PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp.
Our aim was to isolate and characterize white spot syndrome virus (WSSV)-binding proteins from shrimp. After a blot of shrimp hemocyte membrane proteins was overlaid with a recombinant WSSV envelope protein (rVP28), the reactive bands on the blot were detected using anti-VP28 antibody. Among three membrane-associated molecules identified by liquid chromatography-tandem mass spectrometry, there was a 25-kDa protein that bound to both rVP28 and WSSV. Since it had a primary structure with high homology to the small GTP-binding protein Rab7, we named it Penaeus monodon Rab7 (PmRab7). The full-length PmRab7 cDNA was obtained, and results from a glutathione S-transferase pull-down assay confirmed specific binding to rVP28. Reverse transcriptase PCR analysis revealed PmRab7 expression in many tissues, and real-time PCR analysis revealed that expression was constitutive. Binding of PmRab7 to rVP28 or WSSV occurred in a dose-dependent manner and was inhibited by anti-Rab7 antibody. In an in vivo neutralization assay, the number of dead shrimp after challenge with WSSV plus PmRab7 (15%) or WSSV plus anti-Rab7 antibody (5%) was significantly lower than after challenge with WSSV alone (95%). In contrast to the WSSV-injected group, shrimp injected with WSSV plus PmRab7 or WSSV plus anti-Rab7 showed no WSSV-type histopathology. We conclude that PmRab7 is involved in WSSV infection in shrimp. This is the first study to identify a shrimp protein that binds directly to a major viral envelope protein of WSSV.
Sritunyalucksana
Kallaya
K
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand.
Wannapapho
Wanphen
W
Lo
Chu Fang
CF
Flegel
Timothy W
TW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D033561:Penaeidae / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014759:Viral Envelope Proteins / Q000235:genetics / Q000502:physiology*; D014777:Virus Diseases / Q000235:genetics / Q000378:metabolism / Q000821:virology; D046848:White spot syndrome virus 1 / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D002352:Carrier Proteins; D004279:DNA, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D000090802:rab7 GTP-Binding Proteins; D004247:DNA; D020691:rab GTP-Binding Proteins