SARS‐CoV‐2 NSP12 utilizes various host splicing factors for replication and splicing regulation

The RNA‐dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), SARS‐CoV, and Middle East respiratory syndrome coronavirus (MERS‐CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS‐CoV‐2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS‐CoV‐2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS‐CoV‐2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS‐CoV and MERS‐CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS‐CoV‐2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

RNA-dependent RNA polymerase (RdRp) is a critical enzyme for the replication of RNA viruses, including HCoVs.7][8] RdRp is a promising target for antiviral drug research due to its conserved nature across coronaviruses and lack of a human homologue. 9The determined SARS-CoV-2 RdRp complex structure shows that NSP12 contains several domains, including an N-terminal β hairpin domain (31-50 aa), a nucleotide transferase domain (NiRAN, 51-250 aa), an interface domain (251-365 aa), and a RdRp catalytic domain (366-920 aa).NSP7 and NSP8 stabilize the closed conformation of NSP12 and increase its binding to templateprimer RNA. 10,11Recent research has preliminary screened several host proteins, including pre-messenger RNA (mRNA) splicing factor SLU7, peptidyl-prolyl cis-trans isomerase-like 3 (PPIL3), and A-kinase anchor protein 8 (AKAP8 or AKAP95) that interact with SARS-CoV-2 NSP12 by affinity purification mass spectrometry (AP-MS). 12Understanding these interactions between NSP12 and host proteins may be critical for understanding viral replication and disease pathogenesis.
Pre-RNA splicing is a critical step of high-efficiency gene expression, involving the removal of introns and the ligation of exons to form a mature mRNA.This process is carried out in the nucleus by the spliceosome, which is composed of five uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), known as U1, U2, U4, U5 and U6 snRNPs, and non-snRNP associated proteins.
There are two types of splicing: constitutive and alternative splicing (AS), with the latter being regulated by splicing factors (SFs). 13SLU7, PPIL3, and AKAP8 are all splicing factors that play a role in regulating splicing.SLU7 is poised for the selection of the 3′-splice site during the second step of splicing. 14PPIL3 was found in splicing intermediate (B act ) and catalytic (C) complex. 15AKAP8, a splicing regulatory factor, regulates splicing through scaffolding RNAs and RNA processing factors. 16,179][20] SARS-CoV-2 NSP16 has been shown to bind to the mRNA recognition domains of the U1 and U2 splicing RNAs, leading to a suppression of global mRNA splicing.This in turn can reduce the host cells' innate immune response to virus recognition, as certain immune-related genes may not be properly spliced and expressed. 21The RdRp of EV71 has been shown to disrupt the splicing machinery by interfering with the protein Prp8, which is an essential component of the spliceosome.This interference ultimately results in altered splicing patterns in infected cells, which may contribute to viral replication and pathogenesis. 22Dengue virus NS5 bearing the activities of RdRp and methyl transferase intrudes in the cellular spliceosome and modulates splicing, which might result in a less restrictive environment for viral replication. 23Given that SARS-CoV-2 NSP12 interacts with SLU7, PPIL3, and AKAP8, it is possible that it may also modulate splicing to promote viral replication.
In this study, we used co-immunoprecipitation (co-IP) assays and fluorescence microscopy to verify the interactions and colocalizations of SARS-CoV-2 NSP12 with multiple host proteins.The data analyses showed the effects of three host proteins (SLU7, PPIL3, or AKAP8) on SARS-CoV-2 virus entry and RdRp polymerase activity using pseudovirus entry assays, authentic virus infection, RNA extension assays and Gluc (Gaussia luciferase) activity assays.Additionally, the effects of NSP12 and its homologues in SARS-CoV and MERS-CoV on host cell splicing by interacting with the three splicing factors were confirmed using minigene splicing assays.

| Virus and cell lines
The SARS-CoV-2 strain (accession number: NMDCN0000HUI) was provided by the Guangdong Provincial Center for Disease Control and Prevention (Guangzhou, China).This virus was propagated and titrated on African green monkey kidney epithelial cells (Vero E6) (ATCC, no.1586).Human embryonic kidney (HEK) 293T cells provided by Prof. Hai-Yan Ren, ACE2 (angiotensin-converting enzyme 2)-overexpressing HEK293T cells (HEK293T-ACE2) provided by Prof. Xiang-Hui Fu, human lung cancer A549 cells purchased from National Collection of Authenticated Cell Cultures, mouse leukemic monocyte/macrophage cell line Raw264.7 provided by Jia-Yi Xu and Vero E6 cells were grown at 37°C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS).HEK293T cells were used for co-IP assays, Gluc activity assays and minigene splicing assays because of their very high transfection efficiency.Vero E6 cells were used for immunofuorescence assay (IFA) since this cell line is highly susceptible to SARS-CoV-2.A549 cells were used for the fluorescence microscopy, SARS-CoV-2 spike-mediated pseudovirus entry assays and authentic virus infection, since this kind of cells adhered well to the surface of coverslips placed in six-well plates and at the bottom of plates.

| Plasmid construction
Total RNA was extracted from HEK293T cells with Trizol reagent (Invitrogen) and was then used for the synthesis of single-stranded complementary DNA (cDNA) by reverse transcription.Different kinds of nucleic-acid fragments were amplified with the corresponding primers (Table S1).The plasmids used for co-IP, fluorescence microscopy, gene editing, protein expression, Gluc activity assays and minigene splicing assays were constructed.All constructs used in this study were confirmed by DNA sequencing.The operations are as follows: The plasmids pET21b-NSP12, pET21b-NSP7, and pET32a-NSP8 separately expressing SARS-CoV-2 (QSE99647.1)NSP12, NSP7 and NSP8 were provided by Prof. Li-Chuan Gu.The NSP12 and NSP7 gene were cloned into a modified pET21b vector with the C-terminus possessing a 6×His-tag.The NSP8 gene was cloned into the modified pET32a vector with the N-terminus possessing a trx-His 6 -tag and PreScission Protease site.
To construct plasmids for the prokaryotic expression of PPIL3 or SLU7, the ORF of PPIL3 gene was amplified with primers PPIL3-F4/ R4 and cloned into pGEX-6P-1 to produce pGEX-PPIL3.The ORF of SLU7 gene was amplified with primers SLU7-F3/R3 and cloned into pET15b to produce pET15b-SLU7.

| CRISPR/Cas9 gene editing
CRISPR/Cas9 gene editing was performed as previously described with minor modifications. 24A549 cells lacking SLU7, PPIL3 or AKAP8 were generated as follows.Approximately 8 × 10 5 HEK293T cells in six-well plates were cotransfected with 0.51 μg of pMD2-G, 0.78 μg of psPAX2 and 1.20 μg of lentiCRISPRv2-SLU7 sgRNA or other indicated plasmid.At 48 and 72 hpt, the supernatants were collected and mixed.A549 cells in six-well plates were inoculated with 500 μL of harvested supernatant for 24 h and then selected with DMEM supplemented with 10% FBS containing 2 μg/mL puromycin until clones formed.All clones were sorted into 96-well plates by flow cytometer (FACSAria SORP) and each well contained only one cell.
Individual clones formed in 96-well plates were further transferred into six-well plates for culture and then identified by DNA sequencing and Western blot analysis.For DNA sequencing, the genomes of cells in the six-well plates were extracted and amplified by PCR using corresponding primers.The PCR products were sequenced using corresponding primers.

| SARS-CoV-2 spike-mediated pseudovirus entry assay
To analyze the effect of host protein knockout on SARS-CoV-2 pseudovirus entry, 5 × 10 4 A549, A549-SLU7 KO , A549-PPIL3 KO , or A549-AKAP8 KO cells were seeded in 96-well white plates and grown overnight.The pseudovirus infection and measurement of entry efficiency were performed according to a previous report. 24The culture medium was replaced with fresh medium containing 8 μg/mL polybrene for 1 h and then the cells were inoculated with 4E + 6 RLU (relative light units) VSV-SARS-2-S-luc pseudovirus.At 16 hpi, the culture medium was replaced with fresh medium.Entry efficiency was quantified at 48 hpi by measuring the activity of Renilla luciferase in cell lysates using the ONE-Glo™ Luciferase Assay according to the manufacturer's instructions (PekinElmer Envision).The infection experiments were performed under biosafety level 2 (BSL2) laboratory conditions.

| SARS-CoV-2 infection assay
A549 wild-type cells and A549 knockout cells were, respectively, seeded in 24-well plates (500 μL/well) at 1.2 × 10 5 cells/well and grown overnight.Cells were infected with authentic SARS-CoV-2 at an MOI of 5.After 1 h of incubation at 37°C, the cells were collected to extract viral RNA, which was subjected to RT-qPCR analysis.
The infection experiments were performed under biosafety level 3 (BSL3) laboratory conditions.

| RNA-Seq processing and analysis
To investigate whether SLU7 or PPIL3 knockout affects the transcriptions of some genes involved in SARS-CoV-2 entry, the transcriptomes of A549, A549-SLU7 KO , and A549-PPIL3 KO cells were sequenced and analyzed.To analyze the effect of NSP12 on the global cellular AS, the transcriptome of HEK293T cells transfected with pcDNA3.1 or pcDNA3.1-NSP12for 24 h was sequenced and analyzed.RNAs were sequenced using the standard Illumina protocol (LC-Bio Technology CO., Ltd.).Reads were mapped to the genome using HISAT2.Genes differential expression analysis was performed by DESeq. 2 software between two different groups (and by edgeR between two samples).The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥2 were considered differentially expressed genes (DEGs).DEGs were then subjected to enrichment analysis of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.rMATS (version 4.1.1)was used to identify AS events and analyze differential AS events between samples.We identified AS events with FDR < 0.05 in a comparison as significant AS events.The classification of AS is as follows: skipped exon (SE), retained intron (RI), mutually exclusive exon (MXE), alternative 5′ splice site (A5SS) and alternative 3′ splice site (A3SS).

| In vitro RNA primer extension assay using RdRp complex
RNA template-product duplex was designed according to the published SARS-CoV-2 RNA extension assays. 11FAM labeled 13 nt oligonucleotide (FAM-5′-GCUAUGUGAGAUU-3′) and the 23 nt complementary oligonucleotide (3′-CGAUACACUCUAAUUUCAAUUGA-5′) were synthesized for polymerase activity assay.The oligonucleotides were mixed in equal molar ratio in DEPC (diethy pyrocarbonate) water, annealed by heating it to 95°C for 10 min and gradually cooling to room temperature to make the RNA duplex.
To optimize the quantity of RdRp and nucleoside triphosphate (NTP), 2, 3, or 4 μM RdRp and 0.1, 0.5, or 1 mM NTP were added to the reaction buffer (R buffer) containing 200 nM 13 + 23 nt RNA duplex, 20 mM Tris-HCl pH 8.0, 10 mM KCl, 10 mM MgCl 2 , 0.01% Triton-X100, 1 mM DTT.The volume was adjusted to 20 μL by added DEPC-treated ddH 2 O. DEPC-treated water was used for all buffer preparation.The system was reacted at 37°C for 30 min.Eventually, glycerol was added to a concentration of 6.5%.Ten microliters of RNA product for each reaction was resolved on 20% denaturing polyacrylamide-urea gels and imaged with a ChemiDoc™ MP imaging system (BIO-RAD).
To test the effect of SLU7 or PPIL3 on RdRp polymerase in vitro, 4-8 μL of purified SLU7 (3.75 mg/mL, about 11-22 μM) or 2-8 μL of PPIL3 (5.6 mg/mL, about 30-120 μM) and 2 μM RdRp were added to the reaction buffer (R buffer).The volume was adjusted to 20 μL by added DEPC-treated ddH 2 O.The system was reacted for 30 min at 37°C, and then 0.5 mM NTP was added to the reaction system for 30 min at 37°C.The detection of RNA product was as described above.

