Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with 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.
Section snippets
Construction of the ALFPm3 bait vector
The DNA sequence corresponding to a mature peptide of ALFPm3 was amplified by PCR from a pBluescript SK plasmid containing the ALFPm3 gene using a specific primer pair; ALFPm3F and ALFPm3R (Table 1), in which the EcoRI and BamHI recognition sites, respectively, were included at their 5′ ends. The PCR product was cut with EcoRI and BamHI and then cloned in-frame into the pGBKT7 bait vector, digested with the same restriction enzymes. The recombinant plasmid was then isolated and subjected to
Identification of ALFPm3 binding proteins
With regards to the evidence of ALF possessing anti-WSSV activity (Tharntada et al., 2009, Liu et al., 2006), it was of interest to study how ALF performs its activity against WSSV. A yeast two-hybrid assay was employed to identify potential WSSV proteins that could interact with ALFPm3. The gene coding for the mature ALFPm3 was cloned into the pGBKT7 vector and the resulting recombinant plasmid was used as a bait vector for screening for ALFPm3-interacting proteins in the WSSV ORF library.
Discussion
AMPs can possess an antiviral activity as well as an antibacterial activity. The antiviral properties of several AMP families against both enveloped and non-enveloped viruses have been characterized (Findlay et al., 2013), including for ALFPm3 that is an antimicrobial peptide from P. monodon that exhibits an anti-WSSV activity. The interaction between WSSV-binding proteins and viral proteins has been reviewed recently (Sritunyalucksana et al., 2013). Several shrimp secreted and cell-surface
Acknowledgements
We would like to thank Dr. Chen Li-Li from Institute of Marine Biology, National Taiwan Ocean University, for technical support on IEM experiment. This research received financial support from the NRCT-JSPS ASIAN CORE Program in Fishery Science, Project: Development of New Biotechnology for Aquaculture and Risk Management of Aquaculture Products, and from the TRF Senior Research Scholar (RTA5580008), Thailand Research Fund. Student scholarships from the Thailand National Center for Genetic
References (44)
- et al.
Studies on the immunomodulatory effect of extract of Cynodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV)
Fish Shellfish Immunol.
(2008) - et al.
Cationic host defense (antimicrobial) peptides
Curr. Opin. Immunol.
(2006) - et al.
Characterization of a novel envelope protein WSV010 of shrimp white spot syndrome virus and its interaction with a major viral structural protein VP24
Virology
(2007) - et al.
Antimicrobial peptides (AMP) with antiviral activity against fish nodavirus
Fish Shellfish Immunol.
(2010) - et al.
Effect of fucoidan on disease resistance of black tiger shrimp
Aquaculture
(2004) - et al.
Transcriptional profile of genes involved in oxidative stress and antioxidant defense in PC12 cells following treatment with cerium oxide nanoparticles
Biochim. Biophys. Acta
(2014) - et al.
Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells
J. Biol. Chem.
(2008) - et al.
Host defence peptides from invertebrates-emerging antimicrobial strategies
Immunobiology
(2006) - et al.
Effect of the anti-lipopolysaccharide factor isoform 3 (ALFPm3) from Penaeus monodon on Vibrio harveyi cells
Dev. Comp. Immunol.
(2012) - et al.
Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes
Mol. Cell. Proteomics
(2007)
Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp
Virology
Antiviral immunity in crustaceans
Fish Shellfish Immunol.
Interaction between white spot syndrome virus VP26 and hemocyte membrane of shrimp, Fenneropenaeus chinensis
Aquaculture
Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections
Fish Shellfish Immunol.
DNA vaccines encoding viral envelope proteins confer protective immunity against WSSV in black tiger shrimp
Vaccine
Construction and application of a protein interaction map for white spot syndrome virus (WSSV)
Mol. Cell. Proteomics
Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon
Dev. Comp. Immunol.
Localization of anti-lipopolysaccharide factor (ALFPm3) in tissues of the black tiger shrimp, Penaeus monodon, and characterization of its binding properties
Dev. Comp. Immunol.
Reprint of: virus-binding proteins and their roles in shrimp innate immunity
Fish Shellfish Immunol.
Antiviral activities of lactoferrin
Antiviral Res.
A simple and efficient method for purification of intact white spot syndrome virus (WSSV) viral particles
Virus Res.
Interaction of white spot syndrome virus VP26 protein with actin
Virology
Cited by (0)
- 1
These authors contributed equally to this work.