Inferring the potential effect of CIGB-300 treatment on SARS-CoV-2 virus infection based in previous results.
CIGB-300 could alter N-protein localization and its RNA binding capacity
A sequence alignment of N protein from SARS-CoV and SARS-CoV-2 viruses show a high similarity (Fig. 1). N Protein consists of two structural domains (Fig. 1): the N-terminal RNA-binding domain (RBD) (residues 41–186), and the C-terminal dimerization domain (residues 258–361). The rest of the protein is highly disordered (35).
Surjit et al. (37) predicted SARS-CoV N-protein to be heavily phosphorylated. Thr116 and Ser251 were noted as putative phospho-acceptors for CK2 (see Fig. 1), though neither has been corroborated experimentally. We collected a total of 33 phosphorylation sites for SARS-CoV-2 N protein (see Fig. 1 and Additional file 1: Table S1) from four recent mass spectrometry studies (6, 16, 17, 36). Davidson et al. (36) reported two putative CK2 sites at Ser2 and Ser78. Hekman et al. (16) found Ser23 and Ser410 to be phosphorylated by CK2.
Bouhaddou et al. (6) analyzed the impact of phosphorylation in the N-protein surface charges by a 3D structural model of the RNA-binding domain. These changes may modulate the function of N-protein by regulating its RNA binding capacity. One of the phosphorylation sites responsible for these charge changes is the Ser78 (see Fig. 1), a CK2 phospho-acceptor site according to Davison et al. (36). Binding of CIGB-300 to Ser78 would interfere with N-protein RNA binding ability.
On the other hand, N protein is reported to be mainly located in the cytoplasm (37–39). However, a localization analysis of N-expressing cells treated with four different phosphorylation inhibitors found a significant fraction of N protein localized in the nucleus of cells treated with CDK or CK2 inhibitors (37). Additionally, in cell infected by BCoV, CIGB-300 bound N protein, downregulated its expression and significantly reduced the accumulation of viral proteins in the cytoplasm(15).
Bouhaddou et al. (6) found CDK activity to be significantly reduced by SARS-CoV-2 infection while CK2 activity is significantly increased. Consequently, inhibition by CIGB-300 of N protein phosphorylation sites may alter, at least in part, its cytoplasmic localization. Hence, the use of CIGB-300 in Covid-19 patients would interfere the N protein role in viral cell cycle in infected cells as its function in particle assembly happens in cytoplasm.
CIGB-300 could bind ORF6 C-terminus and restore IFNs signaling
One important element of innate immune response to virus infections is the activation of antiviral genes as a consequence of interferon production. After activation of receptors by type I interferons, STAT1 is phosphorylated and forms a complex with STAT2 and IRF9 (40). This complex exposes a nuclear localization signal (NLS) that is bound by KPNA1, and as a last step before entering the nucleus KPNB1 binds KPNA1 and chaperons the complex through the nuclear pore (Fig. 2A) (41).
Several groups have attributed an immune response antagonistic effect to Orf6 protein (41–43). In SARS-CoV experiments, Frieman et al. (41) reported that Orf6 interferes with host immune response by antagonizing STAT1 function. Orf6 binds karyopherin alpha 2 (KPNA2) and retains it in the ER/Golgi membrane. KPNB1 is also retained as it binds KPNA2. In this way, the chaperon function of KPNB1 through the nuclear pore is interfered, and STAT1 signaling is interrupted (Fig. 2B).
Frieman et al. (41) also found that the C-terminal 10 amino acids of SARS-CoV Orf6 are responsible for KPNA2 binding. In Fig. 3 we show the residues of Orf6 involved in the SARS-CoV mutants they generated, Orf649 − 53Ala, Orf654 − 58Ala and Orf659 − 63Ala (author’s nomenclature), by replacing amino acids 49–53, 54–58 and 59–63 with alanines, respectively. The last two mutants, Orf654 − 58Ala and Orf659 − 63Ala, comprising the ten C-terminal amino acids, did not retain KPNA2 and as consequence, STAT1 function was unaffected. The first mutant Orf649 − 53Ala was still able to retain KPNA2. So, the last ten aminoacids were responsible for KPNA2 binding and, as consequence, for KPNB1 recruitment.
