Additional file 1 of Genome-wide analysis of protein–protein interactions and involvement of viral proteins in SARS-CoV-2 replication
Creators: name: Jiang, Yiling
name: Tong, Kuijie
name: Yao, Roubin
name: Zhou, Yuanze
name: Lin, Hanwen
name: Du, Liubing
name: Jin, Yunyun
name: Cao, Liu
name: Tan, Jingquan
name: Zhang, Xing-Ding
name: Guo, Deyin
name: Pan, Ji-An
name: Peng, Xiaoxue
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Biotechnology
subject: 69999 Biological Sciences not elsewhere classified
subject: 80699 Information Systems not elsewhere classified
subject: FOS: Computer and information sciences
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: 110309 Infectious Diseases
subject: Plant Biology
subject: 60506 Virology

description: Additional file 1: Figure S1. The conserved domains of Nsp3 of SARS-CoV-2. The diagram of Nsp3 with indicated conserved domains was produced according to the description of YP_009725299.1 of CDD of NCBI. The separation sites aa 749 for Nsp3.1 and aa 1462 for Nsp3.2 were labelled in the diagram. Figure S2. The expression of all the pM and pVP16 constructs. HEK293T cells were transfected with the indicated pM (A) and pVP16 (B) constructs. 36 h post-transfection, the cells were collected and subjected to WB, and the blots were stained with indicated antibodies. Figure S3. Acid–base analysis for the viral proteins encoded by SARS-CoV-2. Viral proteins encoded by SARS-CoV-2 were listed in the order of PI values. Note that N is the most basic protein and Nsp3.1 is among the most acidic proteins of SARS-CoV-2. Figure S4. The regulatory effect of various Nsps on the replicon of SARS-CoV-2. The cDNA of SARS-CoV-2 genome was inserted in the BAC vector. Its expression was driven by the human Cytomegalovirus (CMV) promoter upstream of its sequence. The bovine growth hormone polyadenylation (BGH) was employed to terminate the transcription and was removed by a hepatitis delta virus ribozyme (RZ) to generate the authentic 3’ terminal sequence of SARS-CoV-2. The S gene was replaced with firefly luciferase to eliminate the generation of live virus and to quantify the replication of replicon. (B) HEK293T cells were transfected with the plasmids expressing indicated viral Nsps, Rep-Luci, and pRL-TK. 48 h post-transfection, the cells were collected and subjected to the Dual-Glo Luciferase Assay. Note that Nsp3.1 drastically inhibited the activity of Rep-luci, the replicon of SARS-CoV-2. (C) HEK293T cells were transfected with Rep-Luci or Rep-Luci with mutations of S759A/D760A/D761A (SDD) in nsp12. 48 h post-transfection, the cells were collected and subjected to quantitative RT-PCR with the common forward primer in leader sequence and indicated reverse primers in various genes at the 3’ proximal end of SARS-CoV-2. Note that deficiency of Nsp12 abolished the expression of subgenomic RNAs of the viral replicon. (D) HEK293T cells were transfected with viral replicon and the plasmids expressing Nsp3.1, Nsp12 or N protein. 48 h post-transfection, the cells were collected and subjected to quantitative RT-PCR for indicated subgenomic RNAs. Note that Nsp3.1 drastically inhibited the subgenomic RNA expression of the viral replicon.