key: cord-1015534-c8u4bmzv
authors: Ferrucci, Veronica; Kong, Dae-Young; Asadzadeh, Fatemeh; Marrone, Laura; Siciliano, Roberto; Cerino, Pellegrino; Criscuolo, Giuseppina; Pisano, Ida; Quarantelli, Fabrizio; Izzo, Barbara; Fusco, Giovanna; Comegna, Marika; Boccia, Angelo; Viscardi, Maurizio; Borriello, Giorgia; Brandi, Sergio; Pierri, Bianca Maria; Tiberio, Claudia; Atripaldi, Luigi; Paolella, Giovanni; Castaldo, Giuseppe; Pascarella, Stefano; Bianchi, Martina; Monica, Rosa Della; Chiariotti, Lorenzo; Yun, Kyong-Seop; Cheong, Jae-Ho; Kim, Hong-Yeoul; Zollo, Massimo
title: Long-chain polyphosphates impair SARS-CoV-2 infection and replication: a route for therapy in man
date: 2020-11-18
journal: bioRxiv
DOI: 10.1101/2020.11.18.388413
sha: 3dd9b189dd7893fc61a994be490fec0d1d081ea5
doc_id: 1015534
cord_uid: c8u4bmzv

Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactions in vitro in SARS-CoV-2–infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.

Coronaviruses (CoVs) contain positive-sense, single-stranded RNA (~30 kb). Four major categories have been reported, with alphaCoV and betaCoV known to infect humans. Those that can replicate in the lower respiratory tract cause pneumonia, which can be fatal (1) , (2) , and these include severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and the new SARS-CoV-2. This last CoV belongs to the betaCoV genus (3) and has resulted in pandemic acute respiratory syndrome in humans (i.e., COVID-19 disease). This can progress to acute respiratory distress syndrome (ARDS), generally around 8 to 9 days after symptom onset (4) . Like the other respiratory CoVs, SARS-CoV-2 is transmitted via respiratory droplets, with possible faecal-oral transmission (5) .

When SARS-CoV-2 infects host cells, it replicates its genomic (g)RNA to produce smaller RNAs known as subgenomic (sg)RNAs, mostly used to synthesise conserved structural viral proteins Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) (6) .

The receptor used by SARS-CoV-2 to enter host cells is angiotensin-converting enzyme 2 (ACE2), which is mainly expressed in the lungs (7) , (8) , together with the cellular serine protease TMPRSS2 (9) .

The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; also, non-structural protein [nsp]12) is a key component of the viral replication/ transcription machinery and contributes to viral genome mutation, and hence to viral adaptation. RdRp is part of a complex machinery (i.e., RdRp/nsp7/nsp8) that is fundamental to transcriptional fidelity (proofreading) (2) . RdRp thus coordinates the replication of both the viral genome (which is used as the template for replication and transcription) and the shorter sgRNAs, according to the prevailing "leader-to-body" fusion model (10) . Of importance, RdRp mutations at position 4 14,408 in European strains of SARS-CoV-2 suggest that the proofreading activity has been affected, thus altering their mutation rates (11) .

Due to the dramatic global spread of COVID-19 and its associated high mortality rate, development of novel therapeutic strategies represents an unmet medical need. We therefore investigated the potential activities of long-chain inorganic polyphosphates (PolyPs) against SARS-CoV-2 viral infection.

The PolyPs are comprised of chains of a few to many hundreds of inorganic phosphates (Pi). They are ubiquitous, with prevalence in peripheral blood mononuclear cells and erythrocytes (12) , and subcellularly in the nucleus, cytoplasm, plasma membranes and mitochondria (13) . PolyPs are involved in of blood coagulation through activation of factor XII (14) and inhibition of the complement terminal coagulation pathway (15) . Furthermore, they are involved in chelation of calcium for bone mineralisation (16) and activation of apoptosis (12) . Furthermore, they act as 'chaperone-like' (17) , and neuronal excitability molecules (18) , (19) , (20) . Of importance, linear PolyPs have been reported to have cytoprotective and antiviral activities against human immunodeficiency virus type 1 (HIV-1) infection in vitro (21) .