Recently Lei et al. (44) carried a similar mutation study of SARS-CoV-2 Orf6 protein. They generated three different mutants; M1, M2 and M3 (author’s nomenclature); by replacing aminoacids 49–52 (YSQL), 53–56 (DEEQ) and 57–61 (PMEID) by alanines, respectively (Orf6 of SARS-CoV-2 lacks the last two amino acids present in SARS-CoV protein). As expected, they obtained similar results: mutant M1 perturbs interferon stimulation as the wild type does, while mutants M2 and M3 lack the inhibitory effect.
In Fig. 3 we show an alignment of Orf6 protein sequences from SARS-CoV and SARS-CoV-2 viruses. The region between amino acids 50–53 with the sequence SELD in SARS-CoV protein and sequence SQLD in SARS-CoV-2, both match the CK2 substrate motif. Additionally, this site in SARS-CoV-2 was experimentally found to be phosphorylated, and predicted by computer analysis to be a phospho-acceptor site of CK2 (17). Ser50, as a CK2 phospho-acceptor site, could be bound by CIGB-300. Mutant M2 of Lei et al. (44) include Asp53 residue at position + 3 relative to Ser50, and this position is known to be important for the recognition of CK2. Therefore, we strongly suggest that the possible binding of CIGB-300 to this phospho-acceptor motif would interfere the interaction of Orf6 C-terminus with KPNA2; avoiding its retention in the ER/Golgi membrane, without interfering KPNA2 chaperon activity of carrying STAT1 complex to the nucleus (Fig. 2C). In this regard, CIGB-300 could exhibit an effect that other CK2 antagonists that target CK2 won’t.
Interfering NUP98 hijacking by CIGB-300 via interaction with Orf6 C-terminus
We analyzed proteomic expression data from Bojkova et al. (20) and found B23 exhibit the highest positive correlation with the expression profile of viral proteins (Additional file 2: Fig. S1). SARS-CoV virus N protein was found to interact with B23 protein (45). Despite that, Gordon et al. (5) did not reported a direct interaction of B23 with viral proteins. Looking for indirect interactions, we intersected the interactors of B23 with the 322 proteins found by Gordon et al. (5) to interact with viral proteins. A total of 21 host proteins resulted from this intersection, among which Nuclear Pore Complex protein 98 (NUP98) shown up as the only one that interact with Orf6, the viral protein with the highest expression correlation to B23.
Bouhaddou et al. (6) determined that phosphorylation at Ser888 of NUP98 increased during viral infection. The sequence around Ser888 is DSDEEE, which fulfills the phospho-acceptor motif of CK2. Additionally, Franchin et al. (46) found the phosphorylation of Ser888 to be altered by a CK2 inhibitor (according to data downloaded from PhosphositePlus web site). NUP98 is part of the Nuclear Pore Complex, responsible for the transport of biomolecules between the nucleus and cytoplasm. Bouhaddou et al. (6) suggested that the SARS-CoV-2 infection-induced phosphorylation of NUP98 may prevent export of mRNAs through the nuclear pore, a similar mechanism to those used by other viruses to increase the translation of viral RNA in the cytoplasm. Binding of CIGB-300 to Ser888 phospho-acceptor site of NUP98 could prevent its phosphorylation and restore host mRNA translocation to cytoplasm.
Also, Gordon et al. (5) found that Met58 and acidic residues Glu55, Glu59 and Asp61 are highly conserved in Orf6 homologs and are part of a putative NUP98/RAE binding motif. Miorin et al. (47) found that SARS-CoV-2 infection blocks the nuclear translocation of STAT1 and STAT2. Orf6 exerts this anti IFN-I activity by hijacking NUP98. Orf6 directly interact with NUP98 at the Nuclear Pore Complex(NPC) via its C-terminal end. A Met58Arg mutant in Orf6 C-terminal region impairs this interaction and abolish the IFN-I antagonistic effect (47).
