White spot syndrome virus (WSSV) is a large, enveloped, double-stranded DNA virus that threatens shrimp aquaculture worldwide. So far, the mechanisms of WSSV-host interactions are ill-defined. Recent studies have revealed that IE1, an immediate-early protein of WSSV, is a multifunctional modulator implicated in virus–host interactions. In this study, the functions of IE1 were further explored by identifying its interacting proteins using GST-pull down and mass spectrometry analysis. A total of 361 host proteins that potentially bind to IE1 were identified. Bioinformatics analysis revealed that the identified IE1-interactors wereinvolved in various signaling pathways such as prophenoloxidase (proPO) system, PI3K-AKT, and MAPK. Among these, the regulatory role of IE1 in shrimp proPO system was further studied. The Co-immunoprecipitation results confirmed that IE1 interacted with the Ig-like domain of Penaeus vannamei proPO or proPO-like protein (hemocyanin). Additionally, we found that knockdown of IE1 reduced viral genes expression and viral loads and increased the hemocytes’ PO activity, whereas recombinant IE1 protein inhibited the PO activity in a dose-dependent manner. Finally, we demonstrated that WSSV could suppress the hemocytes’ PO activity at the early infection stage. Collectively, our current data indicate that IE1 is a novel viral regulator that negatively modulates the shrimp proPO system.

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For DNA viruses, the immediate-early (IE) proteins are generally essential regulators that manipulate the host machinery to support viral replication. Recently, IE1, an IE protein encoded by white spot syndrome virus (WSSV), has been demonstrated to function as a transcription factor. However, the target genes of IE1 during viral infection remain poorly understood. Here, we explored the host target genes of IE1 using RNAi coupled with transcriptome sequencing analysis. A total of 429 differentially expressed genes (DEGs) were identified from penaeid shrimp, of which 284 genes were upregulated and 145 genes were downregulated after IE1 knockdown. GO and KEGG pathway enrichment analysis revealed the identified DEGs are significantly enriched in the minichromosome maintenance (MCM) complex and DNA replication, indicating that IE1 plays a critical role in DNA replication control. In addition, it was found that Penaeus vannamei MCM complex genes were remarkably upregulated after WSSV infection, while RNAi-mediated knockdown of PvMCM2 reduced the expression of viral genes and viral loads at the early infection stage. Finally, we demonstrated that overexpression of IE1 promoted the expression of MCM complex genes as well as cellular DNA synthesis in insect High-Five cells. Collectively, our current data suggest that the WSSV IE1 protein is a viral effector that modulates the host DNA replication machinery for viral replication.