Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

肿瘤坏死因子受体（TNFR）相关因子（TRAFs）是先天免疫应答的重要信号转导蛋白，并参与许多重要的途径，例如NF-κB和干扰素调节因子（IRF）激活的信号通路。在这项研究中，虾的TRAF3直系同源物凡纳滨对虾（LvTRAF3）被克隆并表征。LvTRAF3的转录本为3,865 bp，开放阅读框（ORF）为1,002 bp，编码333个氨基酸的多肽，包括一个保守的TRAF-C结构域。LvTRAF3在肠和血细胞中的表达上调，以应对多聚（I：C）攻击和白斑综合症病毒（WSSV）感染。LvTRAF3的RNAi敲低体内大大增加了WSSV感染虾的WSSV基因转录，病毒载量和死亡率。接下来，我们发现LvTRAF3不能诱导NF-κB通路的激活，这对于介导抗病毒免疫力的抗菌肽（AMPs）的合成至关重要。具体而言，在双荧光素酶报告基因检测中，LvTRAF3无法激活几种具有NF-κB结合位点的启动子，包括来自WSSV基因的启动子（wsv069， wsv056和 wsv403）， 果蝇AMP或虾类AMP。因此，当在WSSV感染期间将TRAF3敲低时，虾AMPs的mRNA水平没有显着改变。相反，我们发现LvTRAF3通过IRF-Vago抗病毒级联信号传递。LvTRAF3在LvIRF的上游起作用，以调节LvIRF的表达。LvVago4 和 LvVago5 在WSSV感染期间 体内。综上所述，这些数据提供了通过IRF-Vago途径而非NF-κB途径激活LvTRAF3参与WSSV宿主防御的实验证据。