SARS-CoV-2-encoded small RNAs are able to repress the host expression of SERINC5 to facilitate viral replication.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.

Interaction between chicken TRIM25 and MDA5 and their role in mediated antiviral activity against IBDV infection.

Infectious Bursal Disease Virus (IBDV) is the causative agent of an immunosuppressive disease that affects domestic chickens (Gallus gallus) severely affecting poultry industry worldwide. IBDV infection is characterized by a rapid depletion of the bursal B cell population by apoptosis and the atrophy of this chief lymphoid organ. Previous results from our laboratory have shown that exposure of infected cells to type I IFN leads to an exacerbated apoptosis, indicating an important role of IFN in IBDV pathogenesis. It has been described that recognition of the dsRNA IBDV genome by MDA5, the only known cytoplasmic pattern recognition receptor for viral RNA in chickens, leads to type I IFN production. Here, we confirm that TRIM25, an E3 ubiquitin ligase that leads to RIG-I activation in mammalian cells, significantly contributes to positively regulate MDA5-mediated activation of the IFN-inducing pathway in chicken DF-1 cells. Ectopic expression of chTRIM25 together with chMDA5 or a deletion mutant version exclusively harboring the CARD domains (chMDA5 2CARD) enhances IFN-ß and NF-ĸB promoter activation. Using co-immunoprecipitation assays, we show that chMDA5 interacts with chTRIM25 through the CARD domains. Moreover, chTRIM25 co-localizes with both chMDA5 and chMDA5 2CARD, but not with chMDA5 mutant proteins partially or totally lacking these domains. On the other hand, ablation of endogenous chTRIM25 expression reduces chMDA5-induced IFN-ß and NF-ĸB promoter activation. Interestingly, ectopic expression of either wild-type chTRIM25, or a mutant version (chTRIM25 C59S/C62S) lacking the E3 ubiquitin ligase activity, restores the co-stimulatory effect of chMDA5 in chTRIM25 knockout cells, suggesting that the E3-ubiquitin ligase activity of chTRIM25 is not required for its downstream IFN-ß and NF-ĸB activating function. Also, IBDV-induced expression of IFN-ß, Mx and OAS genes was reduced in chTRIM25 knockout as compared to wild-type cells, hence contributing to the enhancement of IBDV replication. Enhanced permissiveness to replication of other viruses, such as avian reovirus, Newcastle disease virus and vesicular stomatitis virus was also observed in chTRIM25 knockout cells. Additionally, chTRIM25 knockout also results in reduced MAVS-induced IFN-ß promoter stimulation. Nonetheless, similarly to its mammalian counterpart, chTRIM25 overexpression in wild-type DF-1 cells causes the degradation of ectopically expressed chMAVS.

Type I Interferon acts as a major barrier to the establishment of infectious bursal disease virus (IBDV) persistent infections.

Infectious bursal disease virus (IBDV), the best characterized member of the Birnaviridae family, is a highly relevant avian pathogen causing both acute and persistent infections in different avian hosts. Here, we describe the establishment of clonal, long-term, productive persistent IBDV infections in DF-1 chicken embryonic fibroblasts. Although virus yields in persistently-infected cells are exceedingly lower than those detected in acutely infected cells, the replication fitness of viruses isolated from persistently-infected cells is higher than that of the parental virus. Persistently-infected DF-1 and IBDV-cured cell lines derived from them do not respond to type I interferon (IFN). High-throughput genome sequencing revealed that this defect is due to mutations affecting the IFNα/ß receptor subunit 2 (IFNAR2) gene resulting in the expression of IFNAR2 polypeptides harbouring large C-terminal deletions that abolish the signalling capacity of IFNα/ß receptor complex. Ectopic expression of a recombinant chicken IFNAR2 gene efficiently rescues IFNα responsiveness. IBDV-cured cell lines derived from persistently infected cells exhibit a drastically enhanced susceptibility to establishing new persistent IBDV infections. Additionally, experiments carried out with human HeLa cells lacking the IFNAR2 gene fully recapitulate results obtained with DF-1 cells, exhibiting a highly enhanced capacity to both survive the acute IBDV infection phase and to support the establishment of persistent IBDV infections. Results presented here show that the inactivation of the JAK-STAT signalling pathway significantly reduces the apoptotic response induced by the infection, hence facilitating the establishment and maintenance of IBDV persistent infections.IMPORTANCE Members of the Birnaviridae family, including infectious bursal disease virus (IBDV), exhibit a dual behaviour, causing acute infections that are often followed by the establishment of life-long persistent asymptomatic infections. Indeed, persistently infected specimens might act as efficient virus reservoirs, hence potentially contributing to virus dissemination. Despite the key importance of this biological trait, information about mechanisms triggering IBDV persistency is negligible. Our report evidences the capacity of IBDV, a highly relevant avian pathogen, to establishing long-term, productive, persistent infections in both avian and human cell lines. Data presented here provide novel and direct evidence about the crucial role of type I IFNs on the fate of IBDV-infected cells and their contribution to controlling the establishment of IBDV persistent infections. The use of cell lines unable to respond to type I IFNs opens a promising venue to unveiling additional factors contributing to IBDV persistency.