Generating a host range-expanded recombinant baculovirus

As baculoviruses usually have a narrow insecticidal spectrum, knowing the mechanisms by which they control the host-range is prerequisite for improvement of their applications as pesticides. In this study, from supernatant of culture cells transfected with DNAs of an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant lacking the antiapoptotic gene p35 (vAc∆P35) and a cosmid representing a fragment of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), a viral strain was plaque-purified and named vAcRev. vAcRev had a broader host range than either vAc∆P35 or SeMNPV parental virus, being able to infect not only the permissive hosts of its parental viruses but also a nonpermissive host (Spodoptera litura). Genome sequencing indicated that vAcRev comprises a mixture of two viruses with different circular dsDNA genomes. One virus contains a genome similar to vAc∆P35, while in the other viral genome, a 24.4 kbp-fragment containing 10 essential genesis replaced with a 4 kbp-fragment containing three SeMNPV genes including a truncated Se-iap3 gene. RNA interference and ectopic expression assays found that Se-iap3 is responsible for the host range expansion of vAcRev, suggesting that Se-iap3 inhibits the progression of apoptosis initiated by viral infection and promotes viral propagation in hosts both permissive and non-permissive for AcMNPV and SeMNPV.

Baculoviruses are insect pathogens commonly encountered in nature where they specifically infect insect hosts, mainly lepidopteran insects 1 . As baculoviruses are highly host specific and non-pathogenic to non-target organisms 2,3 , they have been used for integrated pest management as microbial pesticides. However, the fact that baculovirus infection is usually limited to a single or a few closely related insect species makes it less attractive economically. To improve the practical effectiveness of baculoviruses, a variety of studies have been conducted regarding the host range determining factors of baculoviruses and how they work.
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely researched member of the Baculoviridae family. Since the budded form of baculoviruses can enter a wide variety of invertebrate and vertebrate cells, host range is not determined at the level of receptor binding. Instead, earlier studies have reported that several AcMNPV genes such as p143 (helicase), hrf-1, hcf-1, ie2 and p35 are involved in host range determination 4,5 . The blockage of virus infection may vary at different stages in different virus-insect systems, but occur mainly at the steps of transport to the intracellular site of replication, viral gene expression, and generation of viral progeny [6][7][8][9][10][11] .
Caspases are a family of cysteine proteases that play essential roles in apoptosis 19 . During productive infection, baculoviruses interfere with apoptosis by expressing the apoptotic inhibitors P35/P49 or IAPs 20 . P35 is able to inhibit effector caspases, while P49, a P35 homolog, has similar predicted three-dimensional structure to P35 and inhibits initiator and effector caspases by the same mode of action used by p35 to inhibit effector caspases 21 . The other types of baculovirus anti-apoptotic proteins are IAP proteins, which are unrelated to P35/P49. IAPs contain two main motifs: one to three copies of the BIR (baculoviral IAP repeat) domain at their N-termini and a RING domain near their C-termini 6,22,23 .

