Melanoma differentiation‐associated gene 5 in zebrafish provoking higher interferon‐promoter activity through signalling enhancing of its shorter splicing variant

Abstract

SummaryMelanoma differentiation-associated gene 5 (MDA5) is one of the three members in the retinoic acid-inducible gene I-like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up-regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over-expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co-transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

Spring viraemia of carp virus: recent advances

“…In teleost fish, a homologue of human MDA5 has been characterized functionally in rainbow trout and zebrafish, and is responsive to infection by salmon alphavirus, VHSV or SVCV (Chang et al, 2011;Zou et al, 2014). In zebrafish, MDA5 has two splicing variants: MDA5a and MDA5b (Zou et al, 2014).…”

“…In zebrafish, MDA5 has two splicing variants: MDA5a and MDA5b (Zou et al, 2014). Both variants are markedly upregulated in zebrafish embryonic fibroblast cells infected with SVCV (Zou et al, 2014).…”

“…In zebrafish, MDA5 has two splicing variants: MDA5a and MDA5b (Zou et al, 2014). Both variants are markedly upregulated in zebrafish embryonic fibroblast cells infected with SVCV (Zou et al, 2014). Overexpression of MDA5a and MDA5b in cultured fish cells induces a significantly higher type I IFN promoter activity and exerts a protective effect on transfected cells against SVCV (Zou et al, 2014).…”

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish

“…Such short isoforms may compete for signaling molecules or form a direct interaction with their typical type to block downstream signaling as negative regulators [20,39]. However, it was reported in a previous study that a shorter splicing variant of MDA5 in zebrafish acts as an enhancer in MDA5/MAVSmediated signaling activity [29]. In the present study, it was revealed that the insertion variant of RIG-I in zebrafish may also act as a positive regulator in the RIG-I-mediated signaling pathway.…”

“…At 24 h post transfection, the cells were collected and lysed in 500 ml cell extraction buffer as described above. The cellular debris was removed by centrifugation at 12000 g for 10 min at 4 C. The supernatants were transferred to fresh tubes and incubated with antiFlag resin conjugate (FLAG Tagged Protein Immunoprecipitation Kit, Sigma) overnight at 4 C. The resin was washed six times with ice-cold wash solution as described by the manufacturer and eluted with 40 ml 2 Â SDS sample buffer by boiling for 10 min at 95 C. The precipitates were detected by Western blotting with anti-RIG-I and anti-Flag antibodies, as reported in a previous study [29]. Briefly, equal amount of protein samples was electrophoresed on 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membrane with pore size of 0.45 mm (Millipore Corporation).…”