Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication

doi:10.21203/rs.3.rs-337427/v1

PDF

Research

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication

https://doi.org/10.21203/rs.3.rs-337427/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 22 Jul, 2021

Read the published version in Cell & Bioscience →

You are reading this latest preprint version

Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral proteins for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. In summary, our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

Infectious Diseases

Immunology

Allergy & Immune Disorders

SARS-CoV-2

interaction map

Nsp3

replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen for the pandemic of the coronavirus disease 2019 (COVID-19), caused more than 100 million infections and 2 million deaths thus far (WHO). As of now, no anti-SARS-CoV-2 specific drug was available in clinical therapy, except for the FDA’s approval of Veklury (remdesivir). Veklury could only shorten the duration of hospitalization of COVID-19 patients if early administration within 48 hours of hospital admission was applied¹. Vaccines developed using different strategies were authorized by many countries to contain the infection of SARS-CoV-2. However, there are growing concerns about safety like antibody-dependent enhancement (ADE), the duration of the effective anti-SARS-CoV-2 immune response, and the effectiveness against the mutant viruses^2–4. Thus, the anti-SARS-CoV-2 specific drugs are urgently needed, and more drug targets on this deadly virus are waiting to be uncovered.

SARS-CoV-2, the newest member of the genus Betacoronavirus of the Coronaviridae family, encapsulated the known largest single-stranded positive-sense viral RNA, which encoded a 7000-aa polyprotein for its replicase complex and a set of accessory proteins^5–8. The polyprotein is encoded by the open reading frame (ORF) 1a and 1ab, and the latter is translated via the mechanism of -1 ribosomal frameshifting. The ORF1a and ORF1ab are processed by the viral proteases, papain-like protease (PLpro) and 3C-like Protease (3CLpro), into 16 non-structural proteins (Nsps), which compose the replicase complex through an unknown manner. The other 10 ORFs encodes 4 well-known structural proteins, spike (S), nucleocapsid (N), membrane (M) and envelope (E), and a set of accessory proteins without clearly identified functions, ORF3, ORF6, ORF7a, ORF7b, ORF8, and ORF10⁷.

As the largest RNA virus, coronaviruses employ a relatively big number of viral proteins to replicate and transcript their viral RNA and subgenomic RNAs compared with other RNA viruses. This strategy increased the efficiency in many aspects of viral proliferation, including replication fidelity of viral RNAs and viral accessory proteins’ expression. However, the performance of this strategy is highly dependent on the coordination of at least 16 viral non-structural proteins, indicating the disturbance on the assembly of viral protein complexes could be a promising approach to the inhibition of viral replication⁹.

The direct interactions among viral proteins play essential roles in the formation of viral replicase complex, assembly of viral particles, release from the host cells and counter-defense against host immune responses. Compared with the active centers which are mostly inside the structure of proteins, these interactions happen at the peripheral residues of viral proteins. They are likely more vulnerable to small molecules which could competitively bind to the residues which mediate viral protein-protein interactions^{10, 11}.

Several studies focusing on the interactions between viral proteins and the interactions between viral proteins and host proteins were carried out and uncovered many interactions^12–14. Immunoprecipitation in combination with mass spectrometry (IP-MS) and yeast two-hybrid (Y2H) screening were employed in these studies. IP-MS is unable to differentiate indirect interactions from direct ones. It usually needs a relatively high amount of target proteins and thus is likely not sensitive enough to detect weak or transient interactions. The interactions identified by Y2H could be determined to be direct, while due to different intracellular environment from mammalian cells, some interactions, which happen specifically in mammalian cells, could be missed using Y2H screening¹⁵.

Our previous work employed a mammalian two-hybrid system to screen genuine protein interactions for SARS-CoV and identified a few interactions that were not described in other studies using IP-MS or Y2H¹⁵. Many novel interactions, like the interactions of Nsp10/Nsp14 and Nsp10/Nsp16, uncovered in our previous studies, were proven to play essential roles in the replication of viral genomic RNAs.

In this study, we used a mammalian two-hybrid system to examine 784 interaction combinations between 28 SARS-CoV-2 proteins in a pairwise matrix. As a result, 48 interactions were detected, and 14 interactions of 48 have not been reported elsewhere. We identified 19 interactions between Nsps and accessory proteins. To determine the possible roles of accessory proteins involved in viral replication, we investigated the interaction between Nsp3.1 and N proteins. We found the N interacted with the N-terminus of Nsp3.1 through its N-terminus domain, and disruption of the interaction harms viral replication and transcription. We also explored the roles of all the viral proteins in viral replication and transcription and identified ORF3 and Nsp3.2 proteins play positive roles in viral replication and transcription.

Identification of protein-protein interactions of SARS-CoV-2 using mammalian two-hybrid system assays

To analyse the genome-wide protein-protein interactions of SARS-CoV-2, We cloned all the coding sequences of Nsps and ORFs into the pM and pVP16 vectors separately (Table 1). To achieve a better expression, we separated the coding sequence of Nsp3 into 3 parts, Nsp3.1, Nsp3.2, and Nsp3.3, which was designed according to the known functional domains of Nsp3 (Figure S1). S was separated at the cleavage site of furin into S1 and S2. The sensitivity and efficiency of the mammalian two-hybrid system were confirmed by detecting the interaction of p53 and SV40T, which could lead to more than 130 times of increase in the relative expression level of reporter genes compared with negative controls (Figure 1A).

We screened 784 interaction combinations between all the coding sequences of Nsps or ORFs of SARS-CoV-2, and the assays on each combination were repeated at least 3 times (figure 1B). 48 positive interaction combinations were screened out and analyzed using Cytoscape software to visualize molecular interaction networks (figure 1B and 1C). Some interactions were confirmed using co-immunoprecipitation (figure 2). Besides 5 self-interactions, 5 out of 48 interactions between different viral proteins, Nsp1/Nsp3, Nsp3/N, Nsp3/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were examined to be positive bidirectionally and 79.2% interactions could only be detected in one direction, indicating that the fusion domains may influence the interactions, which happened in our previous studies¹⁵. We identified 14 novel interactions, including Nsp1/Nsp7, Nsp1/M, Nsp1/Nsp3.2, Nsp1/ORF7a, Nsp3.1/Nsp10, Nsp3.1/ORF3, Nsp3.2/Nsp10, Nsp3.3/Nsp3.1, Nsp12/M, E/Nsp4, ORF6/Nsp4, ORF6/Nsp10, ORF6/M, and Nsp3.1/N.

Unlike the Nsp1 of SARS-CoV, which barely interacts with other viral proteins, Nsp1 of SARS-CoV-2 has the most interaction partners, 10 Nsps and 7 accessory proteins, among all the viral proteins, and likely functions as the hub of the group of viral proteins (figure 1B and 1C). As the first viral protein being expressed after viral infection, the function of Nsp1 as a hub likely was an advantage for virus to organize the assembly of replicase complex and replication of viral genomic RNAs. Besides its function in suppression of protein expression and defense against host innate immune response¹⁶, the vital role of Nsp1 in the viral replication is supported by the clinical observation that a deletion in the C-terminus of Nsp1 attenuated the viral pathogenicity¹⁷. Nsp3 is one of the most complex proteins and has multiple functional domains¹⁸. The well-known function of Nsp3 is to process viral polyprotein at cleavage sites of Nsp1/2, Nsp2/3 and Nsp3/4. In our interaction screening, Nsp3 was found to interact with Nsp10, Nsp12, Nsp13, and Nsp14, indicating its possible roles in the replication/transcription of viral RNAs besides the process of viral polyprotein. Nsp5 is responsible for the cleavage at the sites separating Nsp4 to Nsp16, spanning 1a and 1b regions. Similar to SARS-CoV, Nsp5 of SARS-CoV-2 interacts with Nsp12, indicating that Nsp12 could be the sites in 1b for Nsp5 to grasp its substrate¹⁵. In agreement with the previous findings that Nsp8 and Nsp12 form the core RNA polymerase complex, Nsp8 of SARS-CoV-2 interacts with Nsp12^{19, 20}. As an RNA binding protein, Nsp8 may facilitate the substrate recognition of Nsp12. We identified 10 interactions Nsp10 involved in, and among them, the interactions of Nsp10/Nsp14 and Nsp10/Nsp16 were also uncovered in our previous work for SARS-CoV¹⁵, indicating a conservative mechanism of the genus Betacoronavirus for regulation of the activity of methyltransferase of Nsp14 and Nsp16. We also identified many interactions between Nsps and accessory proteins, indicating that the possible roles of accessory proteins in the replication/transcription of viral RNAs. 5 self-interactions, Nsp3, Nsp5, Nsp15, and N reported here were also found in SARS-CoV, except for ORF8, which is one of the most distinct ORFs between SARS-CoV and SARS-CoV-2^{21, 22}.

