Isolation of an apoptosis suppressor gene of the Spodoptera littoralis nucleopolyhedrovirus

Abstract

Spodoptera frugiperda SF9 cells infected with mutants of the Autographa californica nucleopolyhedrovirus (AcMNPV) which lack a functional p35 gene undergo apoptosis, aborting the viral infection. The Spodoptera littoralis nucleopolyhedrovirus (SlNPV) was able to suppress apoptosis triggered by vDeltaP35K/pol+, an AcMNPV p35 null mutant. To identify the putative apoptotic suppressor gene of SlNPV, overlapping cosmid clones representing the entire SlNPV genome were individually cotransfected along with genomic DNA of vDeltaP35K/pol+. Using this complementation assay, we isolated a SlNPV DNA fragment that was able to rescue the vDeltaP35K/pol+ infection in SF9 cells. By further subcloning and rescue, we identified a novel SlNPV gene, Slp49. The Slp49 sequence predicted a 49-kDa polypeptide with about 48.8% identity to the AcMNPV apoptotic suppressor P35. SLP49 displays a potential recognition site, TVTDG, for cleavage by death caspases. Recombinant AcMNPVs deficient in p35 bearing the Slp49 gene did not induce apoptosis and showed successful productive infections in SF9 cells, indicating that Slp49 is a functional homologue of p35. A 1.5-kbp Slp49-specific transcript was identified in SF9 cells infected with SlNPV or with vAc496, a vDeltaP35K/pol+-recombinant bearing Slp49. The discovery of Slp49 contributes to the identification of important functional motifs conserved in p35-like apoptotic suppressors and to the future isolation of p35-like genes from other baculoviruses.

FIG. 1

SlNPV infection of SF9 cells prevents induction of apoptosis by an AcMNPV p35 null mutant or by actinomycin D. DNA was extracted from 3 × 10⁵ SF9 cells infected with SlNPV at a multiplicity of infection of 5 (Sl + lanes indicated at the bottom of the figure). (A) vΔ35K/pol⁺ at a multiplicity of infection of 5 was added to the cells (Ac + lanes indicated at the bottom of the figure) at 0, 12, and 24 h after SlNPV infection (lanes 1 and 2, 3 and 4, and 5 and 6, respectively). At 24 h later, a sample for each pair of time points was extracted and analyzed by agarose electrophoresis. (B) Actinomycin D (250 ng/ml) was added at 1, 5, 24, and 48 h after SlNPV infection (lanes 1, 2, 3, and 4, respectively) or at 48 h for mock-infected cells (lane 5). The cells were harvested at 24 h after actinomycin D addition. Size markers in kilobase pairs are indicated on the right.

FIG. 2

Rescue of occluded AcMNPV by cosmids and plasmids bearing SlNPV DNA fragments. (A) NotI linear restriction map of SlNPV. The scales above indicate SlNPV map units (mu) and kilobase pairs. The bars below (C80, C50, C3, and C50) represent the various overlapping cosmids of the genomic SlNPV cosmid library. (B) Restriction map of the cosmid C50 and individual plasmid subclones indicating their ability to rescue or not (+ and −, respectively) the replication of vΔ35K/pol⁺, as detected by the presence of polyhedra in the nuclei of SF9 cells cotransfected with vΔ35K/pol⁺ and plasmid DNA. N, NotI; P, PstI; A, ApaI. (C) Restriction map of the ApaI-PstI region corresponding to SlNPV 31.0 to 39.6 map units. S, SalI; K, KpnI; E, EcoRI. Also, subclones able to rescue vΔ35K/pol⁺ polyhedron formation, as in panel B, are indicated.

FIG. 3

ORF analysis of the SlNPV genome, at map units 37.0 to 38.7, and sequence of the Slp49 gene. (A) Diagram of ORFs in six reading frames. Vertical bars indicate stop codons. The location of the 446-amino-acid (aa) product corresponding to SLP49 is labeled. ORFs encoding products longer than 100 amino acids are indicated by open arrows. (B) Nucleotide and predicted amino acid sequence of SLP49. The early TATA box and a late transcriptional start site are indicated (open squares). A polyadenylation signal is underlined.

