key: cord-0844943-pjsdjdma
authors: Ramón, Ailyn C.; Pérez, George V.; Caballero, Evelin; Rosales, Mauro; Aguilar, Daylén; Vázquez-Blomquist, Dania; Ramos, Yassel; Rodríguez, Arielis; Falcón, Viviana; Rodríguez, María P.; Ke, Yang; Perera, Yasser; Perea, Silvio E.
title: Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection
date: 2021-06-08
journal: bioRxiv
DOI: 10.1101/2021.06.08.447588
sha: 9f119a4b2628ad740f3fd1f9231b72f14f94814c
doc_id: 844943
cord_uid: pjsdjdma

Coronaviruses constitute a global threat to human population since three highly pathogenic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have crossed species to cause severe human respiratory disease. Considering the worldwide emergency status due to the current COVID-19 pandemic, effective pan-coronavirus antiviral drugs are required to tackle the ongoing as well as future (re)emerging virus outbreaks. Protein kinase CK2 has been deemed a promising therapeutic target in COVID-19 supported by its in vitro pharmacologic inhibition and molecular studies on SARS-CoV-2 infected cells. CIGB-325 is a first-in-class synthetic peptide impairing the CK2-mediated signaling whose safety and clinical benefit have been evidenced in Covid-19 and cancer patients after intravenous administration. Here, we explored the putative antiviral effect of CIGB-325 over MDBK cells infected by bovine coronavirus (BCoV) Mebus. Importantly, CIGB-325 inhibited both the cytopathic effect and the number of plaques forming units with a half-inhibitory concentrations IC50 = 3.5 μM and 17.7 μM, respectively. Accordingly, viral protein accumulation at the cytoplasm was clearly reduced by treating BCoV-infected cells with CIGB-325 over time, as determined by immunocytochemistry. Of note, data from pull-down assay followed by western blot and/or mass spectrometry identification revealed physical interaction of CIGB-325 with nucleocapsid (N) protein and a bona fide cellular CK2 substrates. Functional enrichment and network analysis from the CIGB-325 interacting proteins indicated cytoskeleton reorganization and protein folding as the most represented biological processes disturbed by this anti-CK2 peptide. Altogether, our findings not only unveil the direct antiviral activity of CIGB-325 on coronavirus infection but also provide molecular clues underlying such effect. Also, our data reinforce the scientific rationality behind the pharmacologic inhibition of CK2 to treat coronavirus infections.

and infected with 100 µL of virus at a concentration of 70 000 TCID50/well. After 1h of 217 incubation, final volume was completed up to 500 µL and the appropriate drug's concentration 218 was maintained for 16 h and 24 h. Subsequently, the cells were washed with cold PBS three 219 times and fixed in 4% formaldehyde for 10 min at 4 °C. After permeabilization with 0.5% 220 Triton X-100 for 10 min, cells were blocked by incubation with 4% bovine serum albumin 221 (Sigma, MO,USA) in PBS for 30 min at room temperature, washed again, and incubated with 222 30 μg/mL human polyclonal anti-SARS-CoV-2 (CIGB, Cuba) for 2 h at room temperature. 223

Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, MO, USA) was 224 incubated for 1 h at room temperature and washed 3 times with PBS. Finally, AEC substrate 225 (Abcam, Cambridge, United Kingdom) was added and coverglass was mounted using 40% 226 glycerol mounting medium and analyzed using a BX43 upright microscope (Olympus, USA). 227 (Roche, Basel, Switzerland). Cellular lysates were cleared by centrifugation and 300 µg of total 234 protein were incubated with biotin-tagged CIGB-325 (100 μM) or medium alone for 30 min at 235 room temperature and added to 30 µL of pre-equilibrated streptavidin-sepharose matrix (GE 236 Healthcare, IL, USA). Following 1 h at 4 °C, the matrix was collected by centrifugation and 237 extensively washed with cold PBS 1 mM with DTT. CIGB-325 238 interacting proteins were eluted, resolved in an SDS-PAGE gel and processed as described 239