Nsp3 interacts with N protein

N protein is one of four well known structural proteins identified in the viral particles⁵. It forms a long helical nucleocapsid structure which viral RNA was packed inside. This ribonucleoprotein (RNP) complex protects the viral RNA from the attack of host nucleases and recognition of host nucleotide sensors triggering the immune response²³. This structure could also play an essential role in the replication/transcription of viral RNA¹⁵. However, the molecular details about the role of N in replication were obscure. In this screening, the interaction between Nsp3.1 and N was among the strongest ones (figure 1A) and was confirmed by co-immunoprecipitation (figure 2G). Since Nsp3 is the component of the viral replication and transcription complex (RTC), this interaction suggested the N could regulate the replication of viral RNA through the association with Nsp3.

N protein interacts with Nsp3 through its NTD domain

N protein has three major defined domains, N-terminal domain (NTD), serine-arginine-rich (SR) domain, and C-terminal domain (CTD) (figure 3A)^{24, 25}. To determine N protein’s key domains interacting with Nsp3.1, we examined the interactions between various domains of N and Nsp3.1 using co-immunoprecipitation. NTD retains the capability to interact with Nsp3.1, while this capability of N protein is largely lost in CTD (figure 3B and 3C). We also examined the locations of N and Nsp3.1 proteins in the cells. The immunostaining results showed that similar to the wild-type (wt) N, NTD colocalized with Nsp3.1 in the 293T cells, while CTD lost the colocalization with Nsp3.1 (figure 3D, 3E, and 3F). We could not detect the expression of the coding sequence of SR, which happens typically in the expression of proteins with a molecular weight of less than 15 kDa. In our laboratory practice, we increase the size of protein by fusing our target protein with a tag protein, like EGFP, which has a relatively independent structure and unlikely interferes with the function of target protein. As predicted, we detected the decent expression of EGFP-tagged SR. However, Nsp3.1-HA could not be detected in the pull-down samples of EGFP-tagged SR, similar as that of EGFP, while, in contrast, the decent level of Nsp3.1-HA was detected in that of EGFP-tagged N (figure 3G and 3H). Moreover, NTD seems to interact with Nsp3.1 in a stronger manner than N protein, indicating that the other domains of N protein could negatively impact the interaction with Nsp3.1 (figure 3C).

Nsp3 interacts with N through its acidic domain

Thus far, limited knowledge about the functions and structures of Nsp3.1 was available. Based on analysis of the protein sequence, we found a special domain rich in negatively charged amino acids in the N-terminus of Nsp3.1 (figure 4A). As a nucleic acid-binding protein, N protein has a 10.1 of pI and is composed of many positively charged amino acids, which facilitate its interaction with nucleic acids with negative charges and likely also with acidic proteins (figure S2). Accordingly, we first examined the interaction between N protein and the aa 1-235 of N-terminus, which possessed the most acidic amino acids in Nsp3.1. Despite the loss of nearly two-thirds of Nsp3.1, aa 1-235 retained the capability to interact with N protein which was even stronger than that of the full length of Nsp3.1 (figure 4B and 4C). To further narrow down the region that interacts with N protein, we removed aa 1-102, which has the most acidic amino acids, from Nsp3.1 and examined its interaction with N protein. As predicted, the deletion of aa 1-102 largely abolished Nsp3.1/N interaction (figure 4G and 4H). By observing the colocalizations between N and truncated Nsp3.1, we confirmed that only aa 1-235 of Nsp3.1 plays an indispensable role in the interaction between Nsp3.1 and N (figure 4D, 4E, and 4F).

