Interaction of the small GTP-binding protein (Rab7) with β-actin in Litopenaeus vannamei and its role in white spot syndrome virus infection

Abstract In this study, GST Pull-down and mass spectrometry was applied to the precipitation and identification of the small GTP-binding protein (Rab7) interacting protein in hemocyte of Litopenaeus vannamei . According to the search in GenBank with the peptide mass fingerprint, the 45 kDa protein which was pulled down with the GST-tagged Rab7 (GST-Rab7, GTP bound form) was identified to be β-actin with 28% coverage of amino acid sequences. The interaction of Rab7 with β-actin was verified by both GST Pull-down and ELISA in vitro . Meanwhile, confocal microscopic observation showed that Rab7 could be co-localized with β-actin in hemocytes at 12 h post white spot syndrome virus (WSSV) infection (hpi). GST Pull-down and western blotting were used to analyze the cross-interaction between WSSV VP28, Rab7 and β-actin. The results showed that the GST-VP28, His-tagged Rab7 (His-Rab7) and His-β-actin formed a tripartite complex. At 12 hpi, confocal microscopic observation showed that WSSV could be co-localized with Rab7 and β-actin in hemocytes respectively. Furthermore, based on the in vivo neutralization assay, recombinant His-β-actin accelerated the infection of WSSV, conversely, recombinant His-Rab7 delayed WSSV infection in shrimp. These results suggested the interaction of Rab7 with β-actin and this interaction was involved in WSSV infection.