| RT-qPCR
RT-qPCR was performed as previously described with minor modifications. 29Total RNA of transfected HEK293T cells was extracted with RNA isolator Total RNA Extraction Reagent kit (Vazyme Biotech Co., Ltd.).The extracted RNA were converted to plus-strand and minus-strand cDNA with RevertAid™ Master Mix (Thermo Fisher Scientific) using primer plus Gluc-RT and minus Gluc-RT for 1 h at 37°C, respectively.The cDNAs were quantified using Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) in applied system (Bio-Rad).Primers used for PCR are Gluc-RT-F/R.Levels of GAPDH RNA was also quantified with primers GAPDH-RT-F/R, and results were used to normalize the amount of Gluc RNA.
GAPDH was as a negative control.SARS-CoV-2 NSP16 was as a positive control.At 48 hpt, cells were collected and measured by flow cytometry (Cytoflex) and analyzed using Flowjo analysis software.
Three independent biological replicates per condition were prepared.
To analyze the effect of SLU7, PPIL3, or AKAP8 overexpression on the ability of NSP12, NSP12a or NSP12b in inhibiting cellular AS, 1.6 × 10 5 HEK293T were cotransfected with 0.5 μg of pMSCV-IRF7-2A, 0.5 μg of plasmid expressing one of the three virus proteins, 0.5 μg of plasmid expressing one of the three host proteins.At 48 hpt, cells were measured as above.To analyze the effect of SLU7, PPIL3, or AKAP8 knockdown on the ability of NSP12, NSP12a, or NSP12b in inhibiting cellular AS, 1.2 × 10 5 HEK293T transfected with corresponding siRNA for 24 h were cotransfected with 0.5 μg of pMSCV-IRF7-2A and 0.5 μg of plasmid expressing one of the three virus proteins.At 24 hpt, cells were measured as above.
To isolate the protein complexes, an anti-HA affinity gel was used for immunoprecipitation.Western blot analysis was performed on both the cell lysates and immunoprecipitated protein complexes (IP) using anti-HA and anti-Flag antibodies.The results showed that the NSP12-HA protein bands were present in both the cell lysates and immunoprecipitated complexes, indicating that the NSP12-HA fusion protein was effectively immunoprecipitated by the anti-HA affinity gel (Figure 1A).Similarly, the protein bands of SLU7, PPIL3, AKAP8, TCF12, CRTC3, RBM41, and BCKDK were detected in both cell lysates and immunoprecipitated complexes using anti-Flag antibody (Figure 1A).The negative control  using anti-Flag affinity gel.The results confirmed that NSP12 interacted with the seven host proteins (Figure 1B).
The interacting residues between NSP12 and the three splicing factors (SLU7, PPIL3, and AKAP8) were predicted by molecule docking.Six complex models of each splicing factor with NSP12 were obtained (Figures S1-3).The predicted interacting sites were shown in Tables S2-4.The interactions between the three splicing factors and NSP12 mutants (M1~6S, M1~6P, M1~6A) were analyzed by co-IP assays using anti-Flag affinity gel.The results showed that the interactions of SLU7 with M1~6S, the interactions of PPIL3 with M1~6P, and interactions of AKAP8 with M1~6A were still detected, and some interactions were obviously weakened, such as SLU7-M3S (Figure 1C), PPIL3-M3P (Figure 1D) and AKAP8-M2A (Figure 1E), since the expression levels of NSP12, M3S, M3P, and M2A in cell lysates were comparable but the bands of M3S, M3P, and M2A in IP were obviously weakened.Therefore, the three mutants of NSP12 were selected to further analysis of splicing regulation.
As both SLU7 and PPIL3 are involved in cell splicing, the study also tested the interaction between them using co-IP assay, which revealed that SLU7 interacts with PPIL3 as well (Figure 1F).
Whether the two accessory subunits NSP7 and NSP8 of RdRp interact with the three splicing factors were tested.The results showed that NSP7 interacted with AKAP8, but not SLU7 or PPIL3 (Figure 1G).Similarly, NSP8 also interacted with AKAP8, but not the other two host proteins (Figure 1H).The subcellular localization of NSP12 and its colocalization with the three splicing factors in Vero E6 cells infected with authentic SARS-CoV-2 were analyzed by an IFA.Vero E6 cells were infected with authentic SARS-CoV-2 at an MOI of 0.2 for 24 h, and more than 92% cells were infected when the cells were detected using anti-N antibody.Simultaneously, Vero E6 cells alone transfected with pcDNA3.1-NSP12-HAor cotransfected with two indicated plasmids for 24 h were infected with authentic SARS-CoV-2 at an MOI of 0.2 for 24 h.NSP12-HA distributed diffusely in both the cytoplasm and nucleus of the infected cells (Figure 2C).Furthermore, NSP12-HA colocalized with SLU7-Flag or AKAP8-Flag in the nucleus, and colocalized with PPIL3-Flag in the cytoplasm and nucleus of the infected cells (Figure 2D).These findings not only confirm the interactions between NSP12 and the host proteins but also suggest that NSP12 may play a crucial role in the cytoplasm and nucleus by targeting these proteins.