Nsp3 and N formed a protein complex in vitro

Next, we sought to confirm N could directly interact with Nsp3. Since aa 1-102 of Nsp3.1 was indispensable for N-Nsp3.1 interaction, we used aa 2-111 of Nsp3.1 instead of Nsp3.1 to check the interaction in vitro. We co-expressed aa 2-419 of N and aa 2-111 of Nsp3.1 proteins in the bacteria, and the two proteins were purified together. Gel filtration analysis showed aa 2-419 of N and aa 2-111 of Nsp3.1 migrated together, and their molar ratio in the protein complex is close to 1. Through the conversion of elution volume to the approximate molecular weight, at least two aa 2-419 of N proteins could be in the fraction of complex peak, indicating that the interaction sites between Nsp3 and N should be different from the sites for the formation of the oligomer of N and that of Nsp3 (figure 5A), and the recognition of replicase complex on N through Nsp3 should not disturb the structure of N oligomers. Consistent with our IP results, Partial (figure 4B) or complete (figure 4C) deletion of CTD retained the interaction with Nsp3.1.

Sequence analysis indicated that 43 basic amino acids at N-terminus of N were likely dispensable for the interaction if the N-Nsp3.1 interaction depended on the attraction between the acidic and basic domains. Indeed, gel filtration analysis confirmed that the deletion of 43 aa did not impact the interactions and the molar ratio of the interaction (figure 5E and 5F).

The interaction between Nsp3 and N protein played an essential role in the replication and transcription of viral genomic RNAs

As the direct interacting protein with viral genomic RNAs, N protein composed the nucleocapsid structure wrapping viral RNA and thus could be involved in viral replication and transcription^{5, 15}. Nsp3 is processed from the viral polyprotein, which composed RTC and thus it could also join in the viral replication and transcription. Although both could play important roles in viral replication and transcription, whether the association of RTC and N of SARS-CoV-2 was essential for viral replication and transcription was not defined.

We investigated whether inhibition of the interaction between Nsp3 and N could influence the replication and transcription of viral genome. Firstly, we determined whether the interaction could be inhibited. To this end, we constructed the N or Nsp3.1 fused with Nuclear Localization Sequence (NLS). Next, we investigated whether NLS-N or NLS-Nsp3.1 could disturb the interaction between BD-N and AD-Nsp3.1 in the nucleus. Indeed, both NLS-N or NLS-Nsp3.1 could inhibit the interaction between BD-N and AD-Nsp3.1 in a dose-dependent manner (figure 6B). We also confirmed the aa 1-235 of Nsp3.1 could compete with Nsp3.1 in the interaction with N protein using co-immunoprecipitation (figure 6C and 6D).

Since the limited availability of biosafety level 3 (BSL3) laboratory, we utilized the viral replicon instead of live SARS-CoV-2 as a model to study the impact of inhibition of the interaction between Nsp3.1 and N on the viral replication (figure S3). The replicon of SARS-CoV-2 (nCoV-replicon) constructed by our lab expressed the S gene-deleted full-length RNA of viral genome. The deletion of S gene abolished the generation of viruses which raised concerns of biosafety. We replaced the coding sequence of S gene with the reporter gene firefly luciferase, and the replicon with firefly luciferase is named as Rep-Luci. The activity of firefly luciferase could reflect the level of replication and transcription of the replicon (manuscript in submission).

To investigate the role of viral proteins on viral replication and transcription, we expressed plasmids expressing viral proteins, Rep-Luci and RL-TK plasmids in 293T cells and measured the relative luciferase activities (figure 6A). Most of viral proteins promoted the activity of Rep-Luci, but Nsp3.1 inhibited the activity, indicating its potential as an inhibitor for viral replication. We further examined the inhibitory effect of aa 1-80, aa 1-101, or aa 1-235 of Nsp3 on the activity of Rep-Luci (figure 6E). All of the truncated Nsp3.1 proteins, as well as full-length Nsp3.1 protein, inhibited the replication and transcription of replicon, and the inhibitory effect of aa 1-235 of Nsp3 was in a dose-dependent manner (figure 6F). Except that the inhibitory effects of aa 1-80 and aa 1-101 of Nsp3.1 protein were comparable, the inhibitory effects increased in the order of aa 1-101, aa 1-235, and the full-length of Nsp3.1. Interestingly, the coverage of the region of Nsp3.1 of these truncated mutants was also increased in this order. The co-immunoprecipitation results showed that the interaction between aa 1-235 and N was much stronger than that between wt Nsp3.1 and N, indicating that Nsp3.1 could inhibit the function of Nsp3 independent of its interaction with N. As the truncated form of natural Nsp3 protein, Nsp3.1 could exhibit a dominant-negative effect through inactivation of the other function of wt Nsp3 protein.

