The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found

Abstract

One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.

FIG. 1.

(A) SDS-PAGE profile of purified WSSV virion. The uppermost band (VP664; arrow) was excised from the gel and subjected to LC-MS/MS analysis. The peptide sequences suggested that this protein was the translated product of the vp664 ORF. Several other major structural proteins, VP28, VP26, and VP24, are also indicated. (B) Amino acid sequence of VP664. The peptide sequences identified by LC-MS/MS are indicated by underlining. Three bipartite nuclear targeting sequences are boxed. The RGD motif at positions 395 to 397 is indicated by double underlining.

FIG. 2.

Schematic diagram showing the regions of VP664 used for antibody preparation and RT-PCR analysis. The separate shaded boxes represent the amino acid sequences corresponding to the recombinant proteins used for antibody production. The labeling of the RT-PCR products shows the primer sets that were used for amplification (Table 2). The locations of two structural features, the RGD motif and bipartite NLSs, are indicated on the protein, and the WSSV diagnostic PCR primer set developed by our laboratory, pms146F1 and pms146R1 (23), is also indicated.

FIG. 3.

Specificities of six VP664 antibodies as tested by Western blotting. (A) The purified virions were subjected to SDS-PAGE, and the separated viral proteins were transferred onto a PVDF membrane and probed with different antibodies diluted 5,000-fold. (B) Results of probing with the 664-1 antibody at higher dilutions (10,000, 50,000, and 100,000).

FIG. 4.

Electron micrographs of purified virions. (A) The white outlines indicate (i) a complete mature virion with a characteristic tail, (ii) a ruptured mature virion with more than half of the nucleocapsid exposed outside of the envelope, and (iii) a completely exposed mature nucleocapsid. (B) Immature, naked nucleocapsid prior to being enveloped.

FIG. 5.

Immunoelectron microscopy analysis of purified virions probed with VP664 antibody. (A and B) The antibody specifically binds to the nucleocapsid and not to the viral envelope. (C) Most of the gold particles are localized to the perimeter of the nucleocapsid. (D) Occasionally, the gold particles are localized across the top of a nucleocapsid. (E) A preimmune rabbit antibody or gold-conjugated secondary antibody does not bind to virions.

FIG. 6.

Time course analysis of vp664 transcripts by RT-PCR. Total RNAs were extracted from the pleopods of WSSV-infected shrimp and were subjected to RT-PCR analysis with the indicated primers, i.e., vp664, dna pol, and vp28. Shrimp actin was also included as a template control. Lane headings show times postinfection (hours). M, DNA marker.

FIG. 7.

Partial nucleotide sequences of 5′ and 3′ untranslated regions of vp664. (A) Sequencing results for six RT-PCR clones showed that there are two transcription initiation sites for vp664. The putative transcription initiation sites are shown in bold and underlined. The inverted repeats are boxed. (B) Underlining indicates the locations of two polyadenylation signals (AATAAA) downstream of the stop codon. The poly(A) addition site, as determined by 3′ RACE, occurs 12 bp downstream of the first polyadenylation signal and is indicated by a box.

FIG. 8.

Analysis of the entire 18-kb vp664 transcript by RT-PCRs with 15 sets of primer pairs. Lane headings show the primer pairs, as listed in Table 2. Three reactions were carried out for each primer pair as follows: RNA lanes, total RNA as a negative control; vDNA lanes, viral genomic DNA as a positive control; and cDNA lanes, reverse-transcribed cDNA as a PCR template.