Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural proteins

Highlights

• Yeast two-hybrid assay showed that ALFPm3 interacted with 5 WSSV structural proteins.

• Out of ALFPm3-interacting proteins, the interaction between WSSV189 and ALFPm3 was confirmed in vitro.

• The WSSV189 protein could interfere with the WSSV neutralizing activity of rALFPm3.

• For the first time, WSSV189 was identified as a WSSV envelope protein.

• The ALFPm3 performs its anti-WSSV action by binding to the WSSV189 envelope protein and possibly other WSSV proteins.

Abstract

In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24 h and 48 h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter’s neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.

Introduction

White spot syndrome virus (WSSV) is a large enveloped virus causing a severe infectious disease in shrimp as well as in other crustaceans. The WSSV infection in shrimp causes a 100% mortality rate within 7–10 d and results in enormous economic losses in the shrimp farming industry (Flegel, 1997). Thus, several preventive and curative measures have been developed, such as vaccination (Rout et al., 2007, Satoh et al., 2008), immunostimulants (Chotigeat et al., 2004, Balasubramanian et al., 2008), direct neutralization by antiviral proteins (Dupuy et al., 2004, Tharntada et al., 2009) and RNAi (Ongvarrasopone et al., 2008), although they have not been successfully implemented in shrimp farms. In addition, the innate immunity in shrimp has been studied intensively in order to understand the response of shrimp to viral infections, including to WSSV (Liu et al., 2009).

Antimicrobial peptides (AMPs) are effector molecules that play an important role in the innate immune system and function as the first line of defense against invading microorganisms (Hancock et al., 2006). AMPs are active against a large spectrum of microorganisms: bacteria, virus, yeast, parasites and fungi, and even against tumor cells. Some AMPs have been shown to have antimicrobial activity and directly kill pathogens, although others appear to participate in immunoregulatory mechanisms by modulating signal transduction and cytokine production and/or release (Brown and Hancock, 2006, Nagaoka et al., 2012). In response to viral infection, target cells can produce several antiviral factors to control viral invasion, including AMPs that are typically induced in the early innate immune response. AMPs may directly act on viral virions or indirectly suppress viral replication (Mulder et al., 2013). In mammals, it has been reported that lactoferrin, an iron binding glycoprotein, exhibits antimicrobial activity and is involved in the antiviral mechanism. Lactoferrin prevents viral infection of the host cell either by direct binding to virus particles or by binding to the viral receptor or co-receptor molecules of host cells (van der Strate et al., 2001). Indolicidin, an AMP, inactivates HIV-1 by damaging the virion membrane (Robinson et al., 1998), whilst α-defensin peptides are able to inhibit the assembly of polyomavirus particles (Dugan et al., 2008).

Anti-lipopolysaccharide factor (ALF) is an AMP that additionally contains the LPS-binding domain and has been found in many crustaceans, including shrimp. In Penaeus monodon, transcripts of the most abundant isoform (ALFPm3) are upregulated upon Vibrio harveyi or WSSV infection revealing its potential role(s) in the shrimp immune response (Somboonwiwat et al., 2008, Ponprateep et al., 2012). Recombinant ALFPm3 (rALFPm3) protein has been found to exhibit antimicrobial activity against both Gram-negative and Gram-positive bacteria as well as fungi (Somboonwiwat et al., 2005), whilst the antimicrobial action of ALFPm3 against Gram-negative bacteria has been implied to involve bacterial membrane disruption (Jaree et al., 2012). However, ALFPm3 is the only shrimp AMP that is currently reported to exhibit an anti-WSSV activity, where it can efficiently neutralize bacterial pathogens and also protect P. monodon from WSSV infection (Tharntada et al., 2009). The antiviral activity of ALF derivatives has also been reported, where the cyclic synthetic fragment of ALF exhibited antiviral activity against nervous necrosis virus (NNV), a fish non-enveloped viral pathogen, by agglutinating the NNV virions (Chia et al., 2010). In addition, a synthetic ALF based on the LPS-binding domain of Limulus ALF blocked the viral entry of various human pathogenic viruses, such as HIV-1, HCV and HSV1 and 2, by binding to the docking molecule on the host cell surface (Krepstakies et al., 2012, Hoffmann et al., 2014). However, the antiviral mechanism(s) of ALF against WSSV are not understood.

In this study, potential ALFPm3-interacting proteins of the WSSV were identified using yeast two-hybrid screening coupled with in vitro pull-down assay in order to investigate how the ALFPm3 acts on the WSSV. The effect of ALFPm3 binding to interacting WSSV proteins was demonstrated by testing the WSSV-neutralizing activity of the rALFPm3 compared to that pre-incubated with the selected WSSV protein. The direct binding between ALFPm3 and WSSV proteins implied a potential rational for how ALFPm3 neutralizes WSSV.