Cell surface transglutaminase required for nodavirus entry into freshwater prawn hemocytes

To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F₁-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.