As one of the largest RNA viruses, SARS-CoV-2 encodes at least 26 proteins or peptides. Except some of them may function independently, many of them should form protein complex to regulate the replication/transcription of viral genome RNA, the assembly of viral particles, escape from the recognition of host immune defense system, and other functions^{5, 16}. Protein-protein interactions play important roles in the processes mentioned above, and thus the disruption of the critical interactions could result in the inhibition of viral proliferation. The drugs developed on these targets are virus-specific, and unlikely inhibit the functions of host cells. COVID-19 has spread worldwide for more than a year, and anti-SARS-CoV-2 specific drugs were urgently needed. The new essential interactions for viral replication will be helpful for viral drug developments.

An independent intraviral protein-protein interactome of SARS-CoV-2 finished recently by Liang group uncovered 58 interactions using yeast two-hybrid and co-immunoprecipitation. Using different system, we also identified the 34 interactions which were reported by Liang group. Due to the different strategy for screening the protein-protein interactions, we identified 14 interactions which were not detected in the study of Liang group. Among the 14 interactions, the self-interaction of ORF8 and Nsp3-N interaction were as strong as some known combinations, such as Nsp10-Nsp14/Nsp16. This further indicated that the proteins may behave differently in various cell contexts, and thus the interactions that happened in mammalian cells are likely not to be detected in yeast and other cell systems. Indeed, in our previous work, we reported six novel viral protein-protein interactions of SARS-CoV, which were not uncovered in the other studies using yeast system.

Compared with other RNA viruses, SARS-CoV-2 uses a more complex mechanism to replicate and transcribe its viral genome. 16 non-structural proteins and at least one protein encoded by the ORFs in the 3’-proximal end of the viral genome were known involved in this mechanism. Non-structural proteins were processed from the same polyproteins, and thus, their coordination was likely set up while being expressed. As the main interaction protein of viral genome RNA, N protein was believed to play an important role in viral replication and transcription, despite that molecular details for this role were still obscure.

Therefore, after establishing the intraviral protein-protein interactome, we focused on the interactions between Nsps and N protein. In our interaction map, the interaction between N protein and Nsp3.1 was tested to be positive in both directions. The interactions were also confirmed by co-immunoprecipitation after the expression of two proteins in the mammalian cells and by gel-filtration using the purified proteins from bacterial cells.

If Nsp3 was the linker for N to join in the viral RTC, the disruption of this interaction would inhibit the replication/transcription of viral genomic RNAs. To reveal more details, we examined the interactions between different domains of N and Nsp3.1 proteins. We found that N interacts with Nsp3.1 through its N-terminal domain, which contains the most basic amino acids of N protein. The interaction domain in Nsp3.1 is also at its N-terminus, which contains the most acidic amino acid of Nsp3.1 protein. Our results showed the Nsp3.1-N interaction could perform in a manner of charge attraction, indicating a less site-specificity. We found the interaction between NTD and Nsp3.1 was stronger than that of N and Nsp3.1, and CTD, composed of basic residues, negatively impacted the interaction. We hypothesized that CTD itself was incompetent to interact with Nsp3.1 and its basic residues could interfere with the interaction between NTD and Nsp3.1 in a competitive manner.

Due to the limited availability of P3 lab, we used the viral replicon of SARS-CoV-2 to investigate the inhibitory effect of truncated Nsp3.1 on viral replication/transcription. As predicted, the truncated Nsp3.1, which interacted with N protein, markedly decreased the replication/transcription of the replicon. However, Nsp3.1 exhibited more substantial inhibitory effect than truncated Nsp3.1, indicating that as the truncated Nsp3, Nsp3.1 not only disrupted the interaction between Nsp3 and N, but also inhibited some unknown functions of Nsp3. In our design, Nsp3.1 and Nsp3.2 were separated at the linker between Domain Preceding Ubl2 and PL2pro (DPUP) and the ubiquitin-like domain 2 (Ubl2). The inhibitory effect of Nsp3.1 suggested DPUP and Ubl2 mutually regulated their functions in a cis manner, and trans regulator could only exert a dominant-negative regulatory effect on the fusion protein of DPUP and Ubl2. The cis mutual regulation between DPUP and Ubl2 could be the potential target for anti-SARS-CoV-2 design.