(B) Analyzing interactions between NSP12 and seven host proteins by reciprocal co-IP. (C) The interactions between SLU7 and NSP12 mutants (M1~6S). (D) The interactions between PPIL3 and NSP12 mutants (M1~6P). (E)
The interactions between AKAP8 and NSP12 mutants (M1~6A).(F) Analyzing the interaction between PPIL3 and SLU7.(G) Analyzing the interactions between NSP7 and the three splicing factors (SLU7, PPIL3, or AKAP8).(H) Analyzing the interactions between NSP8 and the three splicing factors.edited cells using corresponding primers and then sequenced, confirming that the host gene was edited in the A549 cell line.
Western blot analysis showed that the specific band of host proteins were detected in the control cells using corresponding antibodies, but not in the edited cells (Figure 3A), indicating that the expression of SLU7, PPIL3, or AKAP8 was completely interrupted in A549 cells.
The transcriptomes of A549, A549-SLU7 KO , and A549-PPIL3 KO cells were sequenced and compared to investigate whether SLU7 or PPIL3 knockout affects the transcriptions of genes involved in the entry of SARS-CoV-2.SLU7 knockout significantly changed 1876 genes compared to A549 cells, including 729 upregulated genes and 1147 downregulated genes such as TMPRSS2.Among these genes, the transcriptions of PXDN and TM4SF20 showed the most significant upregulation and downregulation, respectively (Figure S5A).GO enrichment analysis showed that DEGs are involved in many biological processes, such as immune system process and inflammatory response.
KEGG enrichment analysis showed that DEGs are mainly enriched in human diseases, such as herpes simplex virus 1 infection and human papillomavirus infection (Figure S5B).Additionally, 1825 AS events were identified, including 1207 SE, 167 RI, 126 MXE, 158 A5SS, and 167 A3SS (Figure 3E).All genes occurring in RI, MXE, A5SS, A3SS, and top 200 genes of SE were selected for enrichment analysis.These genes were involved in many biological processes, such as mRNA processing.Some genes were also involved in viral infectious disease and immune regulation system (data not shown).
The knockout of PPIL3 resulted in significant changes in 1949 genes, with 637 upregulated genes and 1312 downregulated genes.
Notably, the transcription of MGAM and SLC16A3 showed the most significant upregulation and downregulation, as seen in Figure S5C.These DEGs are involved in various biological processes, including the innate immune response and immune system processes.DEGs are mainly enriched in human diseases, such as herpes simplex virus 1 infection and human papillomavirus infection, as depicted in Figure S5D.In addition, a total of 1825 AS events were identified, including 1812 SE, 243 RI, 157 MXE, 178 A5SS, and 257 A3SS, as illustrated in Figure 3F.All genes involved in RI, MXE, A5SS, A3SS, and the top 200 genes of SE were selected for enrichment analysis, revealing their involvement in various biological processes, such as RNA splicing.Some genes were also found to be involved in virus infection, although specific data was not shown.To determine whether the host proteins have any effects on the polymerase activity of RdRp in vitro, RNA extension assays were performed.In the presence of NTP substrates, RdRp can catalyze the elongation reaction to produce an intact double-stranded RNA product (Figure 4A).NSP7, NSP8, NSP12, SLU7, or PPIL3 was individually expressed and purified, and the RdRp complex was assembled.SDS-PAGE analysis confirmed the high purity of the assembled RdRp complex, SLU7, and PPIL3 (Figure S6A).RNA extension assays showed that the assembled RdRp complex had polymerase activity and could synthetize a 23 nt amplification strand with different concentrations of NTP (Figure S6B).When SLU7 or PPIL3 was added, the intensity of the 23 nt amplification strand was slightly weaker than that in control, indicating that the RdRp activity was dose-dependently reduced by purified SLU7 or PPIL3 in vitro (Figure 4B,C).
Next, we investigate the effect of NSP12 interacting host proteins SLU7, PPIL3, and AKAP8 on polymerase activity using a Gluc reporter system in vivo.In this system, the ORF of Gluc flanked by the 5′ and 3′ UTRs of SARS-CoV-2 is under the CMV (cytomegalovirus) promoter, and the plus-strand RNA (mRNA) of Gluc is first transcribed by the host cellular DNA-dependent RNA polymerase Pol II, which can further translate Gluc.RdRp use mRNA as templates to synthetize negative-sense vRNA, which was transcribed into mRNA to express Gluc (Figure 4D).When cotransfected, the three components (NSP12, NSP7, and NSP8) of RdRp were successfully expressed in HEK293T cells (Figure S6C).Subsequently, HEK293T cells were cotransfected with the plasmids expressing Gluc and NSP12 or RdRp (representing NSP7, NSP8, and NSP12) for 24 h, and the supernatants of the transfected cells were subjected to Gluc activity assay.Compared to control, Gluc activity increased 1.2-fold by NSP12 and increased about 2.1-fold by RdRp (Figure S6D).These findings indicated that the established Gluc reporter system could be used to measure the polymerase activity of RdRp in vivo.
The effect of SLU7, PPIL3, or AKAP8 on the polymerase activity of SARS-CoV-2 RdRp was analyzed by the Gluc reporter system.
When coexpressing Gluc + RdRp + each of host proteins (SLU7, PPIL3, or AKAP8), the Gluc activity increased about twofold with AKAP8 (P = 2E-10), and about 1.2-fold with SLU7 (p = 0.021), but had no significant change with PPIL3 (p = 0.214) compared to the control (Gluc + RdRp + vector) (Figure 4E).The expressions of the three host proteins were knocked down using siRNAs.Western blot analysis showed that all siRNAs decreased the expressions of targeted proteins to varying degrees, with the siSLU7-606, siPPIL3-325, and siAKAP8-1665 having the most significant effect (Figure 4F).Therefore, these siRNAs were selected to further analysis of the polymerase activity of RdRp and splicing regulation.When SLU7, PPIL3, or AKAP8 was knocked down, the polymerase activities of RdRp were significantly reduced (Figure 4G).The effects of overexpressions of the three splicing factors on the levels of plusstrand Gluc RNA and minus-strand Gluc RNA were determined by RT-qPCR.The overexpression of SLU7 or AKAP8 significantly increased the levels of plus-strand Gluc RNA and minus-strand Gluc RNA, consistent with the observed increase in Gluc activity.
However, overexpression of PPIL3 did not have a significant effect on the levels of Gluc RNA (Figure 4H).These results indicated that the three splicing factors played important roles in regulating the polymerase activity of RdRp or replication of SARS-CoV-2.
To assess the possible effect of SLU7, PPIL3, or AKAP8 knockdown on the formation of the RdRp complex, co-IP assays were performed.HEK293T cells were transfected with siNC, siSLU7, siPPIL3, or siAKAP8 for 24 h and then cotransfected with plasmids expressing NSP12-3Flag, NSP7-HA, and NSP8-HA.Cell lysates were incubated with anti-HA affinity gel, and cell lysates and IP were subjected to Western blot analysis using corresponding antibodies.
Compared to the negative control (NC), the amount of NSP12-3Flag in the IP slightly decreased to varying degrees when SLU7, PPIL3, or AKAP8 was knocked down (Figure 4I).

| The mechanism of SARS-CoV-2 infection regulated by SLU7, PPIL3, or AKAP8
Based on the findings that NSP12 interacted with three splicing factors (SLU7, PPIL3, and AKAP8) and primarily colocalized with them in the nucleus, a hypothesis was proposed that NSP12 may modulate the AS of cellular genes in the nucleus by targeting these three proteins.To test this hypothesis, an IRF7-GFP splicing reporter system was used to assess the effect of NSP12 on AS.In this system, EGFP is produced only if the reporter is spliced, but not if the reporter is not spliced due to a stop codon contained in the first intron (Figure 5A).HEK293T were cotransfected with splicing reporter pMSCV-IRF7-2A and plasmid expressing NSP12 for 48 h, and the EGFP intensity was observed under fluorescence microscope (Figure S7) and measured by flow cytometry (Figure 5B).The relative fluorescence intensity was shown in Figure 5C.NSP12 significantly decreased the fluorescence intensity compared with negative control (GAPDH), and as a positive control, NSP16 also significantly decreased the fluorescence intensity.These results indicate that NSP12 significantly suppresses the AS of host genes.Moreover, the NSP12 N-terminal domain (1-356 aa) and C-terminal domain (366-932 aa) also exhibited a strong inhibition effect on the AS of host genes (Figure 5D).Three mutants of NSP12 (M3S, M3P, and M2A) were selected to analyze whether they affect the AS of host genes, since the expression levels of NSP12 and the three mutants are comparable, and these interactions (SLU7-M3S, PPIL3-M3P, and AKAP8-M2A) were obviously weakened, as shown in Figure 1C-E.
Interestingly, the three mutants also exhibited strong inhibition effects on the AS of host genes, and inhibition effects of the three mutants was stronger than that of NSP12 (Figure 5E and Figure S8).The roles of the three host proteins (SLU7, PPIL3, and AKAP8) in cellular AS were further tested using the splicing reporter system.Overexpression of AKAP8 and SLU7 significantly promoted cellular AS, whereas PPIL3 overexpression slightly inhibited cellular AS (Figure 5F).Knockdown of SLU7, PPIL3 or AKAP8 decreased cellular AS (Figure 5G).
The effects of SLU7, PPIL3, or AKAP8 on the ability of NSP12 to inhibit cellular AS were further analyzed.Overexpression of AKAP8 and SLU7 repaired the inhibition effect of NSP12 on cellular AS, while overexpression of PPIL3 enhanced the inhibition effect of NSP12 on cellular AS (Figure 5H).Furthermore, the splicing efficiency of splicing reporter in HEK293T cells transfected with siSLU7, siPPIL3, or siAKAP8 for 24 h and then transfected with NSP12 was lower than that in HEK293T cells transfected with siNC for 24 h and then transfected with NSP12, as shown the pink column of Figure 5I.Since NSP12 and the three splicing factors have abilities to regulate the cellular AS, to more accurately evaluate the effect of NSP12 on cellular AS in the cells knocking down SLU7, PPIL3, or AKAP8, the relative splicing efficiency, namely (R + NSP12)/(R + GAPDH) index (R representing splicing reporter pMSCV-IRF7-2A), were Importantly, the (R + NSP12)/(R + GAPDH) index in HEK293T cells knocked down for SLU7, PPIL3, or AKAP8 was significantly higher than that in the negative control (siNC) (Figure 5I).These results clearly indicated that SLU7, PPIL3, or AKAP8 can regulate the inhibition effect of NSP12 on cellular AS.In other word, the ability of NSP12 to inhibit cellular AS attenuated when three targeting splicing factors were knocked down.
Western blot analysis showed that the expression of NSP12 had no significant effect on the expression of SLU7, PPIL3, or AKAP8 (Figure S9).RNA-seq analysis of HEK293T cells transiently expressing NSP12 revealed significant changes in 26 genes, including 12 upregulated genes and 14 downregulated genes.However, SLU7, PPIL3, or AKAP8 were not among the 26 genes.Among the DEGs, the transcriptions of SAMD4A and PPP2R1A showed the most significant upregulation and downregulation, respectively.GO enrichment analysis revealed that the DEGs were involved in various biological processes, such as DNA replication and negative regulation of gene expression.KEGG pathway enrichment analysis showed that the DEGs were mainly enriched in genetic information processing, such as the spliceosome pathway.Furthermore, a total of 1768 AS events were identified, including 1203 SE, 171 RI, 96 MXE, 122 A5SS, and 176 A3SS events (Figure 5J).Many splicing genes were found to be involved in biological processes such as RNA splicing and viral process.Some splicing genes were also found to be mainly enriched in human diseases, such as viral infectious disease and immune disease (data not shown).

