Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon

Abstract

White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, β2, μ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2β and μ subunits of P. monodon are reported. PmAP-2 β was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2β delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2β co-localized with WSSV particles at plasma membrane. In addition, PmAP-2β-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2β is associated with the JAK/STAT and the Toll pathway.

PmAP2-β depletion enhanced activation of the Toll signaling pathway during yellow head virus infection in the black tiger shrimp Penaeus monodon

“…Next, the RNA interference experiment was carried out in order to investigate the influence of Pm AP2-β on signaling pathways and other immune-related genes during YHV infection. In previous research, we have shown that Pm AP2-β transcript can be efficiently suppressed by Pm AP2-β dsRNA 7 . Interestingly, expression of Pm Spätzle in Pm AP2-β silenced shrimp challenged with YHV was highly up-regulated by 31- and 33-fold at 6 and 12 hpi, compared with non-infected shrimp (Fig.…”

“…Pm AP2-β is a large subunit 2β of the AP-2 complex, which interacts with clathrin. Previously, Pm AP2-β has been characterized and was shown to play a role during WSSV infection 7 .…”

“…Double-strand RNA of Pm AP2-β and GFP were prepared as described previously 7 . In brief, the PCR products ( Pm AP2-β and GFP) were amplified separately by specific primers (Supplementary Information, Table S1 ) with the following conditions: 94 °C for 3 min (denaturation), followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and a final extension at 72 °C for 10 min.…”

“…AP2 consists of 4 subunits, including β2, α, µ2 and σ2. In P. monodon , the β2, µ2 and σ2 subunits of AP-2 have been characterized 4 , 7 .…”

“…Several viruses, including semliki forest virus 10 , vesicular stomatitis virus 11 , influenza A virus 12 , Foot- and mouth disease virus 13 and hepatitis C 14 , hijack the clathrin-mediated endocytosis to enter host cells. Previously, the white spot syndrome virus (WSSV), the most devastating pathogen in shrimp, was also reported to invade host cells via clathrin-dependent endocytic route 7 , 15 , 16 .…”