Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response. Our therapeutic arsenal to treat viral diseases is extremely limited, and there is a critical need for molecules that could be used against multiple viruses. Among possible strategies, there is a growing interest for molecules stimulating cellular defense mechanisms. We recently developed a functional assay to identify stimulators of antiviral genes, and selected compound DD264 from a chemical library using this approach. While searching for its mode of action, we identified this molecule as an inhibitor of pyrimidine biosynthesis, a metabolic pathway that fuels the cell with pyrimidine nucleobases for both DNA and RNA synthesis. Interestingly, it was recently shown that inhibitors of this metabolic pathway prevent the replication of RNA viruses. Here, we established a functional link between pyrimidine biosynthesis pathway and the induction of antiviral genes, and demonstrated that pyrimidine biosynthesis inhibitors like DD264 or brequinar critically rely on cellular immune response to inhibit virus growth. Thus, pyrimidine deprivation is not directly responsible for the antiviral activity of pyrimidine biosynthesis inhibitors, which rather involves the induction of a metabolic stress and subsequent triggering of cellular defense mechanisms.

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPRs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly. MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires two distinct determinants at the carboxyl terminus of the CD-MPR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.

Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors ofVibriospecies; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogenVibrio splendidusLGP32 and show that toxicity is correlated to the presence of a metalloprotease and its correspondingvsmgene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and avsmdeletion mutant (Δvsmmutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Δvsmmutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Δvsmmutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor ofBacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs ofV. splendidus.