To obtain more details about the interaction between Nsp3.1 and N, We co-expressed various truncated Nsp3.1 and N proteins and purified their complex using gel-filtration. Although we obtained crystals containing various complexes, the resolutions were not good enough to determine the accurate structures. Since the interaction between Nsp3 and N protein was dependent on electrostatic forces, we hypothesized that the formation of the complex was in a dynamic process and the complex’s stability was relatively low. Therefore, it is essential to stabilize the complex’s structure using some means before taking the structure’s snapshot.

Collectively, we used a mammalian two-hybrid system to identify the intraviral protein-protein interactions. Together with the previous studies performed by other groups, we established the relative complete intraviral protein interactome. To further validate the potential of interactions as the target for antiviral drug development, we selected Nsp3-N interactions, which was not reported previously, for further investigation and confirmed that potential of Nsp3-N interaction as the target to inhibit the replication of SARS-CoV-2.

Cell culture

HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Thermo Fisher Scientific) at 37 °C with 5% CO₂.

Plasmid construction and transfection

The sequences of open reading frames (ORFs) and Nsps were amplified with primers containing/not containing a sequence of Flag or HA tag using Gold mix (TSINGKE) and cloned into the desired vectors. All clones were validated using Sanger sequencing (TSINGKE).

Hieff Trans^TM Liposomal Transfection Reagent (Yeasen) was used for transfection. One day before transfection, 1x10⁵ cells were plated in 48-well plate. One hour before transfection, the medium was replaced with DMEM without supplements. The plasmids and liposome were incubated in Opti-MEM (Thermo Fisher Scientific) for 5 min before mixed at a ratio of 1:2 (μg:μl). 20 min post-incubation, the mixtures were added in the cell culture dropwise. 6 h post-transfection, the media were replaced with a complete medium. Two days after culture, the cells were subjected to the downstream assays.

Immunoprecipitation and immunoblot analysis

For immunoprecipitation, cells were collected and lysed in 500 µl DISC IP lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol and 1% Triton X-100), supplemented with protease inhibitor cocktail (Roche, 1:100 of dilution), 1 mM PMSF, 1 mM NaVO₄. After centrifugation at 12000 g for 10 min at 4 ℃, the supernatant was recovered. 80 μl of the sample was saved as ‘Input’ control and stored at -80 ℃. The remaining supernatant was incubated with agarose conjugated with appropriate antibodies and was rotated overnight at 4 °C. The next day, the agarose was washed three to five times in DISC IP buffer for 10 min each time at 4°C. The samples bound to the agarose were eluted in 50 µl of the elution buffer (0.1 M Glycine-HCl, pH 3.5) for 5 min and then neutralized in 10 µl of neutralization buffer (0.5 M Tris-HCl, pH 7.4). All samples were stored at -80°C.

The samples’concentration was measured using BCA assay (Thermo Fisher Scientific) and bored with 2x SDS loading buffer for 5 min at 100 °C. Proteins were separated by SDS-PAGE and then transferred to Nitrocellulose (NC) membranes. Membranes were blocked in PBST (0.1% Tween-20 in PBS) containing 5% skim milk and then incubated overnight with indicated primary antibodies at 4 °C. The next day, membranes were washed five times with PBST and incubated with appropriate secondary antibodies for 45 min at room temperature. Then, membranes were washed four to five times with PBST. The final blots were scanned and quantified using Odyssey^®CLx Imaging System (LI-COR Biosciences). Primary antibodies used were anti-HA (1:1000 for WB, Biolegend), anti-Flag (1:1000 for WB, Sigma), and anti-GFP (1:1000 for WB, Proteintech). The secondary antibodies were anti-rabbit Alexa 488 and anti-mouse Alexa 594 (1:10000 for WB, LI-COR Biosciences).