We show here that long-chain inorganic PolyPs have anti-viral activities against SARS-CoV-2 in different cellular models (i.e., Vero E6, Caco2 and human primary nasal epithelial cells). We demonstrate that intracellularly, PolyPs enhance proteasome-mediated ACE2 down-regulation, and impair transcriptional activity of RpRd during its synthesis of the major structural sgRNAs and proteins (i.e., S, E, M, N). As PolyPs are already known not to be toxic, these data open the door to clinical trials with PolyPs in humans.

Anti-viral activity of linear PolyPs with average chain lengths of 15, 34, 91 Pi residues has been previously shown on HIV-infected cells (21) .

To investigate potential cytoprotective effects of long-chain PolyPs, PolyP120 was used here, initially with epithelial cells derived from an African green monkey kidney (i.e., Vero E6 cells; ATCC CRL-1586), because of their high expression of ACE2 (22) . The Vero cells were first incubated with 37.5 μM PolyP120, and after 30 min they were infected with SARS-CoV-2 viral particles (multiplicity of infection [MOI] , 0.01) obtained from a swab of an Italian patient affected by COVID-19 ( Fig. S1A ; viral sequence shown in Fig. S1B ).

Following treatment of these cells with PolyP120, there were significantly decreased expression levels of the viral N gene (as fragments N1-3) 12 h after viral infection, compared to the vehicle control (Fig. S1C) . Moreover, quantitative immunofluorescence of the same PolyP120-pretreated cells show reduced levels of the SARS-CoV-2 S-, E-and N-proteins ( Fig. S1D and E) . However, the RdRp protein was not down-regulated here ( Fig. S1D and   E) . These data suggest that the anti-viral activity of PolyP120 can be ascribed to reduced SARS-CoV-2 entry into host cells (which is mainly mediated by ACE2), and/or inhibition of RdRp transcriptional activity.

To address these hypotheses, potential binding sites for PolyP120 on both ACE2 and the viral RdRP proteins were investigated through molecular docking. These data showed the potential for binding between ACE2 and short-chain PolyPs (e.g., PolyP20), which was mostly mediated by four ACE2 amino-acid residues (Arg514, His401, His378 and Arg393; 1A ).

The colocalisation between PolyPs and ACE2 in Vero cells was then examined. For this, the Vero cells were treated with a short-chain PolyP (i.e., PolyP8) conjugated with a fluorescent dye (i.e., 4-methylumbelliferone; MU). The immunofluorescence indicated colocalisation between PolyP8-MU and ACE2 in these treated cells (Fig. 1B, Fig. S2A and   B ). Furthermore, we investigated whether the long-chain PolyP120 colocalised with ACE2.

Here, a DAPI-based approach was used to label PolyP120 (as previously described (23)), with colocalisation also seen between the intracellular PolyP120 and ACE2 (Fig. S2C ) in these Vero cells. This was thus indicative of an interaction between PolyPs and ACE2.

As a 'scaffold-like' activity has been previously described for PolyPs (17), we investigated whether ACE2 protein expression can be modulated by PolyPs. Due to the high ACE2 expression levels in nasal epithelial cells (24) , we used human primary cells obtained from nasal brushings of healthy subjects (Fig. S2D) . These were treated with increasing concentrations of PolyP120. Immunoblotting demonstrated a dose-dependent reduction in ACE2 protein levels in these cells after 24 h treatments with PolyP120 (4.2-112 μM) (Fig.   1C ). In Vero cells, the addition of 37.5 μM PolyP120 also showed a time-dependent ACE2 down-regulation (Fig. 1D) . Of interest, the decrease in ACE2 protein levels was dependent on the number of phosphates in the PolyPs (Fig. 1E) . Thus, to reduce the levels of ACE2 in Vero cells, they needed to be treated with PolyPs with >8 phosphate residues. Furthermore, longchain PolyPs treatment showed greater accumulation of ACE2 upon addition of the proteasome inhibitor MG-132, compared to the vehicle controls (Fig. 1F) , thus suggesting that the PolyPs-mediated downregulation of ACE2 occurs via proteasome. To further validate that PolyPs affect the ubiquitin-proteasome system, PolyP120 was shown not only to increase the levels of ACE2 in presence of MG-132 ( Fig. 1G ) but also to reduce its levels upon addition of the protein synthesis inhibitor cycloheximide (Fig. 1H) . These data are further confirmed using transfection of an HA-ubiquitin plasmid construct in HEK293 cells (Fig. 1I,   Fig. S2E ).

