Current needs in coronavirus research

Identification of cell-signaling pathways involved in CoV replication and pathology. Understanding how CoV spreads in the body at a molecular level can help select antiviral drugs that inhibit these pathways. We study PBM-PDZ protein-protein interactions and viral accessory genes involved in the innate immune and inflammatory responses, since activation of these pathways is responsible for virulence.

Activation of p38 MAPK by syntenin. During SARS-CoV infection, the envelope (E) protein PBM motif interacts with syntenin PDZ motif, redistributing syntenin to the cytoplasm, activating p38 MAPK and triggering the overexpression of pro-inflammatory cytokines. This interaction is a novel therapeutic target, as administration to a p38 MAPK inhibitor in vivo, led to a significant increase in mice survival after SARS-CoV infection.

To determine the relevance of post-transcriptional regulation of gene expression to the inflammatory pathology. We study the contribution to dysregulated inflammation of small non-coding RNAs (host miRNAs and virus-derived RNAs) and RNA-protein complexes. RNAs and proteins involved in these regulatory networks represent potential antiviral targets.

Contribution of host and viral small non-coding RNAs to SARS-CoV lung pathology. Our group described that small viral RNAs (svRNAs) derived from the SARS-CoV genome during infection contribute to lung pathology by enhancing the expression of pro-inflammatory cytokines. Most likely, this effect is mediated by silencing the expression of inhibitory factors in RNA interference silencing complexes (RISC) (left part of the graph). Infection with a virulent SARS-CoV induces the expression of host microRNA-223, which downregulates the expression of several host target mRNAs, including the cystic fibrosis transmembrane conductance regulator (CFTR), involved in edema resolution. Silencing of CFTR by miRNA-223 contributed to lung edema (right part of the graph). Administration of antisense RNAs that inhibited svRNA-N or miRNA-223 to mice significantly reduced SARS-CoV-induced pulmonary pathology, which represents a novel antiviral strategy.

Development of next generation of SARS-CoV-2 vaccines consisting in replication-competent propagation-deficient RNA replicons and to determine their efficacy in animal model systems. Vaccine development includes:

1. Engineering the SARS-CoV-2 RNA-replicons by deleting or modifying viral genes responsible for propagation and virulence, using reverse genetics
2. Development of packaging cell lines that efficiently complement the generation of virus-like particles (VLPs)
3. Identification of RNA-replicon delivery systems
4. Engineering simplified and safer versions by reducing the replicase size.

RNA replicon vaccines to protect against highly pathogenic human coronaviruses. Two types of replicon delivery systems have been designed: (i) a chemically synthesized one that includes two components, the RNA replicon and a cationic polymer to form nanoparticles. (ii) formation of virus like particles (VLPs) complemented in packaging cells lines with the proteins required for propagation from cell to cell.

To understand the mechanisms of PASC, two main hypotheses are being investigated. The presence of defective RNA genomes that reduce viral dissemination, inducing proinflammatory reactions in specific tissues, is being studied in human samples and in a mouse model for PASC. Different mechanisms, previously shown by our group to modulate innate responses and inflammation in acute infection are being analyzed using complementary technologies. Host factors interacting with virus determinants of virulence that mediate cystic fibrosis transmembrane conductance regulator (CFTR) reduction are being identified as potential targets for antiviral drugs. The contribution of SARS-CoV-2 accessory genes 6, 7a, 7b and 8 are being studied using virus deletion mutants. Post-transcriptional regulation of gene expression either mediated by viral and host small non-coding RNAs or by RNA-protein complexes including viral N and host MOV10 proteins are being analyzed. This information will serve as the basis for new therapeutic interventions.

Lung edema resolution mechanism. Airway epithelium acts as a barrier between the alveolar space and the tissue interstitium. There is an asymmetric distribution of ion channels, such as the epithelial sodium channel (ENaC), located in the apical part together with the cystic fibrosis transmembrane conductance regulator (CFTR), and the Na⁺/K⁺ APTase pump (NKA) in the basolateral part. These ionic transporters generate an osmotic gradient that controls the water levels in the alveolar space. Water moves due to the generated osmotic gradient in a paracellular or transcellular way through specific channels. Human pathogenic CoVs E protein decreases CFTR activity, avoiding edema resolution and increasing lethality.