| The general mechanism of NSP12 modulate the cellular genome AS
The protein sequence of SARS-CoV-2 NSP12 shows 96.3% and 71.2% similarity to NSP12 from SARS-CoV (NSP12a) and NSP12 from MERS-CoV (NSP12b), respectively.Fluorescence microscopy was used to observe the distribution of NSP12a-EGFP and NSP12b-EGFP in the cytoplasm and nucleus of A549 cells (data not shown).
Co-IP assays were then performed to determine whether the NSP12 homologs also interacted with three splicing factors.The results showed that NSP12a strongly interacted with SLU7 and AKAP8, but not PPIL3 (Figure 6A).NSP12b, otherwise, strongly interacted with AKAP8 and only weakly with SLU7, but not PPIL3 (Figure 6B).
To investigate whether NSP12a or NSP12b also modulate AS of cellular genes, the splicing reporter system was used.The results showed that NSP12 and its two homologs all repressed cellular AS, with NSP12b showing the most significant inhibitory effect, and NSP12a having the weakest inhibition effect (Figure 6C).
Further analysis was conducted to examine the effects of SLU7, PPIL3, or AKAP8 on the ability of NSP12a or NSP12b to inhibit cellular AS.Overexpression of AKAP8 or SLU7 was found to repair the inhibition effect of NSP12a on cellular AS, while overexpression of PPIL3 enhanced the inhibition effect of NSP12a on cellular AS (Figure 6D), which is consistent with NSP12 on cellular AS.
Overexpression of SLU7, PPIL3, or AKAP8 had only a slight influence on the ability of NSP12b to inhibit cellular AS (Figure 6E).Knockdown of SLU7, PPIL3, or AKAP8 in HEK293T cells increased the (R + NSP12a)/(R + GAPDH) index, compared to the negative control (siNC) (Figure 6F).These results suggested the ability of NSP12a to inhibit cellular AS also attenuated when three splicing factors were knocked down.Similar effects were observed in NSP12b, although the indices varied (Figure 6G).The interactions between NSP12 and seven host proteins were confirmed by co-IP and reciprocal co-IP assays.The interactions between NSP12 and endogenous SLU7, PPIL3, and AKAP8 during SARS-CoV-2 infection were also detected by co-IP.HEK293T-ACE2 transfected with pcDNA3.1-NSP12-HAwere infected with authentic SARS-CoV-2 at an MOI of 1 for 24 h.An anti-HA affinity gel was used for immunoprecipitation.As shown in Figure S10, NSP12-HA, SLU7, PPIL3, and AKAP8 were detected in cell lysates, but only very weak band of PPIL3 was detected in IP, and no band of SLU7 or AKAP8 was detected in IP.Although SLU7, PPIL3, and AKAP8 that interact with NSP12 were detected by affinity purification mass spectrometry (AP-MS), 12 it is possible that the proteins SLU7 and AKAP8 in IP were too weak to detect by Western blot using corresponding antibodies.Although the complexes of NSP12 with splicing factors were predicted by molecule docking, whether direct interactions exist between NSP12 and the splicing factors remains to be further determined.These seven host proteins have different functions, with SLU7 being involved in the selection of the 3′-splice site during the second step of splicing and SLU7 knockdown inhibiting early stages of human immunodeficiency virus type 1 (HIV-1) replication. 14,30PPIL3, a member of peptidyl-prolyl isomerase (PPIase) family, has been found to be present in spliceosome B act and C complexes. 15AKAP8 regulates splicing through scaffolding RNAs and RNA processing factors. 16TCF12, belonging to the basic helixloop-helix (bHLH) subfamily of transcription factors, is known to have a crucial regulatory role in certain types of cancers. 31CRTC3 plays important roles in adipose development and energy metabolism. 32topic expression of CRTC3 in hepatoma cells stimulated the activity of the preS2/S promoter of hepatitis B virus (HBV). 33CRTC3 also inhibited HIV-1 infection. 34RBM41 is a protein that is not well studied or characterized.On the other hand, BCKDK is a key enzyme involved in the metabolism of branched-chain amino acids and has been implicated in promoting colorectal cancer tumorigenesis.
Additionally, it has been linked to various hereditary diseases, including Huntington disease, autism, and metabolic disorders like obesity. 35These findings suggest that the interactions between NSP12 and these host proteins may be closely related to virus replication and host pathogenesis.
Despite coronavirus genome replication and transcription occurring in the cytoplasm, these interactions may still have significant effects on the virus life cycle and pathogenesis. 36FP-fused NSP12 and two subunits NSP7, NSP8 were diffusely distributed throughout the cytoplasm and nucleus of A549 cells, which is consistent with previous reports. 37NSP12-EGFP was observed to colocalize with some host proteins, such as SLU7, PPIL3, and AKAP8, in the nucleus.NSP12-HA distributed diffusely in both the cytoplasm and nucleus, and colocalized with the three splicing factors in the nucleus of Vero E6 cells infected with authentic SARS-CoV-2 (Figure 2D).Other studies have reported that NSP12 distributed diffusely and some exhibited puncta-like structures or inclusion bodies in the cytoplasm and nucleus of Vero E6 cells infected with SARS-CoV-2 as determined by IFA using anti-NSP12 monoclonal antibody. 38These findings suggest that NSP12 may have novel roles in the nucleus by interacting with these host proteins, possibly in regulating splicing.
It has been reported that some splicing factors played important role in virus infection and replication, 39 as demonstrated in studies involving SLU7 and HIV-1 replication. 30Moreover, Cyclophilin A of PPIase family is a key factor in coronavirus replication. 40,41Our study focused on the effects of three splicing factors, SLU7, PPIL3, and AKAP8 on the subcellular localization of NSP12, virus infection and the polymerase activity of RdRp.SLU7, PPIL3, or AKAP8 knockout did not have a significant effect on the localization of NSP12, but it did decreased the entry efficiency of SARS-CoV-2 pseudovirus and authentic virus into A549 with different degrees.To investigate the possible reasons why SLU7 or PPIL3 knockout significantly decreased the efficiency of virus entry, transcriptome analysis was conducted.
The results showed that SLU7 or PPIL3 knockout caused a large number upregulated or downregulated genes, including some genes involved in SARS-CoV-2 infection, such as TMPRSS2. 42This suggested that the knockout of SLU7 or PPIL3 could affect the virus entry efficiency due to changes in the expression of genes related to viral infection.Moreover, SLU7 or PPIL3 knockout resulted in a large number of genes undergoing AS events, and some of these genes are involved in viral infectious disease and immune system.These PPIL3 knockout affects virus entry but also indicated that SLU7 and PPIL3 are both involved in all of the common modes of AS, and that SE is the most frequent target for their regulation.