The Orf6 interactions with KPNA2 and NUP98 have been both reported to interfere with IFN signaling. In both cases the C-terminal domain of Orf6 was responsible for the interaction, mutations in this region abolished the anti-IFN activity. The binding of CIGB-300 to the CK2 phospho-acceptor site Ser50 in Orf6 could impair the interaction with both KPNA2 and NUP98 and in some extend restore IFN signaling.
CIGB-300 downregulate host proteins phosphosites consistently activated by SARS-CoV-2
We now compare the phosphoproteomics studies of SARS-CoV-2 infection in Vero E6 (6), Caco-2 (17), iAT2 (16) and A549 (18) cell lines with that of Perera et al. (10) on CIGB-300 kinase antagonistic effect in H125.
First, we combined results of the four studies at the level of phosphorylation sites and found a total of 8642 different sites that were upregulated in at least one of the studies (Venn diagram in Fig. 4A). As noted by Hekman et al. (16), there are few proteins differentially regulated that coincide in all the four studies. Indeed, we found only six phosphosites that were upregulated by SARS-CoV-2 infection in the four cell lines.
Next, we intersected the data on SARS-CoV-2 infection with that of phosphorylation sites down regulated by the treatment of H125 cell line with CIGB-300, resulting in a total of 364 sites (see Fig. 4B). Of the six sites that were found upregulated in the four phosphoproteomics studies, half were downregulated by CIGB-300. These three sites, MATR3_S188, SQSTM1_S272 and DIDO1_S1456, have in common to have tens of Phosphorylation sites. We envisage that these three phospho-acceptor sites, can be targeted by a CIGB-300 treatment in Covid-19 patients.
MATR3 is a nuclear matrix protein with 36 phosphosites according to UniProt annotations. MATR3 plays multiple functions in DNA/RNA processing, it contains two RNA recognition Motifs and two Zinc Finger domains. It was proposed to stabilize mRNA species, to play a role in the regulation of DNA virus-mediated innate immune response (48) and to be associated to splicing regulation (49). In HIV-infected cells, Sarracino et al. (50) found MATR3 to be essential for RNA processing. MATR3 phoshorylation was found to greatly enhance its DNA binding ability (51, 52). It is well documented its implications in Amyotrophic lateral sclerosis (53), a disease causing muscle weakness and respiratory failures, symptoms common in Covid-19 patients. CIGB-300 interference on virus-infection induced phosphorylation of MATR3 may play a role diminishing its effects in immune response attenuation and its implications in viral RNA processing.
SQSTM1 exhibit several phosphorylation sites, of these, Ser272 is the only one significantly activated by Sars-CoV-2 in the four phosphoproteomic studies. Zhang et al. (54) found that phosphorylation of SQSTM1 at Thr269 and Ser272 by MAPK13 promotes the microaggregates transport to the microtubule organizing center (MTOC) to form aggresomes which are later degraded through autophagy. Gao et al. (55) also showed that SQSTM1 phosphorylation increases its ability to sequester ubiquitinated proteins into aggresomes playing an important role in aggresome formation. Stukalov et al. (18) revealed significant reduction of autophagy flux by ORF3 which combined with the augmented microaggregates transport due to SQSTM1 phosphorylation conduces to the accumulation of aggresomes.
Several studies have reported the role of SQSTM1 accumulation and aggresome formation in lung related diseases. Tran et al. (56) demonstrated the role of aggresome formation induced by cigarette smoke in chronic obstructive pulmonary disease (COPD). They found a significant higher accumulation of SQSTM1 in smokers as compared to nonsmokers, and an increased severity of COPD. Wu et al. (57) found that the accumulation of SQSTM1 plays a critical role in airway inflammation induced by nanoparticles.
Cystic fibrosis (CF), is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in defective autophagy, causing the accumulation of CFTR containing aggregates (58). SQSTM1 knockdown favoured the clearance of defective CFTR aggregates (59).
Inhibition by CIGB-300 of SQSTM1 phosphorylation at Ser272 may reduce the accumulation of aggresomes and this way attenuates lung inflammation and fibrosis induced by viral infection.
DIDO1 (death inducer-obliterator 1 or death-associated transcription factor DATF1) is a protein involved in apoptosis and have been also implicated in the progression of several type of cancer (60–63). DIDO1 possess 92 phosphosites, according to data we downloaded from Phosphosite. Of these sites we found 16 that match the CK2 phospho-acceptor motif described by Pinna (64). It is not clear the implications of DIDO1 in the course of viral infection, but it is known the induction of apoptosis by viral proteins and then DIDO1 may be activated by apoptosis pathway through phosphorylation. CIGB-300 may interfere this activation.
CIGB-300 at early Stage of SARS-CoV-2 Infection
We examine kinase activity from the earliest stages of the viral infection by analyzing phosphoproteomics data of Bouhaddou et al. (6) at 2h and 4h time points, and from Hekman et al. (16), at 1h and 3h time points.
GSEA analysis with proteins sets ranked by phosphorylation changes was performed to identify enriched REACTOME pathways. In Fig. 5A we show the plots of the normalized enrichment scores (NES) vs. FDR q-values and Nominal p-values of Reactome pathways. The most significant pathways (FDR < 5%) at 1h are predominantly down regulated (negative NES) while at 2h and 3h are up regulated (positive NES). Figure 5B shows the heat map of statistical significant pathways at each time point. After one hour of infection we observed a clear initial inhibition of host protein synthesis machinery, reflected in the inactivation of several phosphorylation sites of proteins involved in RNA metabolism events, such as “mRNA Splicing” and the “Pre-processing of capped intron containing mRNA” (Fig. 5B, light-green color in the heat map). This inactivation is reverted by the activation of these same biological events at 2h and 3h (Fig. 5B and 5E). The phosphosites listed are those belonging to proteins from the core enrichment set (CES) of each enriched pathway and those that were identified to be inactivated by CIGB-300 in Perera et al. (10). The Venn diagram in Fig. 5C shows the comparison of phosphosylation sites from proteins in “mRNA Splicing/Processing of capped intron containing pre-mRNA” pathway that were regulated at 1h, 2h, 3h after infection or inhibited by CIGB300 action. Of those, SRSF1_S199 was the only site up-regulated at 2h and 3h. SRSF1_S201 was up-regulated at 3h as well as some other SRSF’s proteins phosphosites (Fig. 5C). The interaction networks of proteins in these CESs for each time point are shown in Fig. 5D. SRSF proteins are highly connected and predominant in the three networks.
SRSFs are RBP splicing factors that belong to the family of S/R rich proteins. Rogan et al. (65) proposed a molecular mechanism for viral-RNA pulmonary infections based on protein expression and RBP binding site pattern analysis. They compared the distribution of RBP binding motifs in several viral genomes including SARS_CoV-2, Influenza A, HIV-1 and Dengue. These authors identified strong RBPs binding sites in SARS-CoV-2 genome. After infection, as the number of SARS-CoV-2 genomes increase, the proportion of SRSFs bound to viral genome versus host transcriptome also increases. As the virus replicates in cytoplasm, newly synthetized SRSF1 molecules are bound by viral RNA and retained there, resulting in the formation of R-loops in the nucleus due to a reduction of RBP import. Rogan et al. (65) suggested that R-loop induced apoptosis could contribute to the spreading of viral particles to neighboring pneumocytes causing a deterioration of lung functions.
Phosphorylation plays an important role in SRSF proteins function. SRPK1 kinase was shown to phosphorylate multiple serine residues in SR rich domain of SRSF1 (66, 67), promoting its nuclear import where it plays an important role in RNA stability (68) and alternative splicing (69). CK2 was found to be the major kinase that phosphorylate SRPK1 and this phosphorylation occurs mainly at Ser51 and Ser555, resulting in 6-fold activation of the enzyme (70). After SARS-CoV-2 infection of AT2 cell, Ser51 is activated at 3h and 6h (16).