later. For in vivo pull-down assays, BCoV-Mebus infected and uninfected MDBK cells were 240 treated with biotin-tagged CIGB-325 (100 μM) or medium alone for 30 min at 37° C in 5% 241 CO2. Subsequently, cells were collected and pull-down assay was conducted as above 242 mentioned. Proteins bound to streptavidin-sepharose were eluted, resolved in a 12 % SDS-243 PAGE gel, and transferred onto nitrocellulose membranes. For western blot analysis, a human 244 polyclonal IgG anti-SARS-CoV-2 (CIGB, Cuba) and a mouse monoclonal antibody against 245 CK2α (Abcam, Cambridge, United Kingdom) were used as primary antibodies. Detection was 246 performed with peroxidase-conjugated anti-mouse IgG 1:5000 (Sigma, MO, USA) and anti-247 human IgG 1:100 (Jackson ImmunoResearch, USA). In parallel, untreated cells were subjected to the same experimental procedure to identify those proteins non-specifically bound to 249 streptavidin-sepharose matrix. 250

Proteins bound to streptavidin-sepharose matrix of both, CIGB325 treated sample and 252 background control were processed by the FASP method using Microcon 30k centrifugal 253 ultrafiltration units (Merck, Darmstadt) [29] . A volume corresponding to 4 µg of the peptide 254 mixture was separated by a Proxeon Easy-nLC System (Thermo Fisher Scientific) using an 255 RP-C18-A2 column (3 μm), 75 μm i.d. × 10 mm (Thermo Fisher Scientific) connected online 256 to a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTof-2 (Water, MA, 257 USA). Differences between groups were determined using one-way ANOVA followed by Dunnett's 297 multiple comparisons test. Analyses were performed in GraphPad Prism (v6.01) software for 298

Windows (GraphPad Software, Inc, San Diego, CA, USA). All experiments were conducted at 299 least in triplicates and differences were considered significant for a p-value < 0.05.

To test the in vitro CIGB-325 effect, we analyzed the CPE of BCoV-Mebus using a cell-based 303 assay. MDBK cells appeared as rounded and multinucleated cells within 48 h; beyond 72 h of 304 incubation a strong cell monolayer damage was observed. CIGB-325 elicited a very potent 305 inhibition of the virus CPE with an IC50=3.5 μM just some significant cytotoxicity emerges at 306 higher concentrations (CC50=150 μM) (Figure 1A ), hence indicating a selectivity index 307 (SI=42). Specificity of the CIGB-325 antiviral effect was corroborated using the F20-2 peptide 308 which contains the same cell-penetrating peptide as CIGB-325 but lacks the CK2 inhibitory 309 domain [13] . Interestingly, testing of another small molecule inhibiting CK2 activity (CX-310 4945), also reduced significantly the BCoV-Mebus CPE as well as IFN alpha-2b, which was 311 used as an antiviral drug reference ( Figure 1B) . Members of the family of proteins including actin, tubulin, myosin, heat shock protein 70 368 (HSP70), heat shock protein 90 (HSP90), tyrosine 3-monooxygenase/tryptophan 5-369 monooxygenase activation protein as well as others showed in Table S2 were identified as host 370 CIGB-325 interacting proteins. In agreement with CIGB-325 interactome from tumor cells [14, 371 35] , nucleolar protein B23/NPM1 was also identified in our study. 372

To envisage the putative biological processes that might be perturbed by CK2 targeting, 373 enrichment analysis of the CIGB-325 interacting host proteins was performed. Of note, protein 374 folding and the response to unfolded protein, cytoskeleton organization and cell cycle were 375 significantly represented above all biological processes in the interactomic profile ( Figure S4) . (Table S2) . To better understand the global putative connection 381 among the CIGB-325 interactome on infected MDBK cells, we constructed a protein-protein 382 interaction (PPI) network with the identified proteins using information annotated in STRING 383 database ( Figure 4A) . In PPI network, we detected two functional physiological complexes 384 corresponding to proteins involved in cytoskeleton reorganization and protein folding, 385 consistently with the most represented biological processes. 386

