidence for the management of convalescent COVID-19 patients.

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