Altogether, these data indicate that PolyP120 can bind to ACE2 and enhance its proteasome-dependent degradation, thus indicating its anti-viral mechanism of action. Of importance, the potential cytotoxicity of PolyPs were also evaluated when administered at different doses to the human primary cells, using 'real-time' cell proliferation assays (i.e., cell index). These data showed that only the highest dose of 112 μM PolyP120 had cytotoxic effects, as seen by decreased cell proliferation rate (Fig. S2F) . However, Caspase 3 enzymatic assays did not show activation of the apoptotic cascade by PolyP120 (Fig. S2G) , thus further confirming the lack of toxic effects of PolyP120.

To gain further insight into the anti-viral mechanisms of PolyPs in their inhibition of viral gene transcription, we investigated potential interactions with RdRp. Molecular docking analysis showed putative binding sites for PolyP15 on the RdRp protein ( Fig. S3A and Table   1 ). Further docking studies performed with RdRp in its active replicating form (i.e., RdRp/nsp7/nsp8 complex; Table 1 Whether the downregulation of the viral genes mediated by PolyPs was due to inhibition of the viral transcription process was also investigated. Thus, we used total RNA samples from oropharyngeal swabs of eight Italian patients with COVID-19 ( Table 2 ) and examined modulation of amplification of the viral N1-3 gene fragments in the presence of PolyP120 (Fig. S3D) . These data showed increase in Ct values for both N1 and N3, and a total inhibition of the N2 gene fragment (i.e., undetermined Ct) when the reactions were performed in presence of 37.5 μM PolyP120 (Fig. S3E) . Of interest, addition of PolyP120 did not alter amplification of the N gene fragments when external S RNA controls (N1/2/3-O 2methyl) were used (Fig. S3F) . These in-vitro data indicated that PolyP120 requires the host proteins and/or carriers for inhibition of viral gene transcription.

Then, to further dissect out transcriptional inhibition mediated by PolyP120 on RdRp, we focused on its discontinuous transcription, taking into account four structural sgRNAs (i.e., sgS, sgE, sgM, sgN). For this purpose, SARS-CoV-2-infected Vero cells were treated with PolyP120 and then analysed for transcription of these sgRNAs (Fig. S4A) . These data confirmed a reduction in the N gene fragments, RdRp and S transcripts in PolyP120-treated cells ( Fig. S4B and C) . Additional data were obtained using Nanopore technology and direct RNA sequencing of the RNAs extracted from viral infection of these Vero cells showing decreased levels of the sgRNAs ( Fig. 2C and Table S2 ; see Methods and CEINGE portal).

Furthermore, using RT-PCR with SYBR-Green, we also confirmed these data showing decreased levels of sgS, sgE, sgM and sgN (Fig. 2D) . Moreover, immunoblotting performed with PolyP120-treated SARS-CoV-2-infected Vero cells also showed dramatic reductions in both ACE2 and N-protein levels (Fig. S4D) .

This 'therapeutic' anti-viral action of PolyP120 was also confirmed by immunofluorescence, which showed significant reduction of ACE2 and the SARS-CoV-2 proteins (i.e., N, E, S) in these PolyP120-treated Vero cells (Fig. 2E, Fig. S4E and F, and Altogether, these data show the mechanisms of action of the PolyPs through reduction in the transcriptional activity of RdRp, which appears to be due to their binding to its active enzymatic core (see Fig. 2A ).

Of interest, we also noted down-regulation of ACE2 at the transcriptional level (Fig.   S4C ). Treatment with the transcriptional inhibitor actinomycin D showed significant decrease in ACE2 mRNA expression levels in the Vero cells after 24 h (Fig. S4G) , thus also suggesting transcriptional inhibition mediated by PolyPs.

To investigate the potential 'therapeutic' effectiveness of PolyPs, additional studies were performed in which PolyPs at different chain length were added after SARS-CoV-2 infection.

For this purpose, SARS-CoV-2-infected Vero cells were incubated with PolyPs (starting from the 120 phosphate residues of PolyP120, and including PolyP126 and PolyP189), and then analysed for expression levels of the viral N gene (Fig. S6A) . No significant differences were seen between these different chain length PolyPs for the decreased expression of the viral N gene (N1-3), compared to the vehicle control (Fig. S6B) .