Figure 6 show the expression profile of CK2 and the levels of phosphorylation of SRPK1 S51 site, according to data from Hekman et al. (16). A clear correlation is observed between the amount of CK2 kinase and the phosphorylation activation of this phospho-acceptor site, an additional argument supporting the role of CK2 on the activation of SRPK1 during SARS-CoV-2 infection.
These results are consistent with previous reports predicting an extensive reshaping of splicing pathways by SARS-CoV-2 infection (17, 20). SRSF1 is an important element of this splicing machinery that is clearly used by SARS-CoV-2 for its own replication and translation.
The increasing amount of SRSF1 bound to viral genome as the infection progress is a clear indication of its role in viral RNA processing. Phosphorylation is an important mechanism that control SRSF1 function.
Taking all these together, we suggest that CIGB-300 intervene SRSF1 role in SARS-CoV-2 protein synthesis interfering its phosphorylation by SRPK1 kinase.
Infection-induced protein-protein interactions could be perturbed by CIGB-300
Next, we compared the host-viral PPIs reported by Gordon et al. (5) with phosphoproteomic data from Perera et al. (10) on the identification of CK2 substrates significantly inhibited by the CIGB-300. Figure 7 show virus-host interactions from Gordon et al. (5) in which host proteins contain phospho-acceptor sites that were inhibited by CIGB-300 treatment (highlighted in yellow). In this network several proteins are relate to RNA processing and transcription (LARP1, LARP7, LARP4B), supporting the results already mentioned. Binding of CIGB-300 to phospho-acceptor sites of host proteins inhibiting its phosphorylation may perturb the binding by viral proteins and consequently the viral life cycle.
Evaluating how CIGB-300 may interfere host-host protein interactions implicated in virus-induced mechanisms, we found that 68 proteins (SC2_300 set from now on) have activated phospho-acceptor sites in at least two of the four phosphoproteomic studies, which were inhibited by CIGB-300 (see Additional file 3: Table S2). The PPI network built with these proteins is shown in Fig. 8. A majority of the nodes in the network are interconnected indicating potential functional relations among them of biological significance. Proteins are grouped by mRNA metabolism, Cell Cycle, and Selective Autophagy pathways, identified as significant by a Reactome enrichment analysis (see Additional file 4: Table S3). The five proteins with higher degree are also highlighted. Among them are HNRNPA1, HSPB1, SRRM2, and SRRM1, which are implicated in mRNA metabolism, corroborating the potential impact of CIGB-300 in viral replication and transcription. The fifth protein was B23/NPM1, identified as a major target of CIGB-300 in cancer cells, but also as a relevant target for antiviral therapies (11–13).
In this network HSPB1 heat shock protein (alias HSP27) is one of the highest degree nodes. HSPB1 was found to be overexpressed in idiopathic pulmonary fibrosis (IPF) patients. It activates pro-fibrotic mechanisms and consequently has been suggested as a target to treat IPF (71, 72). In tumor cells, Ivermectin inhibits the phosphorylation of Ser78 and Ser82 of HSP27, while Ser15 is only slightly inhibited (73). Also Ivermectin have shown to be on inhibitor of SARS-CoV-2 with a significant reduction of viral RNA levels (74) and increase the clinical recovery of mild and severe Covid-19 patients (75, 76). SARS-CoV-2 activates HSPB1 Ser15 and Ser82 during infection while CIGB-300 inhibits both phospho-acceptor sites (10). This is an additional argument in favor of using CIGB-300 in Covid-19 patients aiming to reduce pulmonary lesions as it was evidenced in a phase I/II clinical trial (14).
