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2.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1356909

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
3.
J Virol ; 95(17): e0074721, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1350002

ABSTRACT

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 nonstructural protein 12 (NSP12) was able to suppress interferon-ß (IFN-ß) activation in IFN-ß promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-ß promoter-mediated luciferase activity was reduced during coexpression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-ß production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, the type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon-stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-ß promoter luciferase assays. In conclusion, unlike as previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-ß antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. IMPORTANCE Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-ß activation. However, most of these results were generated from IFN-ß promoter luciferase reporter assay and have not been validated functionally. In our study, we found that, although NSP12 could suppress IFN-ß promoter luciferase activity, it showed no inhibitory effect on IFN-ß production or its downstream signaling. Further study revealed that contradictory results could be generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-ß signaling. On the other hand, our study suggests that caution needs to be taken with the interpretation of SARS-CoV-2-related luciferase assays.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase , Interferon-beta , Promoter Regions, Genetic , SARS-CoV-2 , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-beta/antagonists & inhibitors , Interferon-beta/biosynthesis , Interferon-beta/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
4.
Ann Palliat Med ; 10(7): 7270-7279, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1311481

ABSTRACT

BACKGROUND: We aim to investigate the clinical characteristics and survival rate of coronavirus disease 2019 (COVID-19) patients. METHODS: Ninety-seven COVID-19 patients were enrolled. The laboratory results, lung imaging and medical treatment were compared. Patients were followed up after 1 year, and the Kaplan-Meier test was used for survival analysis. RESULTS: Compared with the non-severe group, the age of the severe group was older, and the proportion of concomitant diseases were higher. As fever was the primary clinical manifestation, dyspnea and anorexia were more common in severe patients. Lung imaging manifestations and laboratory indicators were worse in the severe group. Accordingly, the treatment of glucocorticoid, antibiotics, and advanced life support were in high proportion. Of the 97 patients with COVID-19, 4 severe patients died within one month during the 1-year follow-up, with the median survival time of 47.0 weeks (95% CI: 45.1-48.9). CONCLUSIONS: Severe cases of COVID-19 are characterized by advanced age, more concomitant diseases and complications, which lead to a decreased short-term survival rate. However, there were no deaths after one month, which implied a good prognosis if the risk period were passed smoothly.


Subject(s)
COVID-19 , Humans , Lung , Retrospective Studies , SARS-CoV-2 , Survival Analysis
5.
Microbiol Spectr ; 9(1): e0016921, 2021 09 03.
Article in English | MEDLINE | ID: covidwho-1270881

ABSTRACT

Nonstructural protein 1 (Nsp1) of severe acute respiratory syndrome coronaviruses (SARS-CoVs) is an important pathogenic factor that inhibits host protein translation by means of its C terminus. However, its N-terminal function remains elusive. Here, we determined the crystal structure of the N terminus (amino acids [aa] 11 to 125) of SARS-CoV-2 Nsp1 at a 1.25-Å resolution. Further functional assays showed that the N terminus of SARS-CoVs Nsp1 alone loses the ability to colocalize with ribosomes and inhibit protein translation. The C terminus of Nsp1 can colocalize with ribosomes, but its protein translation inhibition ability is significantly weakened. Interestingly, fusing the C terminus of Nsp1 with enhanced green fluorescent protein (EGFP) or other proteins in place of its N terminus restored the protein translation inhibitory ability to a level equivalent to that of full-length Nsp1. Thus, our results suggest that the N terminus of Nsp1 is able to stabilize the binding of the Nsp1 C terminus to ribosomes and act as a nonspecific barrier to block the mRNA channel, thus abrogating host mRNA translation.


Subject(s)
SARS-CoV-2/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , COVID-19 , Crystallography, X-Ray , HEK293 Cells , Humans , Protein Biosynthesis , Protein Conformation , Protein Domains , RNA, Messenger , Sequence Analysis, Protein , Viral Nonstructural Proteins/metabolism
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