Furthermore, similar data were obtained for Vero cells infected with SARS-CoV-2 particles obtained from a Korean patient (EPI_ISL_407193; Fig. S1B ), by measuring viral RdRp and E genes expression upon addition of PolyP120 at different concentrations (9.38, 18 .75, 37.50 μΜ) ( Fig. S6C and D) . The increased Ct values for the RdRp gene in these Vero cells treated with PolyP120 indicated inhibition of SARS-CoV-2 infection, including for the lowest PolyP120 concentration used (Fig. S6D) .

As different mutations have been identified in Europe for the RdRp, S and ORF1ab genes (11), we also performed sequencing analysis focusing on the viral regions that flank these hot-spot mutation sites between the Italian and Korean SARS-CoV-2. These data showed non-synonymous variants in these genes (i.e., RdRp, S, ORF1ab) in the viral genome from an Italian patient compared to that from the Korean patient (Fig. S2A, red arrows) .

Furthermore, these variants were conserved across Italian viral sequences, compared to Asian viral sequences (Fig. S2A) , as previously described (25) . Importantly, the mutation in RdRp (at position 14408) was associated with an increased mutation rate, which would appear to be due to an alteration in the proofreading that is finely regulated by the RdRp/nsp7/nsp8 machinery complex (11) . Our data show that PolyP120 can decrease the expression of these viral genes in the infected Vero cells also in the presence of this SARS-CoV-2 point mutation.

In this regard, our docking analysis suggested that the mutated site in RdRp falls outside of its RNA-dependent transcriptional activity domain and its potential binding sites with the PolyPs (Fig. S6E) . This thus supports the hypothesis that the PolyPs impair the RNA-dependent transcriptional activity rather than the proofreading activity.

To determine further whether increased PolyPs size, in terms of Pi residues, has any effects on their antiviral activity, human primary cells were pretreated with PolyP120 and PolyP126, then infected with SARS-CoV-2 at the different viral loads of 0.01 and 0.16 MOI, and analysed for expression levels of the viral N gene (Fig. S7A) . These data indicated that PolyP126 shows similar anti-viral activity to PolyP120 in these human cells infected with SARS-CoV-2 (Fig. S7B) . Altogether, these data suggest that following the pretreatment of human primary nasal epithelial cells, the longer-chain PolyPs (e.g., PolyP120, PolyP126) have anti-viral effects through inhibition of N gene transcription. This preventive antiviral action was also confirmed in SARS-CoV-2-infected human colorectal carcinoma cells (i.e., Caco2 cells), where PolyP120 pre-treatment showed a decrease for the viral genes (N, RdRp) ( Fig. S7C to E) and ACE2 protein (Fig. S7F) . These data thus suggest that the mechanism of action of PolyP120 is conserved across different human cellular models.

Furthermore, we also tested the 'therapeutic' effectiveness of PolyP120 in SARS-CoV-2-infected human primary nasal epithelial cells (Fig. 3A, Fig. S8A ). The PolyP120 treatment showed decreased levels of the N1-3 gene fragments (Fig. S8B) , and reductions in the other viral genes (i.e., RdRp, S, E) and ACE2 (Fig. S8C) . Furthermore, immunoblotting confirmed total inhibition of the ACE2 and N protein levels in these treated human primary nasal epithelial cells (Fig. 3B) .

Patients with COVID-19 also show increased circulating levels of inflammatory cytokines, including interferon-γ and interleukin (IL)-6, IL-10 and IL-12 (26) . As the PolyPs have been previously described as having anti-inflammatory functions (14) , we investigated the potential modulation by PolyP120 of these cytokines in human primary cells infected with SARS-CoV-2. Indeed, real-time PCR analyses showed significant reductions in interferon-γ, IL-6, IL-10 IL-12 and tumour necrosis factor-α (Fig. 3c) . The anti-inflammatory action exerted by PolyPs could be ascribed to the inhibition of NF-kB pathway, as shown by reduced levels of phosphorylated (Ser311)-p65 in SARS-CoV-2-infected Vero cells after treatment with PolyP120 (Fig. 3D) .

As the PolyPs have been previously reported to be associated with the inflammatory pathways that are mainly regulated by NF-κB (27) and mTOR (28) , we hypothesise that this down-regulation of the cytokines can be ascribed to modulation of the inflammatory cascades in an autocrine and paracrine manner by PolyP120.