FIG. 4

Comparison of Slp49 and p35 predicted amino acid sequences. The alignment was performed with the GAP program (11). The P35 sequence was reported previously (13). Horizontal dots indicate gaps made to optimize the alignment. Vertical bars, identical amino acids; vertical dots, similar amino acids.

FIG. 5

Contribution of the Slp49 3′ end and SLP49 C terminus to the rescue of the replication of vΔ35K/pol⁺. The plasmids, pES2 bearing the complete Slp49 ORF, pESΔS1-2 and pKSΔS1-2, with the Slp49 3′ end deleted, and pESterm, displaying a frameshift causing a mutation in SLP49 amino acid 352 resulting in termination of the peptide at amino acid 364, were cotransfected separately with vΔP35K/pol⁺ DNA. Success (+) or failure (−) to rescue vΔ35K/pol⁺ replication was monitored as indicated in Fig. 2. S, SalI; K, KpnI; E, EcoRI; P, PstI.

FIG. 6

Infection of SF9 cells with a recombinant virus bearing the Slp49 gene. (A) Extracts from SF9 cells (4 × 10⁵) were mock infected (lane 1) or infected at a multiplicity of infection of 10 with vΔ35K/pol⁺, vAcp496, or AcMNPV (lanes 2, 3, and 4, respectively), harvested at 48 h after infection, and subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis with anti-polyhedrin antiserum (7). (B) Extracts from SF9 cells were mock infected (lanes 1 and 2) or infected at a multiplicity of infection of 10 with vΔP35K/pol⁺ (lanes 3 and 6) or vAcp496 (lanes 4 and 7) or AcMNPV (lanes 5 and 8). The cells were harvested 12 and 24 h later (lanes 1 and 3 to 5 and lanes 2 and 6 to 8, respectively) and analyzed as in panel A with anti-P35 antiserum. Molecular mass markers are indicated on the right.

FIG. 7

vAc-Slp49 recombinants bear the Slp49 gene. Restriction enzyme digestion (A and C) and Southern blot analysis (B and D). (A and B) HindIII or PstI-digested DNA (indicated at the top of the figure) from AcMNPV (lanes 1 and 18), vΔ35K/pol⁺ (lanes 2 and 17), SlNPV (lanes 3 and 16), or polyhedron-positive-phenotype recombinants vAcp491 to vAcp496 (lanes 4 to 9 and lanes 10 to 15). (C and D) BglII-SalI-digested DNA from AcMNPV (lane 1), vΔ35K/LacZ (lane 2), vΔ35K/pol⁺ (lane 3), pES (lane 4), SlNPV (lane 5), or polyhedron-positive-phenotype recombinants vAcp491 to vAcp496 (lanes 6 to 11). A BglII-SalI 856-bp ³²P-labeled Slp49 fragment (Fig. 3A) was used for hybridization.

FIG. 8

vAcp496 suppresses apoptosis in SF9 cells. DNA extracted from the cells infected with vΔ35K/pol⁺, AcMNPV, or vAcp496 (indicated at the top of each lane) was analyzed by agarose gel electrophoresis. mi, mock-infected cells. Size markers are indicated on the left.

FIG. 9

Temporal analysis of Slp49 transcription in SlNPV- and vAcp496-infected SF9 cells. A Northern blot of total RNA extracted from mock-infected cells (lane 1) or cells infected with AcMNPV (lanes 2 and 3), vΔ35K/pol⁺ (lanes 4 and 5), SlNPV (lanes 6 to 8), or vAcp496 (lanes 9 to 14) is shown. RNA was extracted at 3 h (lane 9), 6 h (lane 10), 9 h (lane 11), 12 h (lanes 2, 4, 6, and 12), 18 h (lanes 7 and 13), and 24 h (lanes 3, 5, 8, and 14) after infection. The ³²P-labeled Slp49 fragment used for hybridization was the same as in Fig. 7. Size markers (in kilobase pairs) are indicated on the right.