Protein expression and purification

The fragment of Nsp3.1 (aa 2-243, aa 3-180, aa 3-111 and aa 106-180) and N protein (aa 2-419, aa 2-175, aa 2-203, aa 2-254, aa 2-365, aa 43-365 and aa 43-419) were cloned into a pGEX-6p-1 vector with GST tag and a pRSFDuet-1 vector with a 6xHis tag and a PreScission protease site (LEVLFQ’GP) at the N-terminus, respectively. The Nsp3.1-N protein complex was co-expressed in Escherichia coli BL21 (DE3). Briefly, the overnight cultures were transferred into fresh LB medium containing 50 μg/mL kanamycin and 100 μg/mL ampicillin, and induced with 0.1 mM IPTG when OD600 reached to 0.8 and cultured at 20℃ about 16 h. The cells were harvested by centrifugation, and the pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol and 5mM MgCl₂). The cells were then disrupted by the high pressure cracker (UH-24, Union-biotech), and cell debris was removed by centrifugation. The supernatant was mixed with glutathione Sepharose-4B beads (GE Healthcare), and rocked for 2 h at 4°C. Subsequently, the glutathione Sepharose-4B beads were transferred into a column and washed with lysis buffer about 10 volumes. Then, the protein was eluted with lysis buffer containing 15 mM reduced glutathione. The protein products were digested with PreScission protease and dialysis against reduced glutathione in lysis buffer to rebind the glutathione Sepharose-4B beads. The Nsp3.1-N protein complex was mainly collected in flow-through sample, and maybe few GST tag contaminant could be detected. Finally, the Nsp3.1-N protein complex was further purified and analysed by gel-filtration chromatography. The fractions were collected and subjected to SDS-PAGE, followed by coomassie blue staining.

Mammalian two-hybrid and dual-luciferase reporter assays

The details of the assay were described previously¹⁵. In brief, cells in each well of 48-well plate were transfected with 300 µg DB fusion genes in pM, 300 µg AD fusion genes in pVP16, 100 µg pG5-Luc as reporter construct and 100 µg pRL-TK (Promega) as an internal control.

48 hours post-transfection, the cells were washed with PBS and lysed in 100 µl of passive lysis buffer (PLB). 20 µl of cell lysate from each sample was subjected to Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The values of firefly luciferase and renilla luciferase were measured in Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek) and the ratios of the two luciferases’ values were calculated to obtain the relative luciferase activity. A total of 784 assays, 28 x 28, were performed and each assay was repeated 3 times at least. The combination of pM and AD fusion genes in pVP16 and that of pVP16 and DB fusion genes in pM were used as negative controls.

Immunofluorescence

HEK293T cells were seeded on coverslips in 24-well plate. 48 h post-transfection with indicated plasmids, cells in each well were washed with 200 μl PBS and fixed with PBS containing 4% paraformaldehyde at room temperature for 15 minutes. Then cells were washed once with PBS and permeabilized with PBS containing 0.1% Triton X-100 at room temperature for 10 minutes. After being washed with PBS and PBST (PBS containing 0.1% Tween), cells were incubated in 500 µl of PBST containing 5% goat serum (blocking buffer) for 1 hour and then left in a blocking buffer containing indicated antibodies overnight at 4 °C. The next day, the cells were washed 4 times in PBST at room temperature for 10 minutes each time and then incubated in a blocking buffer containing appropriate secondary antibodies for 1 h. After being washed three times with PBST and one time with PBS, the cells were stained with DAPI solution (1 μg/ml DAPI in PBS) for 5 min and left in PBS. The coverslip was mounted on slides with ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). The slides were observed under Nikon Eclipse Ti2E (Tokai Hit STX stagetop incubator).

RNA extraction and Reverse Transcription

TRIzol^® Reagent (Thermo Fisher Scientific) was used to extract total RNA from cells. Briefly, 0.5 ml of TRIzol reagent was added to cells in 6 cm plate. After 5 min of incubation, 0.1 ml chloroform was added to the lysate. The sample was mixed thoroughly and centrifuged for 15 min at 12,000 x g at 4 °C. The clear aqueous phase was recovered and mixed with 0.25 ml of isopropanol. After 10 min of incubation at 4 °C, the RNA was precipitated using 10 min of centrifugation at 12,000 x g at 4 °C. The RNA pellet was washed once with 70% ethanol and dissolved in RNase-free water after air-dry. 1 μg of total RNA was reversely transcribed with oligo(dT) primer using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa).

Statistics

Two-tailed Student's t-test was used to analyse the significance of the differences between two groups. Results were considered significant when p-value was less than 0.05.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

Conflict of interest

The authors declare that they have no conflict of interest.

Authors’ contributions

JAP and XXP conceived the ideas and designed the experiments. XXP and JAP wrote the paper. All authors performed experiments or data analysis.

Acknowledgements

We thank Prof. Sheng-Ce Tao (Shanghai Jiaotong University) for providing the cDNAs of SARS-CoV-2. This work was supported by grants (Natural Science Foundation of China #31971161 and #31900546); Natural Science Foundation of Guangdong 2019A1515011332; Shenzhen Science and Technology Program (JCYJ20190807160615255 and JCYJ20190807153203560).

