In this study, we characterize a novel white spot syndrome virus (WSSV) structural protein, VP51A (WSSV-TW open reading frame 294), identified from a previous mass spectrometry study. Temporal-transcription analysis showed that vp51A is expressed in the late stage of WSSV infection. Gene structure analysis showed that the transcription initiation site of vp51A was 135 bp upstream of the translation start codon. The poly(A) addition signal overlapped with the translation stop codon, TAA, and the poly(A) tail was 23 bp downstream of the TAA. Western blot analysis of viral protein fractions and immunoelectron microscopy both suggested that VP51A is a viral envelope protein. Western blotting of the total proteins extracted from WSSV virions detected a band that was close to the predicted 51-kDa mass, but the strongest signal was around 72 kDa. We concluded that this 72-kDa band was in fact the full-length VP51A protein. Membrane topology assays demonstrated that the VP51A 72-kDa protein is a type II transmembrane protein with a highly hydrophobic transmembrane domain on its N terminus and a C terminus that is exposed on the surface of the virion. Coimmunoprecipitation, colocalization, and yeast two-hybrid assays revealed that VP51A associated directly with VP26 and indirectly with VP28, with VP26 acting as a linker protein in the formation of a VP51A-VP26-VP28 complex.Viral structural proteins, especially the envelope proteins, are important, not only because they are involved in virion morphogenesis, but also because they are the first molecules to interact with the host. The structural proteins often play vital roles in cell targeting, virus entry, assembly, and budding (1, 2, 21, 22, 24), as well as triggering host antiviral defenses (26). In the case of white spot syndrome virus (WSSV) (genus Whispovirus, family Nimaviridae) (37), a double-stranded DNA virus that has caused severe mortality and huge economic losses to the shrimp farming industry globally for more than a decade (5, 19), proteomic methods have helped to identify a total of 58 structural proteins, over 30 of which are currently recognized as envelope proteins (13,31,44,47). Some of the WSSV envelope proteins involved in shrimp infection have been identified (12,14,34,36,41,43), and these envelope and other WSSV structural proteins have been used in various studies, including RNA interference-based gene knockdown to silence viral structural-protein gene expression (8, 27, 45), DNA and protein vaccination to elevate host immunity (25,29,36,39), and antibody neutralization techniques that neutralize the virus by preventing envelope proteins from interacting with host cell receptors (12,34,41,43).In the present paper, we characterize and investigate the functionality of a WSSV structural protein that was first reported by Tsai et al. (32). This protein, designated VP51A, corresponds to open reading frame 294 of the WSSV-TW isolate, and its gene encodes a polypeptide of 486 amino acids (aa) with a theoretical mass of 51 kDa. A method was recently establ...