RNA extension assays showed that purified SLU7 or PPIL3 with high concentration slightly inhibited the polymerase activity of RdRp in vitro.Gluc activity assays and RT-qPCR indicated that these three host proteins play roles in the polymerase activity of RdRp or replication of SARS-CoV-2.However, the reason why the results from in vitro and in vivo are inconsistent, and the effects of these host proteins on the replication of authentic SARS-CoV-2 require further investigation.Co-IP assays showed that SLU7, PPIL3 or AKAP8 knockdown decreased the stability of RdRp.It was also reported that other host protein cyclin-dependent kinase 2 (CDK2) knockdown also decreased the stability of RdRp. 43Whether the stability or assembly efficiency of RdRp affected by the three splicing factors is reason why they regulate the polymerase activity of RdRp in vivo needs more evidence.
Two types of RNA virus polymerases have been found to regulate cellular AS by targeting spliceosome proteins.For instance, RdRp of enterovirus 71 (EV71) interferes with Prp8 and disrupts the intracellular splicing machinery by entering the nucleus. 22Similarly, the dengue virus NS5 with RdRp and methyl transferase intrudes in the cellular spliceosome and modulates splicing. 23A hypothesis was proposed that NSP12 could regulate splicing by targeting three splicing factors (SLU7, PPIL3, and AKAP8).Splicing reporter assays indicated that NSP12 along with its N-terminal domain (1-356 aa), C-terminal domain (366-932 aa) and three mutants (M3S, M3P, and M2A) repressed cellular AS.Surprisedly, inhibition effects of the three mutants was stronger than that of NSP12, although these interactions (SLU7-M3S, PPIL3-M3P, and AKAP8-M2A) were obviously weakened.It could be results from two reasons.One, these mutations of predicted interacting residues change other characteristics of NSP12, such as the ability to target other key splicing factors, since other splicing factors interacting with NSP12 and its homologues NSP12a and NSP12b were detected by our group using AP-MS, such as spliceosome RNA helicase DDX39B, serine/arginine-rich splicing factor 3 and splicing factor 1 (data not shown).Two, these splicing factors also could antagonize the inhibition effect of NSP12 on cellular AS.When the interactions of three mutants with these splicing factors were weakened, the ability of these splicing factors to antagonize the inhibition effect of these mutants on cellular AS were weakened, leading to stronger inhibition effects of these mutants.
The roles of the three splicing factors (SLU7, PPIL3, and AKAP8) in cellular AS were further confirmed, with SLU7 and AKAP8 identified as positive regulators of minigene splicing.Interestingly, AKAP8 was previously identified as a positive regulator in other minigene splicing experiments. 16PPIL3 was first discovered in spliceosome B act complex and C complex, 15 but its specific role in cellular AS remains unclear.Nonetheless, the results of the study indicate that the expression level of PPIL3 is crucial for normal cellular AS.Further analysis was conducted to investigate whether the process of NSP12 regulating cellular AS was influenced by three host proteins SLU7, PPIL3, and AKAP8.Our data indicated that each of these host proteins had the ability to regulate the inhibition effect of NSP12 on cellular AS.When the three splicing factors were knocked down, the inhibition effect of NSP12 on cellular AS is diminished (Figure 5I), supporting that NSP12 inhibits cellular AS by targeting these splicing factors.Viruses have been known to modulate cellular splicing through multiple mechanisms, such as interacting with spliceosomes to inhibit splicing or modulating splicing factor localization, levels, and posttranslational modifications. 20,44,45In this study, we found that NSP12 had no significant effect on the localization, transcription or expression level of SLU7, PPIL3, or AKAP8.This suggests that NSP12 might affect other characteristics or functions of these host proteins or other components of spliceosome by interacting with them, thereby regulating cellular AS.However, more experimental evidence are needed to support this hypothesis.Our results also showed that NSP12 transient expression significantly changed the expression of 26 genes and led to a total of 1768 AS events.These genes were enriched in biological processes, such as RNA splicing, which not only further verified that NSP12 regulates cellular AS, but also suggested that NSP12 had a slight impact on gene transcription but a significant effect on cellular AS.
The ability of NSP12 homologues from other HCoVs to interact with the three splicing factors (SLU7, PPIL3, and AKAP8) and regulate cellular AS was further analyzed.Interestingly, NSP12a was found to strongly interact with SLU7 and AKAP8, but not with PPIL3.NSP12b, on the other hand, strongly interacts with AKAP8 and very weakly with SLU7, but not with PPIL3.These results indicated that NSP12 and its homologues in SARS-CoV and MERS-CoV have different ability to interact with PPIL3 or SLU7.Comparison of NSP12 and its homologues in other HCoVs showed that NSP12b have the most obvious inhibition effect, while the ability of NSP12a was the weakest.Furthermore, our data indicated that the ability of NSP12a to inhibit cellular AS also attenuated when three splicing factors were knocked down, and similar observations was made with NSP12b, albeit with varying degrees of influence.In summary, our findings suggest that NSP12 and its homologues from other HCoVs possess different abilities to regulate cellular AS, and this regulation is influenced by the three splicing factors (SLU7, PPIL3, and AKAP8) with different degrees of interaction.In summary, our results supported the model that SARS-CoV-2 NSP12 utilizes three splicing factors (SLU7, PPIL3 and AKAP8) to regulate virus entry, replication and cellular AS.This may be a common mechanism among HCoVs, as suggested by the analysis of NSP12 homologues (Figure 7).In this model, the transcriptions and AS of certain host genes involved in virus entry are regulated by SLU7 or PPIL3 when the virus infects, which may facilitate virus infection.After virus entry, RdRp primarily performs viral RNA replication in the cytoplasm, and its stability and the polymerase activity are directly or indirectly regulated by SLU7, PPIL3, or AKAP8, affecting the replication of SARS-CoV-2.Additionally, partial NSP12 enters the nucleus and interacts with the three splicing factors (SLU7, PPIL3, and AKAP8) or other components of spliceosome, potentially interfering the yet-to-be defined characteristics or functions of spliceosome, ultimately inhibiting cellular AS, which may contribute to viral replication and pathogenesis.However, the exact reason why NSP12 regulates cellular AS remains unclear and requires further investigation.Our study not only reveals a novel role of human coronavirus NSP12 as a splicing modulator but also provides insights into the interactions between HCoVs and their hosts.This may potentially lead to the development of antiviral strategies targeting NSP12-mediated splicing regulation.