Aiswarya, D. et al. Use of Remdesivir in Patients with Covid-19 on Hemodialysis- a Study of Safety and Tolerance. Kidney Int Rep (2020).
Ho, D. et al. Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization. Res Sq (2021).
Widge, A.T. et al. Durability of Responses after SARS-CoV-2 mRNA-1273 Vaccination. N Engl J Med 384, 80-82 (2021).
Lee, W.S., Wheatley, A.K., Kent, S.J. & DeKosky, B.J. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nat Microbiol 5, 1185-1191 (2020).
Chen, Y., Liu, Q. & Guo, D. Emerging coronaviruses: Genome structure, replication, and pathogenesis. J Med Virol (2020).
Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature (2020).
Chan, J.F. et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 9, 221-236 (2020).
Wang, C., Horby, P.W., Hayden, F.G. & Gao, G.F. A novel coronavirus outbreak of global health concern. Lancet 395, 470-473 (2020).
Ke, M. et al. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex. Virus Res 167, 322-328 (2012).
Wang, Y. et al. Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis. J Virol 89, 8416-8427 (2015).
Chen, Y. et al. Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS Pathog 7, e1002294 (2011).
Samavarchi-Tehrani, P. et al. A SARS-CoV-2 – host proximity interactome. 2020.2009.2003.282103 (2020).
Gordon, D.E. et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing. bioRxiv (2020).
Li, J. et al. Virus-Host Interactome and Proteomic Survey Reveal Potential Virulence Factors Influencing SARS-CoV-2 Pathogenesis. Med (N Y) 2, 99-112.e117 (2021).
Pan, J. et al. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV replication. PLoS One 3, e3299 (2008).
Thoms, M. et al. Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369, 1249-1255 (2020).
Benedetti, F. et al. Emerging of a SARS-CoV-2 viral strain with a deletion in nsp1. J Transl Med 18, 329 (2020).
Klemm, T. et al. Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2. Embo j 39, e106275 (2020).
Peng, Q. et al. Structural and Biochemical Characterization of the nsp12-nsp7-nsp8 Core Polymerase Complex from SARS-CoV-2. Cell Rep 31, 107774 (2020).
Chen, J. et al. Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex. Cell 182, 1560-1573.e1513 (2020).
Alkhansa, A., Lakkis, G. & El Zein, L. Mutational analysis of SARS-CoV-2 ORF8 during six months of COVID-19 pandemic. Gene Rep 23, 101024 (2021).
Su, Y.C.F. et al. Discovery and Genomic Characterization of a 382-Nucleotide Deletion in ORF7b and ORF8 during the Early Evolution of SARS-CoV-2. mBio 11 (2020).
Lu, X., Pan, J., Tao, J. & Guo, D. SARS-CoV nucleocapsid protein antagonizes IFN-beta response by targeting initial step of IFN-beta induction pathway, and its C-terminal region is critical for the antagonism. Virus Genes 42, 37-45 (2011).
Kang, S. et al. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites. Acta Pharm Sin B 10, 1228-1238 (2020).
Cong, Y. et al. Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle. J Virol 94 (2020).

Due to technical limitations, table 1 is only available as a download in the Supplemental Files section.

FigS1.png
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 labeled in the diagram.
FigS2.png
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.
FigS3.png
The plasmid map of 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.
Table1.png

PDF

Journal Publication

published 22 Jul, 2021

Read the published version in Cell & Bioscience →

Editorial decision: Major revision
11 Apr, 2021
Review #4 received at journal
11 Apr, 2021
Review #3 received at journal
11 Apr, 2021
Review #1 received at journal
25 Mar, 2021
Review #2 received at journal
25 Mar, 2021
Reviewer #4 agreed at journal
21 Mar, 2021
Reviewer #3 agreed at journal
20 Mar, 2021
Reviewer #2 agreed at journal
16 Mar, 2021
First submitted to journal
16 Mar, 2021
Editor assigned by journal
16 Mar, 2021
Reviewers invited by journal
16 Mar, 2021
Reviewer #1 agreed at journal
16 Mar, 2021
Submission checks completed at journal
16 Mar, 2021
Editor invited by journal
16 Mar, 2021

You are reading this latest preprint version

Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Discussion

Materials And Methods

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1