Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo. (preprint)

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of extra- or intra-cellular Ca2+ content have been suggested to block SARS-CoV-2 replication. Here, we demonstrate that a newly nontoxic caloxin-derivative compound (PI-7) inhibits ATP2B1, reduces the extra- and intra-cellular Ca2+ levels and impairs SARS-CoV-2 replication and propagation (VOCs: Delta and Omicron 2), as also measured by inhibition of syncytia in vitro. Furthermore, a FOXO3 transcriptional site of regulation of expression at the 5 end of the ATP2B1 locus, together with a rare homozygous intronic variant in the ATP2B1 locus (rs11337717; chr12:89643729, T>C), are shown to be associated with severity of COVID19 (symptomatic versus asymptomatic patients). Here, we identify the mechanism of action during SARS-CoV-2 infection, which involves the PI3K/Akt signaling pathway, inactivation of FOXO3 (i.e., phosphorylation), and inhibition of transcriptional control of both membrane and reticulum Ca2+ pumps (ATP2B1 and ATP2A1 [i.e., SERCA1], respectively). The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 shows a lack of toxicity, its prophylactic use as a therapy against the COVID19 pandemic is here proposed.

Common Variants at 21q22.3 Locus Influence MX1 Gene Expression and Susceptibility to Severe COVID-19 (preprint)

The COVID-19 disease, caused by the SARS-Cov-2, presents a heterogeneous clinical spectrum. The risk factors do not fully explain the wide spectrum of disease manifestations, so genetic factors could account for novel insights into its pathogenesis.In our previous study, we hypothesized that common variants on chromosome 21, near TMPRSS2 and MX1 genes, may be genetic risk factors associated with the different clinical manifestations of COVID-19. Here, we performed an in-depth genetic analysis of chromosome 21 exploiting the genome-wide association study data including 6,406 individuals hospitalized for COVID-19 and 902,088 controls with European genetic ancestry from the COVID-19 Host Genetics Initiative. We found that five single nucleotide polymorphisms (SNPs) within TMPRSS2 and near MX1 gene show suggestive associations (P≤1x10-5) with severe COVID-19. All five SNPs replicated the association in two independent cohorts of Asian subjects while two and one out of the 5 SNPs replicated in African and Italian populations, respectively (P≤0.05). The minor alleles of the five SNPs correlated with a reduced risk of developing severe COVID-19 and an increased level of MX1 expression in blood. One (rs12329760, V197M) of these SNPs resided in the exon 6 of TMPRSS2 and was also predicted to alter the protein conformation.Our findings provide further evidence that host genetic factors can contribute to determining the different clinical presentations of COVID-19 and that MX1, an antiviral effector of type I and III interferon pathway, could be a potential therapeutic target.Funding: This study was supported by the project "CEINGE TASK-FORCE COVID19", codeD64I200003800 by Regione Campania for the fight against Covid-19 (DGR n. 140 del 17 Marzo 2020).Conflict of Interest: Nothing to disclose.

Common variants at 21q22.3 locus influence MX1 gene expression and susceptibility to severe COVID-19 (preprint)

The COVID-19 disease, caused by the SARS-Cov-2, presents a heterogeneous clinical spectrum. The risk factors do not fully explain the wide spectrum of disease manifestations, so it is possible that genetic factors could account for novel insights into its pathogenesis. In our previous study, we hypothesized that common variants on chromosome 21, near TMPRSS2 and MX1 genes, may be genetic risk factors associated to the different clinical manifestations of COVID-19. Here, we performed an in-depth genetic analysis of chromosome 21 exploiting the genome-wide association study data including 6,406 individuals hospitalized for COVID-19 and 902,088 controls with European genetic ancestry from COVID-19 Host Genetics Initiative. We found that five single nucleotide polymorphisms (SNPs) within TMPRSS2 and near MX1 gene show suggestive associations (P[≤]1x10-5) with severe COVID-19. All five SNPs replicated the association in two independent cohorts of Asian subjects while two and one out of the 5 SNPs replicated in African and Italian populations, respectively (P[≤]0.05). The minor alleles of these five SNPs correlated with a reduced risk of developing severe COVID-19 and increased level of MX1 expression in blood. Our findings provide further evidence that host genetic factors can contribute to determine the different clinical presentations of COVID-19 and that MX1, an antiviral effector of type I and III interferon pathway, may be a potential therapeutic target.

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2 (preprint)

The Spike (S)-protein of SARS-CoV-2 binds host-cell receptor ACE2 and requires proteolytic 'priming' (S1/S2) and 'fusion-activation' (S2') for viral entry. The S-protein furin-like motifs PRRAR685{downarrow} and KPSKR815{downarrow} indicated that proprotein convertases promote virus entry. We demonstrate that furin and PC5A induce cleavage at both sites, ACE2 enhances S2' processing, and their pharmacological inhibition (BOS-inhibitors) block endogenous cleavages. S1/S2-mutations (S1/S2) limit S-protein-mediated cell-to-cell fusion, similarly to BOS-inhibitors. Unexpectedly, TMPRSS2 does not cleave at S1/S2 or S2', but it can: (i) cleave/inactivate S-protein into S2a/S2b; (ii) shed ACE2; (iii) cleave S1-subunit into secreted S1', activities inhibited by Camostat. In lung-derived Calu-3 cells, BOS-inhibitors and S1/S2 severely curtail 'pH-independent' viral entry, and BOS-inhibitors alone/with Camostat potently reduce infectious viral titer and cytopathic effects. Overall, our results show that: furin plays a critical role in generating fusion-competent S-protein, and indirectly, TMPRSS2 promotes viral entry, supporting furin and TMPRSS2 inhibitors as potential antivirals against SARS-CoV-2

SARS-CoV-2 protein ORF3a is pathogenic in Drosophila and causes phenotypes associated with COVID-19 post-viral syndrome (preprint)

The Coronavirus Disease 2019 (COVID-19) pandemic has caused millions of deaths and will continue to exact incalculable tolls worldwide. While great strides have been made toward understanding and combating the mechanisms of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, relatively little is known about the individual SARS-CoV-2 proteins that contribute to pathogenicity during infection and that cause neurological sequela after viral clearance. We used Drosophila to develop an in vivo model that characterizes mechanisms of SARS-CoV-2 pathogenicity, and found ORF3a adversely affects longevity and motor function by inducing apoptosis and inflammation in the nervous system. Chloroquine alleviated ORF3a induced phenotypes in the CNS, arguing our Drosophila model is amenable to high throughput drug screening. Our work provides novel insights into the pathogenic nature of SARS-CoV-2 in the nervous system that can be used to develop new treatment strategies for post-viral syndrome.

Long-chain polyphosphates impair SARS-CoV-2 infection and replication: a route for therapy in man (preprint)

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.