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INTRODUCTION Coronaviruses, belonging to the family Coronaviridae, are enveloped viruses that have a single-stranded positive-sense RNA genome approximately 26-32 kilobases in size.They can cause intestinal and respiratory infections in various birds and mammals.Seven human coronaviruses (HCoVs) have been identified, four of which cause mild respiratory infections (HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1), while severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have relatively high mortality rates and emerged in 2002 and 2012, respectively.

pH 7 . 4 ,
150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Sigma) and 1 mM phenylmethylsulfonyl fluoride for 1 h.The cell lysates were centrifuged to remove cell debris and the lysate supernatants were collected and divided into two parts.Five hundred microliters of lysate supernatant was used to analyze the coexpression of NSP12 and other proteins by Western blot analysis using anti-HA and anti-Flag antibodies.Other 500 μL of lysate supernatant was incubated with red anti-HA affinity gel (Sigma) or anti-Flag affinity gel (Sigma) overnight at 4°C.The precipitates were collected by centrifugation, washed three times with ice-cold RIPA buffer, eluted with 40 μL of phosphate-buffered saline (PBS), and finally subjected to Western blot analysis.
2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 16 h.Harvested cell pellets were resuspended in buffer A (25 mM Tris-HCl pH 8.0, 250 mM NaCl, 4 mM MgCl 2 , 10% Glycerol).The cell lysate was applied to a nickel-affinity column (Ni-NTA; GE Healthcare) for purification.NSP12 and NSP7 were eluted with buffer B (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 4 mM MgCl 2 , 250 mM Imidazole).NSP8 was subjected to on-column tag cleavage by PPase (PreScission Protease) and then eluted by the buffer A. Next, the three proteins were purified by ion exchange chromatography (Source Q, GE Healthcare) and size exclusion chromatography (GE Healthcare) with buffer C (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 4 mM MgCl 2 ).For RdRp complex assembly, protein samples were mixed with the molar ratio of NSP7:NSP8:NSP12 = 2:2:1 at 4°C overnight.The incubated RdRp complex was then concentrated with a 100 kDa molecular weight cut-off centrifugal filter unit (Millipore Corporation) and further purified by size exclusion chromatography in buffer C with 5 mM DTT.The plasmid pGEX-PPIL3 was transformed into E. coli BL21 (DE3).PPIL3 was then expressed after induction with 0.1 mM of IPTG.Harvested cell pellets were resuspended in buffer D (25 mM Tris-HCl pH 7.5, 250 mM NaCl, 3 mM DTT).The cell lysate was then loaded onto glutathione-coated Sepharose resin (GE Healthcare) for purification.GST-PPIL3 was subjected to on-column tag cleavage by PPase and then eluted by buffer D. PPIL3 was purified by size exclusion chromatography with buffer E (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 3 mM DTT).The plasmid pET15b-SLU7 was transformed into E. coli BL21 (DE3).SLU7 was then expressed after induction with 0.2 mM of IPTG.Harvested cell pellets were resuspended in buffer F (25 mM Tris-HCl pH 7.5, 250 mM NaCl).The cell lysate was applied to a nickel-affinity column and eluted with elution buffer G (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 300 mM Imidazole).SLU7 was purified by ion exchange chromatography and size exclusion chromatography with buffer H (25 mM Tris-HCl pH 7.5, 100 mM NaCl).

(
pcDNA3.1) showed no band in both the cell lysate and immunoprecipitated complex, further confirming the strong interactions between NSP12 and the seven host proteins.The interactions between NSP12 and the seven host proteins were further analyzed by conducting reciprocal co-IP experiments F I G U R E 1 (See caption on next page).

3. 2 |
Localizations of SARS-CoV-2 NSP12, NSP7, NSP8, and host proteins Fluorescence microscopy was used to visualize the subcellular localization of various fusion proteins in A549 cells.NSP12, NSP7, and NSP8 fused with enhanced green fluorescent protein (EGFP) were found distributed diffusely in both the cytoplasm and nucleus of the cells.SLU7 fused with red fluorescent protein (SLU7-RFP) was mainly distributed in the nucleus, with little distribution in the cytoplasm.PPIL3-RFP showed diffuse distribution in both the cytoplasm and nucleus, while AKAP8-RFP, TCF12-RFP, and RBM41-RFP were found only in the nucleus.CRTC3-RFP showed a mainly nucleus distribution with some distribution in the cytoplasm, and BCKDK-RFP was found only in the cytoplasm with some small granular aggregation (Figure 2A).Coexpression of NSP12-EGFP with various RFP-fused host proteins revealed distinct patterns of colocalization.NSP12-EGFP mainly colocalized with SLU7-RFP in the nucleus, resulting in the aggregation of NSP12-EGFP into small granules in some cells.NSP12-EGFP diffusely colocalized with PPIL3-RFP in both the cytoplasm and nucleus.In the nucleus, NSP12-EGFP colocalized with AKAP8-RFP, TCF12-RFP, or RBM41-RFP.NSP12-EGFP mainly colocalized with CRTC3-RFP in the nucleus and small yellow fluorescent granules were observed in the nucleus of some cells.NSP12-EGFP also colocalized with BCKDK-RFP in the cytoplasm, and small yellow fluorescent granules were observed.Additionally, PPIL3-EGFP mainly diffusely colocalized with SLU7-RFP in the nucleus (Figure 2B).

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I G U R E 2 Fluorescence micrographs of cells expressing a single protein or co-expressing two proteins.(A) Expressing protein NSP12-EGFP or other indicated proteins alone in A549 cells.Scale bar: 10 μm.(B) Co-expressing two proteins, such as NSP12-EGFP + SLU7-RFP.EGFP fused proteins, such as NSP12-EGFP, are green fluorescence.RFP fused proteins, such as SLU7-RFP, present red fluorescence.Nucleus and colocalization are blue and yellow, respectively.Scale bar: 10 μm.(C) Expressing protein NSP12-HA alone in Vero E6 cells infected with authentic SARS-CoV-2.Scale bar: 100 μm.(D) Co-expressing two proteins in Vero E6 cells infected with authentic SARS-CoV-2.Scale bar: 100 μm.EGFP, enhanced green fluorescent protein; RFP, red fluorescent protein.

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I G U R E 2 (Continued) F I G U R E 3 The effects of SLU7, PPIL3, or AKAP8 knockout on the localization of NSP12 and virus infection.(A) Validation of SLU7, PPIL3, or AKAP8 gene editing by CRISPR-Cas9.Host protein expression in gene-disrupted A549 cells were separately determined by Western blot analysis using corresponding antibodies.(B) Expressing protein NSP12-EGFP alone in A549 or host gene-disrupted A549 cells.Scale bar: 10 μm.(C) The effects of SLU7, PPIL3, or AKAP8 knockout on pseudovirus entry.Cells were infected with an equal number of SARS-CoV-2 pseudotyped virions and the entry efficiencies were quantified at 48 hpi by measuring luciferase activity (in relative light units, RLU) (n = 3).SLU7 or PPIL3 knockout in A549 cells significantly reduced pseudovirus infection.Error bars indicate standard deviations.**Represents p < 0.01.(D) The effects of SLU7, PPIL3, or AKAP8 knockout on the infection of SARS-CoV-2 authentic virus.(E) The effects of SLU7 knockout on cellular AS.(F) The effects of PPIL3 knockout on cellular AS.AS, alternative splicing.

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I G U R E 4 (See caption on next page).

F I G U R E 4
The effects of SLU7, PPIL3, or AKAP8 on polymerase activity and stability of RdRp.(A) The 13 + 23 nt RNA template-product duplex.Arrowhead indicated the direction of RNA extension.The 5′ end of the RNA product contains a FAM fluorescent label.(B) Analyzing the effect of SLU7 on the polymerase activity of RdRp by denaturing urea polyacrylamide gel electrophoresis (urea PAGE).(C) Analyzing the effect of PPIL3 on the polymerase activity of RdRp by urea PAGE.(D) Schematic diagram of the Gluc reporter system.The ORF of Gluc is flanked by the 5′ UTR and 3′ UTR of SARS-CoV-2.The plus-strand RNA (mRNA) of Gluc was first transcribed by the host cellular DNA-dependent RNA polymerase Pol II, which can further translate Gluc.RdRp use mRNA as templates to synthetize negative-sense vRNA, which was transcribed into mRNA to express Gluc.(E) The effect of SLU7, PPIL3 or AKAP8 overexpression on polymerase activity of RdRp.Vector represents vector pcDNA3.1(+).NS represents no significance.(F) Validation of SLU7, PPIL3 or AKAP8 knockdown by siRNA.HEK293T cells transfected with four chemically synthesized siRNAs for 24 h were subjected to Western blot analysis.(G) The effect of SLU7, PPIL3 or AKAP8 knockdown on polymerase activity of RdRp.(H) Levels of both plus-strand and minus-strand CoV-Gluc RNA were determined by RT-qPCR from HEK293T cells transfected with the indicated plasmid DNA.Results shown are the average of three independent experiments.R represents reporter pCoV-Gluc.Vector represents vector pcDNA3.1(+).(I) The effects of SLU7, PPIL3, or AKAP8 knockdown on stability of RdRp.mRNA, messenger RNA; RdRp, RNA-dependent RNA polymerase; UTR, untranslated region.F I G U R E 5 (See caption on next page).
YANG ET AL.|15 of 21   The aim of this study was to investigate the mechanisms and biological significances of the interactions between NSP12 and multiple host proteins.Our findings confirmed that NSP12 interacted and colocalized with seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8).The three splicing factors were found to regulate virus infection and the polymerase activity of SARS-CoV-2 RdRp to varying degrees.Additionally, NSP12 and homologues from SARS-CoV and MERS-CoV were shown to inhibit cellular AS, which was influenced by the three splicing factors.
findings not only provided clues to reveal the reason why SLU7 or F I G U R E 5 Examine the impact of NSP12 on cellular AS.(A) Schematic of fluorescence reporter used to assay mRNA splicing.(B) EGFP intensity plot of HEK293T cells coexpressing the splicing reporter and NSP12 (blue).GAPDH (red) was as a control.(C) The effect of NSP12 on the AS.GAPDH was as a negative control.NSP16 was as a positive control.R represents splicing reporter pMSCV-IRF7-2A.(D) The effects of Nterminal domain (1-356 aa) and C-terminal domain (366-932 aa) of NSP12 on the AS.(E) The effects of three mutants (M3S, M3P, and M2A) of NSP12 on the AS.(F) The effect of SLU7, PPIL3 or AKAP8 overexpression on the AS.(G) The effect of SLU7, PPIL3, or AKAP8 knockdown on the AS.(H) The effect of NSP12 on cellular AS in the cells overexpressing SLU7, PPIL3, or AKAP8.(I) The effect of NSP12 on cellular AS in the cells knocking down SLU7, PPIL3, or AKAP8.(J) The effect of NSP12 on cellular AS by transcriptome analysis.AS, alternative splicing; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.F I G U R E 6 The effect of SARS-CoV NSP12 (NSP12a) or MERS-CoV NSP12 (NSP12b) on the AS of cellular genes.(A) Interaction between NSP12a and SLU7, PPIL3, or AKAP8.(B) Interaction between NSP12b and SLU7, PPIL3, or AKAP8.(C) Comparative analysis of the effects of NSP12, NSP12a, or NSP12b on cellular AS.R represents splicing reporter pMSCV-IRF7-2A.(D) The effect of NSP12a on cellular AS in the cells overexpressing SLU7, PPIL3, or AKAP8.(E) The of NSP12b on cellular AS in the cells overexpressing SLU7, PPIL3 or AKAP8.(F) The effect of NSP12a on cellular AS in SLU7, PPIL3, or AKAP8 knockdown cells.(G) The effect of NSP12b on cellular AS in SLU7, PPIL3, or AKAP8 knockdown cells.