Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural 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 (24h and 48h 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.

RNAi screening identifies a new Toll from shrimp that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

“…Previous reports show that shrimp ALF can protect against WSSV infection via interfering with viral replication in vitro and in vivo in crayfish Pacifastacus leniusculus (59) and CqALF can disrupt WSSV envelope integrity that leads to the decrease of WSSV infectivity in the red claw crayfish Cherax quadricarinatus (60). Furthermore, an ALF isoform 3 from P. monodon has performed its anti-WSSV action by binding to several viral structural proteins such as wsv131 (WSSV186), wsv134 (WSSV189) and wsv339 (WSSV395) (61). On the other hand, lysozyme is a key effector of the innate immune system and kills bacteria by catalytic hydrolysis of cell wall peptidoglycan, but it also exhibits catalysis-independent antimicrobial properties.…”

Antiviral action of the antimicrobial peptide ALFPm3 from Penaeus monodon against white spot syndrome virus

“…In this research, four recombinant ALFPm3-interacting proteins from WSSV, WSSV186 (rWSSV186), WSSV395 (rWSSV395), WSSV458 (rWSSV458), and WSSV471 (rWSSV471) were expressed in the Escherichia coli expression system and purified as described in supplementary data. The purified rALFPm3 protein was prepared according to the method described by Suraprasit et al, 2014. To produce the WSSV proteins tagged with 6× His-tag (rWSSV186, rWSSV395, rWSSV458, and rWSSV471), each recombinant clone was constructed. Expression and purification of each rWSSV protein with hexahistidine (His6) at its N-terminus were carried out under native conditions as previously described (Suraprasit et al, 2014).…”

“…The purified rALFPm3 protein was prepared according to the method described by Suraprasit et al, 2014. To produce the WSSV proteins tagged with 6× His-tag (rWSSV186, rWSSV395, rWSSV458, and rWSSV471), each recombinant clone was constructed. Expression and purification of each rWSSV protein with hexahistidine (His6) at its N-terminus were carried out under native conditions as previously described (Suraprasit et al, 2014). The rWSSV186 and rWSSV395 proteins were dialyzed and further purified using DEAE Sepharose fast flow (GE Healthcare).…”

“…In vitro pull-down assays were modified on the basis of described protocols (Suraprasit et al, 2014). For one in vitro reaction, 300 g of rWSSV395 protein was incubated with DEAE Sepharose fast flow (GE Healthcare) at 4 °C for 1 h with gentle rocking motion on a rotating platform with the DEAE beads pre-equilibrated with the wash solution (50 mM Tris-HCl, pH 7.4 containing 200 mM NaCl).…”

“…Previously, yeast-two hybrid screening was performed to elucidate the anti-WSSV action of ALFPm3 (Suraprasit et al, 2014). Several WSSV proteins such as WSSV186, WSSV189, WSSV395, WSSV458, and WSSV471 could interact with ALFPm3 but only WSSV189 was characterized, leaving the other WSSV proteins for further studies.…”