Previous reports from our group have indicated that CIGB-325 also impairs CK2 signaling by 387 direct binding to CK2 catalytic subunit in tumor cells [14, 15] . Therefore, we finally move 388 forward to determine if this direct peptide-enzyme interaction could also take place within the 389

with biotinylated-CIGB-325 followed by western blot with a commercial anti-CK2 391 monoclonal antibody, it was possible to demonstrate the presence of CK2 in the pull-down 392 fraction ( Figure 4B) . To corroborate whether this interaction could impact any of the CK2-393 mediated signaling pathways, we also evaluated the effect of CIGB-325 over the PI3K/Akt 394 pathway by measuring the downstream phosphorylation of the RPS6 protein, as biomarker of the PI3K/Akt pathway activation which in turns, represents one of the CK2 mediated signaling 396 pathways. Importantly, CIGB-325 treatment during 45 min clearly impaired the RPS6 397 phosphorylation (i.e.; at residues S235/236) at 24 h post-BCoV infection on MDBK cells 398 ( Figure 4C ). However, a significant increase in RPS6 phosphorylation was observed upon 399

BCoV-Mebus infection in these cells which reinforces the relevance of this pathway for the 400 viral replication processes in the coronavirus biology [36, 37] . 401

During the last 20 years, three zoonotic CoVs, SARS-CoV, MERS-CoV and SARS-CoV-2 403 have evidenced that novel highly pathogenic coronavirus could spread into human populations 404 [38] . Therefore, the development of an effective antiviral drug against already-known and new 405 coronaviruses that may emerge from animal reservoir hosts constitutes an urgent necessity. 406

Host-directed drugs represent a therapeutic strategy with broad-spectrum activity since viruses 407 commonly hijack cellular factors and signaling pathways for their own replication [9, 39] . were the most representative from the constructed protein-protein interaction network as well 461 as functional enrichment analysis. Interestingly, these three cellular processes are all relevant 462 for the coronavirus life cycle [12, 40, 41] . 463 Furthermore, we interrogated the CIGB-325 interactomic profile for the presence of CK2 464 substrates and found that 30% (15 proteins) corresponded to previously validated CK2 465 substrates [31, 32] . A priori, these data reinforce the notion that inhibition of the CK2-mediated 466 phosphorylation could play a central role mediating the CIGB-325 antiviral activity during 467

BCoV-Mebus infection. However, three different CK2 substrates targeted by CIGB-325 might 468 be of particular relevance concerning the antiviral activity on BCoV-Mebus infection due to its 469 already known proviral function. That is the case of B23/NPM protein, a major target for 470 CIGB-325 in solid tumor cells [42] , that plays an important role as a proviral chaperone in 471 animal and human virus infections [43] [44] [45] . Moreover, the myosin heavy chain 9 (MYL9) is 472 another bonafide CK2 substrate whose phosphorylation at the S1943 is upregulated during 473 infection of . Likewise, the heat shock protein 90 alpha 474 family class B member 1 (HSP90AB1) has been identified as a viral RNA interacting protein 475 significantly up-regulated during the SARS-CoV-2 replicative cycle in the RNA-bound 476 proteome on infected lung cells. Consistently, compounds targeting this protein showed a 477 strong inhibition of SARS-CoV-2 protein production [46] . Therefore, B23/NPM, MYH9 and 478 HSP90AB1 represent cellular CK2 substrates whose inhibition by CIGB-325 along with that 479 on viral N protein, may impact the BCoV-Mebus viral machinery eliciting antiviral activity. 480

However, our data do not preclude that other CK2 substrates unveiled among CIGB-325 481 interacting proteins might be also involved in the antiviral activity. 482

The CIGB-325 interactomic data originated from different modalities of pull-down 483 experiments here is rather linked to the ability to target the phosphoaceptor domain at the CK2 484 substrates initially described for this peptide inhibitor [13] However an alternative mechanism 485 of inhibiting the CK2-mediated phosphorylation by direct targeting the CK2 catalytic subunit 486 of the enzyme was recently described for . Importantly, our in vivo pull-down 487 experiments revealed that CIGB-325 can also interact with CK2 catalytic subunit in MDBK 488 cells as it has been previously evidenced in lung cancer and leukemia cells [14, 15] . In line 489 with the abovementioned, the treatment with CIGB-325 showed an impairment of the 490 PI3K/AKT pathway, a CK2 mediated signaling pathway, evaluated by the phosphorylation of 491 downstream signaling protein RPS6. Noteworthy, whether this dual anti-CK2 inhibitory 492 mechanism described for CIGB-325 in other cellular contexts could also be running in parallel, 493 remains to be elucidated. 494 

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Figure 1. In vitro antiviral activity of CIGB-325 against BCoV-Mebus. MDBK cells were pre-treated with 752

CIGB-325 at the indicated doses for 1 h, and virus (14 000 TCID50/well) was then added to allow attachment for 753

Afterward, the cells were incubated in the presence of the indicated drug concentration for 4 days. A. IC50 754 (left axis) and CC50 (right axis) were estimated from the fitted dose-response curves based on treatment with five 755

Crystal violet stain wells for each 756 experimental condition in the CPE inhibition assay (right section). B. Antiviral effect of positive

CX-4945) and negative controls (F20.2) were determined by cell viability assay using crystal violet stain. Cells 758 were infected as previously described and incubated with CIGB-325 (30 µM)

Uninfected cells with the vehicle were set to 100%. C. Progeny titer of the cell 760 cultures supernatants collected from CPE inhibition experiments were assessed using plaque assay

are shown as mean ± SD, n = 3. Statistically significant differences between vehicle and drug treatment 762 are represented as **p < 0.01 and *** p < 0.001 determined using one-way ANOVA

Statistically significant differences between vehicle and drug 771 treatment are represented as **p < 0.01 and ***p < 0.001 determined using one-way ANOVA followed Dunnett 772 post-test. Cell preparations for western blot and Immunocytochemistry were carried out as described in Materials 773 and Methods. A human anti-SARS-CoV-2 polyclonal antibody from one COVID-19 convalescent patient was 774 used for identification of BCoV-Mebus M and N protein

In vitro pull-779 down was performed with cellular lysates from infected MDBK cells incubated 30 min with biotin-tagged CIGB-780 325 (100 μM). Subsequently, 30 µL of streptavidin-sepharose were added to each reaction and the CIGB-325 781 interacting proteins were eluted, resolved in 12%-SDS-PAGE and subjected to western blot to identify the viral 782 N protein. For in vivo pull-down, MDBK cells were treated 30 min

subsequently lysed and processed as indicated above. PD: pull-down fractions; PASS: flow-through fractions

Representative images 785 obtained by confocal microscopy showing the co-localization of CIGB-325-F with BCoV-Mebus N protein after 786 30 min of incubation on infected MDBK cells. Red fluorescence: N protein-derived signal; Green: CIGB-325-787 derived signal

Protein-protein interaction network associated with CIGB-325 host interacting proteins on 790 infected MDBK cells and subsequent effect over CK2. A Network was generated using information gathered 791 from STRING database. Biological processes retrieved from Gene Ontology database are indicated and CK2 bona 792 fide substrates are indicated with an asterisk in red. B. In vivo pull-down was performed with biotinylated CIGB-793 325 as bait to capture CK2 in MDBK infected cells. Interacting proteins were then resolved by 12% SDS-PAGE, 794 transferred, and each fraction was inspected by western blot with the antibody against CK2α. C. Effect of CIGB-795 325 over phosphorylated and total protein levels of RPS6

After 24 h of incubation, CIGB-325 was incubated for 45 min and preparation of cell extracts for 797 western blot was carried out as referred to in Material and Methods

MDBK cells were infected 800 with (14 000 TCID50/well), 16 h and 26 h post-infection CIGB-325 (30 µM) was added. Afterward, the cells 801 were incubated for 3 days and the antiviral effect was determined by crystal violet stain. IFN-alpha 2b represents 802 the positive control and was added 1h pre-infection as earlier described

Statistically significant differences between vehicle and drug treatment are represented as **p < 0.01 and *** p < 804 0.001 determined using one-way ANOVA followed by Dunnett post-test