Due to the respiratory failure experienced by patients with COVID-19, new drug formulations that are appropriate for aerosol inhalation, and thus more direct lung delivery, represent an unmet medical need. For this reason, we tested the delivery of PolyP120 into Vero cells via a nebuliser system. Here, 37.5 μM PolyP120 was nebulised for 2 min (condensation rate, 1 mL/min) into the tissue culture flask containing the attached Vero cells in the absence of medium. After nebulising, medium was added back to the cells, and the flasks were left for 12 h under growth conditions (see Video 1, Supplemental Material). Entrance of the PolyP120 into the cells was confirmed by immunofluorescence using DAPI, thus showing the efficacy of this delivery for the nebulised PolyP120 into the cells (Fig. S8D) .

We then investigated whether this PolyP120 aerosol formulation maintains the antiviral activity of PolyP120 on Vero cells. Thus, the nebulised PolyP120 was similarly applied to Vero cells that were then infected with SARS-CoV-2 viral particles (Fig. S8E ).

Real-time PCR analysis indeed showed down-regulation of the N gene fragments and reduced levels of ACE 2 and the viral genes, and all of the structural sgRNAs (Fig. 3E) . This thus indicated that PolyP120 delivered in this aerosol formulation maintained its anti-viral action against SARS-CoV-2 infection.

Here, we have shown that long chain length PolyPs (i.e., PolyP120, with 120 phosphate residues) can inhibit the transcription of SARS-CoV-2 viral genes ( Fig. 4A) impairing the replication of the virus in the host cell. Future diagnostic use of this technology in vivo will be of great impact monitoring the disease status.

Of importance, we have described two intracellular mechanisms of action behind this anti-viral function of PolyPs, as summarised in Fig. 4B . The first mechanism involves timeand dose-dependent proteasome-mediated degradation of ACE2 protein in the presence of long chain length PolyPs. This appears to be due to the binding of the PolyPs to ACE2, which should be mainly mediated by four amino-acid residues of ACE2 that are also conserved across different species: Arg514, His 401, His 378 and Arg393. This is of importance to avoid further spillover. We also note the role of PolyPs as a source of Pi to increase the pool of ATP available to enhance the ubiquitin-proteasome system (29) .

Interestingly, we have also shown that the SARS-CoV-2-infected cells have reduced ACE mRNA expression levels after treatment with PolyP120, we think this is because the expression of ACE2 is regulated by the HNF1α transcriptional factor (30) and epigenetically by SIRT1 (31) . These phenomena need further investigations.

Of note, in directing any treatment here at ACE2 as the receptor for SARS-CoV-2 entry into cells, the use of ACE inhibitors has been discouraged for patients with COVID-19. This is because of the consequent increased levels of ACE2 mRNA, which might facilitate engagement and entry of SARS-CoV-2 into cells (32) . However, the future use of PolyPs might indeed overcome this side effect of the use of ACE inhibitors. Thus, the combination of ACE inhibitors with PolyPs can be postulated for the treatment of patients with COVID-19.

14 The second mechanism of action presented here involves transcriptional inhibition of RdRP (Fig. 4B) . The PolyPs appear to bind to viral RdRp from SARS-CoV-2, mainly through seven of its amino-acid residues located within the RdRp RNA-binding site. PolyP120 appears to decrease the expression of the main SARS-CoV-2 viral genes through this mechanism. Of importance, the transcription of the main viral sgRNAs is also impaired confirming their inhibitory actions on RdRp transcription. This inhibition also occurs in the The various concentrations of PolyP120 tested here are also shown not to have any toxic effects, as seen by monitoring of the caspase 3 enzymatic activation in human primary nasal epithelial cells. However, the highest concentration of PolyP120 tested (i.e., 112 μM) blocked proliferation of these primary cells. This anti-proliferative effect might be due to the inhibition of the MAP-kinase cascade (via ERK), which has previously been reported for inorganic PolyPs (33).

PolyPs have also been shown to be key modulators of inflammatory pathways, with actions mainly via the NF-κB cascade (27) . Here, we have shown that PolyP120 decreases the levels of the main cytokines (i.e., interferon-γ, IL-6, IL-10, IL-12) that are part of the cytokine storm often seen for patients with COVID-19 (26) by affecting NF-kB cascade. Among these cytokines, IL-6 levels in patients with COVID-19 are significantly elevated, and are associated with adverse clinical outcomes. While inhibition of IL-6 levels with tocilizumab appears to be effective and safe in preliminary investigations, results need to be evaluated of ongoing clinical trials to better define the role of tocilizumab in COVID-19 and its routine clinical application (34) .

