Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS c...Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL pro , following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. Methods The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.Results The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. Conclusions The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL pro of SARS-CoV urbani strain, 3CL pro of transmissible gastroenteritis virus and 3CL pro of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL pro .展开更多
Retinoic acid-inducible gene Ⅰ (RIG-Ⅰ) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses.Herein we investigate their functions in human epithelial cells...Retinoic acid-inducible gene Ⅰ (RIG-Ⅰ) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses.Herein we investigate their functions in human epithelial cells,the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).A deficiency in MDA5,RIG-Ⅰ or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication.The expression of the type I/III interferon(IFN) during infection was impaired in MDA5;and MAVS;,but not in RIG-Ⅰ;,when compared to wild type (WT) cells.The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2),the cellular entry receptor for SARS-CoV-2,was approximately 2.5-fold higher in RIG-Ⅰ;than WT cells.These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor,IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-Ⅰ in epithelial cells.展开更多
文摘Background The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL pro , following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. Methods The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein.Results The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. Conclusions The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL pro of SARS-CoV urbani strain, 3CL pro of transmissible gastroenteritis virus and 3CL pro of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL pro .
基金supported by a National Institutes of Health grant (No. R01AI132526)a UConn Health Startup fund to Wang P。
文摘Retinoic acid-inducible gene Ⅰ (RIG-Ⅰ) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses.Herein we investigate their functions in human epithelial cells,the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).A deficiency in MDA5,RIG-Ⅰ or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication.The expression of the type I/III interferon(IFN) during infection was impaired in MDA5;and MAVS;,but not in RIG-Ⅰ;,when compared to wild type (WT) cells.The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2),the cellular entry receptor for SARS-CoV-2,was approximately 2.5-fold higher in RIG-Ⅰ;than WT cells.These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor,IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-Ⅰ in epithelial cells.