Human phenotypes involving kinase activity induced by SARS-CoV-2, potentially targeted by CIGB-300
We build a network with the top 20 human phenotypes most enriched in the set SC2_300, using GeneCodis tool (see Fig. 9 and Additional file 5: Table S4). The network can be divided in two main subnetworks, one related to muscular disorder phenotypes that include Paralysis, Distal muscle weakness, Rimmed Vacuoles, Mildly elevated creatine kinase and Fatigue. The second subnetwork groups phenotypes related to respiratory(Exertional dyspnea, difuse alveolar hemorraghe), bleeding (Metrorrhagia, oral cavity bleeding) and coagulation disorders(Disseminated intravascular coagulation).
The phenotypes in the first subnetwork are all associated to HNRNPA1, MATR3 and SQSTM1 genes, and have been also reported as Covid-19 symptoms (77–81). For example, elevated creatine kinase levels is associated to a poor outcome prediction (77) and persistent fatigue is a common symptom in Covid-19 patients (80). As we mentioned MATR3 and SQSTM1 possess phosphosites that were activated in all four phosphoproteomic studies we analyzed, and HNRNPA1 was the node with the highest degree in the PPI network built. Rimmed Vacuoles, the most significant of the enriched phenotypes, are found in areas of destruction of muscle fibers. Fatigue phenotype is located somewhere in the interface between the two subnetworks and is connected to the three genes mentioned, and also to the three genes that are in the core of the second subnetwork: B23/NPM1, FIP1L1 and NUMA1. The phenotypes in the second subnetwork have been all identified in Covid-19 patients (82, 83).
Table 2
Summary of main findings.
Analysis Type
|
Subject
|
Identity
|
Working Hypothesis
|
Experimental Clues
|
Reference
|
Individual CK2 sites
|
Viral proteins
|
N
|
CK2 phosphosite inhibition/blockage by CIGB-300 impairs viral replication and IFN signaling
|
Interaction, co-localization, N mRNA and protein expression inhibition by CIGB-300 in a subrogate model
|
(15)
|
Individual CK2 sites
|
Viral proteins
|
ORF6
|
CK2 phosphosite inhibition/blockage by CIGB-300 impairs IFN signaling
|
Additive/Synergistic profile of CIGB-300 plus IFN alpha
|
Unpublished
|
Co-Expression and network propagation
|
Host and Viral Proteins
|
NUP98, ORF6, B23
|
CK2 phosphosite inhibition/blockage by CIGB-300 impairs IFN signaling
|
Additive/Synergistic profile of CIGB-300 plus IFN alpha (unpublished)
|
Unpublished
|
Phosphoproteome overlap
|
Host Proteins
|
MATR3, SQSTM1, DIDO1
|
CIGB-300 Multitarget effect impairing viral transcription/splicing, inflammation, immunoresponse and apoptosis
|
Pulmonary lesions resolution in CT Phase I
|
(14)
|
Kinase activity profiles
|
Host proteins
|
SRPK1 (SRSF1,2,6,10)
|
CIGB-300 impairs Viral RNA splicing
|
None, to be evaluated in preclincial seetings
|
NA
|
PPIs vs H125 Phosphoproteome
|
Host and Viral Proteins
|
Several proteins (see text and Fig. 7) [LARP1,LARP7,LARP4B]
|
CIGB-300 impairs Viral RNA processing and transcription
|
None, to be evaluated in preclincial seetings
|
NA
|
SARS-CoV-2 vs H125 Phosphoproteome overlap, Network and Enrichment analysis
|
Host proteins
|
Several proteins (see text and Fig. 8)
[HNRNPA1, HSPB1, SRRM2, SRRM1, B23]
|
CIGB-300 Multitarget effect impairing on mRNA metabolism cell cycle and Autophagy pathways
|
Pulmonary lesions resolution in CT Phase I
|
(14)
|
Human Phenotypes
|
Host proteins
|
HNPRNPA1, MATR3, SQSTM1, B23, FIP1L1, NUMA1
|
CIGB-300 might relief COVID19 clinical symptoms
|
To be evaluated in CT Phase II
|
NA
|