Pre-existing T-cell immunity to SARS-CoV-2 might be relevant also, because it might influence COVID-19 disease severity. It is possible that people with high levels of preexisting memory CD4+ T-cells that recognize SARS-CoV-2 can mount a faster and stronger immune response upon exposure to SARS-CoV-2, and thereby limit the disease severity. 

Vero cells were kept in DMEM (41966-029; Gibco) with 10% FBS (10270-106; Gibco), 2 mM L-glutamine (25030-024; Gibco), and 1% penicillin/streptomycin (P0781; Sigma).

Freshly isolated human nasal epithelial cells were collected by nasal brushing of healthy donors (as previously described (38) 

Vero cells or human primary nasal epithelial cells were plated in T25 flasks (6 ×10 5 cells/flask) for treatment with 37.5 μM PolyPs (from P94 to P126). After 20 min, these pretreated cells were infected with SarsCov2 viral particles obtained from a frozen swab from an Italian patient positive for COVID-19. Non-infected cells were used as the negative control. 

To carry out the direct sequencing of the RNA with nanopore technology, we prepared the RNA library using direct RNA sequencing kits (SQK-RNA002; Oxford Nanopore kits), following the manufacturer instructions. Briefly, we used poly(dT) adapter and SuperScript II Reverse Transcriptase (Thermo Fisher Scientific) to generate RNA-DNA hybrids. Agencourt RNAClean XP magnetic beads (BECKMAN) were used to purify the RNA-DNA duplexes.

Subsequently, RNA-DNA hybrids were ligated to Nanopore sequencing adapters (RMX) using T4 DNA ligase (NEB E6056) prior to sequencing. After another step of purification using Agencourt RNAClean XP magnetic beads, the library concentration was estimated with Qubit assay (Thermo Fisher). Then, the Nanopore direct RNA library was loaded into the flow cell (R9.06) using MinION for 26 h. The motor protein RMX pulls the 3'-end of the RNA strand into the Nanopore channel (39) . Changes in the ionic current are then detected at each pore by a sensor.

Reads from Nanopore direct sequencing were initially identified by alignment to a set of reference sequences using Minimap2 version 2.17 (40) , with the 'long-read spliced alignment' preset. The reference sequences include SARS-CoV-2 viral genome (GenBank, NC_045512), the Chlorocebus sabaeus host genome (assembly ChlSab 1.1), and the human ribosomal DNA repeat (GenBank, U13369.1). Reads identified as viral were then realigned to the same viral genome sequence using Minimap2, with the following options: -k 8 -w 1 -g 30000 --max-chain-skip 40 -p 0.7 -N 32 --frag=no --splice -G 30000 -C 0 -u n --splice-flank=no -A 1 -B 2 -O 2,24 -E 1,0 -z 400,200 --no-end-flt. Reads were assigned to each canonical sgRNA by selecting those which overlapped the genomic interval 54-86 (leader), did not overlap the genomic interval 100-21546 (ORF1ab), and contained the specific downstream transcription regulatory sequence preceding each ORF (see Supplementary Table   2 for list of genomic intervals). Mapped reads filtering and counting were carried out by using Samtools version 1.9 (http://htslib.org).

The sequence alignments were realized using the Clustal Omega software (https://www.uniprot.org/align/).

For the real-time PCR assays, Cq values of N1, N2 and N3 are reported as means ±standard deviation, as the ratios to the internal control detected with the CLONIT quanty COVID-19 kits (Ref. RT-25; IVD approved). Statistical significance was defined as a p value <0.05 versus the vehicle control. Immunofluorescence data were analysed by two-sample t-tests using the SPSS software, version 16.0. A probability (P) <0.05 was considered statistically significant.

Electrostatic potential calculations were carried out with Adaptive Poisson-Boltzmann Solver software (41) within the AutoDock Tools environment (42) using the default parameters. RdRp/nsp7/nsp8 -4.8 ± 0.2 Table 2 . Clinical information and Ct values for the cohort of Italian patients with COVID-19.

All of the samples were from oropharyngeal swabs that originated from Naples, Italy. 

Arg5228 Asp5237 Arg5241

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Epithelial Nasal cells Supplementary Fig. 8 

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We would like to thank Prof. David Schlessinger Scientist Emeritus Intramural Research