Singapore grouper iridovirus VP128 inhibits STING-TBK1 mediated signaling to evade antiviral immunity.

Singapore grouper iridovirus (SGIV) belongs to the family Iridoviridae and the genus Ranavirus, which is a large cytoplasmic DNA virus. Infection of grouper with SGIV can cause hemorrhage and swelling of the spleen of the fish. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. In the present study, the protein encoded by SGIV ORF128 (VP128) was identified. VP128 is predominantly localized within the endoplasmic reticulum (ER). Overexpression of VP128 significantly promoted SGIV replication. VP128 inhibited the interferon (IFN)-3 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), and TANK-binding kinase 1 (EcTBK1). Moreover, VP128 interacted with EcSTING and EcTBK1. The interaction between VP128 and EcSTING was independent of any specific structural domain of EcSTING. Together, our results demonstrated that SGIV VP128 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion.

Antiviral role of grouper FoxO1 against RGNNV and SGIV infection.

As a key regulator of the innate immune system, FoxO1 has a variety of activities in biological organisms. In the present study, grouper FoxO1 (EcFoxO1) was cloned and the antiviral activity in red grouper neuron necrosis virus (RGNNV) and Singapore grouper iridescent virus (SGIV) was examined. The open reading frame (ORF) of EcFoxO1 contains 2,034 base pairs that encode a protein of 677 amino acids with a predicted molecular weight of 73.21 kDa. EcFoxO1 was shown to be broadly distributed in healthy grouper tissues, and was up-regulated in vitro in response to stimulation by RGNNV and SGIV. EcFoxO1 has a whole-cell distribution in grouper spleen (GS) cells. EcFoxO1 decreased the replication of RGNNV and SGIV, and activated interferon (IFN) 3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB) promoter activities. EcFoxO1 could interact with EcIRF3. Together, the results demonstrated that EcFoxO1 might be an important regulator of grouper innate immune response against RGNNV and SGIV infection.

Grouper Atg14 promotes Singapore grouper iridovirus (SGIV) replication by inhibiting the host innate immune response.

As one of the important members of the autophagy-related protein family, Atg14 plays a key role in the formation and maturation of autophagosomes. However, little is known about the potential roles of fish Atg14 and its roles in virus infection. In the present study, the homolog of Atg14 (EcAtg14) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) of EcAtg14 consists of 1530 nucleotides, encoding 509 amino acids, with a predicted molecular weight of 56.9 kDa. EcAtg14 was distributed in all tested tissues, with higher expression in liver, blood and spleen. The expression of EcAtg14 was increased in grouper spleen (GS) cells after Singapore grouper iridovirus (SGIV) infection. EcAtg14 was distributed in the cytoplasm of GS cells. Overexpression of EcAtg14 promoted SGIV replication in GS cells and inhibited IFN3, ISRE and NF-κB promoter activities. Co-immunoprecipitation results showed that there was an interaction between EcAtg14 and EcBeclin. EcAtg14 also promoted the synthesis of LC3-II in GS cells. These findings provide a basis for understanding the innate immune mechanism of grouper against viral infection.

Singapore grouper iridovirus VP122 targets grouper STING to evade the interferon immune response.

Singapore grouper iridovirus (SGIV) is a highly pathogenic Iridoviridae that causes hemorrhage and spleen enlargement in grouper. Despite previous genome annotation efforts, many open reading frames (ORFs) in SGIV remain uncharacterized, with largely unknown functions. In this study, we identified the protein encoded by SGIV ORF122, now referred to as VP122. Notably, overexpression of VP122 promoted SGIV replication. Moreover, VP122 exhibited antagonistic effects on the natural antiviral immune response through the cGAS-STING signaling pathway. It specifically inhibited the cGAS-STING-triggered transcription of various immune-related genes, including IFN1, IFN2, ISG15, ISG56, PKR, and TNF-α in GS cells. Additionally, VP122 significantly inhibited the activation of the ISRE promoter mediated by EccGAS and EcSTING but had no effect on EccGAS or EcSTING alone. Immunoprecipitation and Western blotting experiments revealed that VP122 specifically interacts with EcSTING but not EccGAS. Notably, this interaction between VP122 and EcSTING was independent of any specific domain of EcSTING. Furthermore, VP122 inhibited the self-interaction of EcSTING. Interestingly, VP122 did not affect the recruitment of EcTBK1 and EcIRF3 to the EcSTING complex. Collectively, our results demonstrate that SGIV VP122 targets EcSTING to evade the type I interferon immune response, revealing a crucial role for VP122 in modulating the host-virus interaction.