Suppression of Shrimp Melanization during White Spot Syndrome Virus Infection

Kornsunee Phiwsaiya,Anchalee Tassanakajon,Walaiporn Charoensapsri,Piti Amparyup,Jantiwan Sutthangkul,Saengchan Senapin

doi:10.1074/jbc.m114.605568

Abstract

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

Highlights

Melanization plays a major role in invertebrate defense
We show the importance of the P. monodon melanization reaction in the defense against white spot syndrome virus (WSSV)
In vivo gene co-silencing of PmproPO1 and PmproPO2 followed by WSSV infection resulted in a higher mortality rate of shrimp at 1–3 dpi compared with the control shrimp (Fig. 1), implicating the role of proPO system in defense against virus

Summary

Background

Melanization plays a major role in invertebrate defense. Results: Suppression of shrimp melanization increased the WSSV susceptibility. The control and disabling of the host melanization was reported in M. sexta [31], where oviposition of the wasp Microplitis demolitor in M. sexta larvae uses the M. demolitor bracovirus (MdBV) that encodes for epidermal growth factor-like motif (Egf) proteins Egf1.0 and Egf1.5 These small serine proteinase inhibitors are capable of suppressing the melanization response of host insects by inhibiting the PPAEs and so inhibit the proteolytic cleaving of proPO into the active PO [32, 33]. This study provides the first demonstration that melanization could have an antiviral role in shrimp and that as part of WSSV pathogenesis, the virus might have the ability to suppress the melanization response of the shrimp via the inhibition of the proPO proteinase cascade, analogous to that found in insects. This study reveals the crucial hostpathogen interaction during WSSV infection

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION