Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis

Abstract

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.

FIG. 1.

Organization of the SGIV genome. The SGIV genome is shown in a linear format. A total of 162 ORFs, predicted by the FGENESV program (available through: http://www.softberry.com), supplemented with Vector NTI suite 7.1, are indicated by their locations, orientations, and putative sizes. Blue arrows represent ORFs with known function, while red arrows represent ORFs detected by RT-PCR. “M” represents an ORF whose expressed product was identified by MALDI-TOF mass spectrometry. Yellow lines represent repetitive sequence regions. The scale is in 5 kbp.

FIG. 2.

Sequence alignment of selective SGIV ORF060R, ORF152L, and ORF076L with other known proteins. The homologous regions are shaded (black represents identical, grey represents conservative). The positions of the amino acid sequence are indicated on the left of the sequence. (A) Alignment of deduced amino acids of SGIV, ORF060R, accession no. AAS18075; TFV, ORF009L, accession no. NP_571991; ATV, ORF007L, accession no. AAP33184; RRV, accession no. AAK53744; LCDV, ORF132L, accession no. NP_078720; ISKNV, ORF063L, accession no. NP_612285; and CIV, ORF022L, accession no. NP_149485. (B) Alignment of deduced amino acids of SGIV, ORF152R, accession no. AAS18167; ATV, ORF050R, accession no. AAP33229; EHNV, accession no. CAB37349; TFV, ORF056L, accession no. NP_571999; and CIV, ORF161L, accession no. NP_149624. (C) Alignment of deduced amino acids of SGIV, ORF076L, AAS18091; human, Homo sapiens, accession no. NP_000261; cattle, Bos taurus, accession no. AAB34886; and mouse, Mus musculus, accession no. BAB25491.

FIG. 3.

Phylogenetic relationship of SGIV with representative iridoviruses. The analysis was based on the multiple alignments of the protein sequences of the major capsid protein and ATPase of iridoviruses. (A) SGIV, ORF072R, accession no. AAS18087; ATV ORF014L, accession no. AAP33191; ISKNV, ORF006L, accession no. AAL72276; TFV, ORF096R, accession no. AAK55105; FV3, accession no. AAB01722; EHNV, accession no. AAO32315; LCDV-1, ORF147L, accession no. AAC24486; CIV ORF274L, accession no. AAK82135; CZIV, accession no. AAB82569; RSBI, accession no. AAP74204; WIV, accession no. AAB82568; TIV, accession no. VCXFTI; and SIV, accession no. VCXFSI. (B) SGIV, ORF134L, accession no. AAS18149; TFV, ORF016R, accession no. AAL77796; ATV, ORF083L, accession no. AAP33264; FV3, accession no. AAA43823; SOV, accession no. AAN77575; ISKNV, ORF122R, accession no. 98847; GIV, accession no. AAL68652; RSBI, accession no. BAA28670; SCV, accession no. AAL73346; LCDV, ORF054R, accession no. NP_078656; and CIV, 075L, accession no. AAB94422.

FIG. 4.

Conserved segments between the SGIV and ATV genomes. Both genomes are linearized and shifted genes encoding MCP as the start point. Only linked genes or annotated ORFs are indicated. Straight lines represent the gene linkages between two species. Black bars indicate the conserved syntenic regions of both genomes.

FIG. 5.

SDS-PAGE of SGIV proteins. Viral proteins were purified and separated via one-dimensional SDS-PAGE. Thirty-nine visible gel-separated protein bands were excised and digested enzymatically, and their mass spectra were obtained and automatically searched against the SGIV ORF database. Twenty-six proteins were identified by MALDI-TOF mass spectrometry. However, peptide signals from bands 26, 35, 36, 37, 38, and 39 were too low to give satisfactory identification.

FIG. 6.

Amplification of 20 novel genes of SGIV via RT-PCR. Total RNA (harvested after 48 h of infection) was isolated by using the RNeasy Mini kit and amplified by using the OneStep RT-PCR kit. Full lengths of 14 genes were amplified (A and B). Partial sequences were acquired from another six genes (C). Lanes C, control; lane M, 1-kb DNA ladder (Promega).