Results
Obtaining a viral strain with a broader in vitro host range. To study the potential of the SeMNPV genome sustaining the replication of vAc ∆P35 , vAc ∆P35 DNA and a SeMNPV cosmid library, which consists of 5 cosmids and represents the entire viral genome 28 , were cotransfected into Sf9 cells. Although most of the transfected cells underwent apoptosis, polyhedral inclusion bodies (PIBs) were observed in a few cells, indicating that productive infection was established in these cells (Fig. 1Aa, arrowhead). By contrast, vAc ∆P35 -transfected Sf9 cells underwent apoptosis severely and no PIBs were formed (Fig. 1Ab); whereas no cytopathic effects were observed in the SeMNPV cosmid library-transfected cells 5 days post transfection (p.t.) (Fig. 1Ac). Surprisingly, when Sf9, SpLi-221, and Se301 cells were inoculated with the supernatants from the cotransfected cells, PIBs were produced in a few cells of all three cell lines (Fig. 1B, arrowheads), although most cells underwent apoptosis. These results indicated that progeny budded virions (BVs) were present in the supernatants. Because, consistent with previous studies, vAc ∆P35 infection induced apoptosis of Sf9, SpLi-221, and Se301 cells, but not Hi5 cells (Fig. 1D, upper panels), and SeMNPV infection induces apoptosis of SpLi-221 cells, the productive infection of these cells suggested that a viral strain with a broader host range was generated in the cotransfected cells. With 10 undiluted serial passages of the supernatant in Sf9, SpLi-221 and Se301 cells, the percentage of PIB-containing cells increased, and apoptosis was alleviated gradually (data not shown). Apparently, the PIB-positive viral strain became predominant and the proportion of apoptosis-inducing viruses decreased upon serial passage.
Sf9 cells were also cotransfected with vAc ∆P35 DNA and individual cosmids of the SeMNPV cosmid library. Only cosmid 22 could rescue the replication of vAc ∆P35 in Sf9 cells, while cells underwent severe apoptosis upon the cotransfection of vAc ∆P35 DNA with the other individual cosmids (data not shown). The results indicated that one or more genes in cosmid 22 could not only substitute the function of p35 to block apoptosis, but also allow virus replication in the semi-or non-permissive cell lines for AcMNPV.
Subsequently, plaque purification was carried out to isolate the PIB-positive viral strain. During the purification, three types of plaques were observed: Type I, PIB-positive; Type II, PIB-negative but with cytopathic effect; Type III, cells in the plaque underwent apoptosis (Fig. 1C). Notably, if the inoculum was diluted too much, only Type П and/or Type III plaques were formed. After four rounds of plaque purification focusing on Type I plaque, a viral strain was obtained and designated as vAcRev. vAcRev could stably establish productive infection in Sf9, Se301, SpLi-221 and Hi5 cell lines (Fig. 1D,

Comparisons of replication between vAcRev and wild type viruses in vitro and in vivo.
To determine any differences in the replication kinetics between vAcRev and wild type (wt) viruses in corresponding cells permissive to the wt viruses, growth curve assays were performed. There was no significant difference in replication kinetics among vAcRev, vAc WT and vAc ∆P35 in Hi5 cells ( Fig. 2A). The replication kinetics between vAcRev and vAc WT were similar in Sf9 cells (Fig. 2B). In contrast, the yields of infectious BV of SeMNPV and SpltNPV were around two logs lower than that of vAcRev in Se301 (Fig. 2C) and SpLi-221 (Fig. 2D) cells respectively, suggesting that the replication of vAcRev is higher than the two wt viruses in their host cell lines.
Bioassays showed that vAcRev could orally infect T. ni larvae; however, different symptoms were observed among the larvae infected with vAcRev, vAc ∆P35 and AcMNPV. The larvae that died of vAcRev or AcMNPV infection showed typical symptoms of baculovirus infection, but the larvae infected with vAc ∆P35 did not undergo Scientific RepoRts | 6:28072 | DOI: 10.1038/srep28072 liquefaction, which is consistent with a previous study 29,30 . The LD 50 values of vAcRev, vAc ∆P35 and AcMNPV were 82, 141.1 and 111.1 PIBs/larvae respectively, and the values were not significantly different between AcMNPV and vAc ∆P35 (P = 0.1256) or AcMNPV and vAcRev (P = 0.2914) ( Table 1). However, the LD 50 value for vAcRev was significantly lower than that for vAc ∆P35 (P = 0.0166).
Bioassays showed that vAcRev could orally infect S. exigua larvae, and no significant difference in infectivity was observed between AcMNPV, vAc ∆P35 and vAcRev, with the LD 50 values being 4219.4, 6214.2 and 5183.8 PIBs/larvae, respectively (Table 1). However, these viruses were not as infectious in S. exigua larvae as SeMNPV, which had an LD 50 value of 166.0 PIBs/larvae. Larvae that died after infection with AcMNPV or SeMNPV showed typical symptoms of baculovirus infection. In contrast, the dead larvae infected with vAcRev and vAc ∆P35 did not undergo liquefaction, but exhibited other symptoms of baculovirus infection.
The vAcRev genome was further sequenced by a massive parallel pyrosequencing technology (454 GS-FLX). A total of 127,575 high-quality reads with an average read length of 251 bp were produced. The total number of sequenced bases was 32,058,578 bp, which provided more than 100-fold coverage of the AcMNPV-C6 genome. Assembly performed by the Newbler software of the 454 suite package (454 Life Sciences) resulted in 9 large (defined as > 500 bp) contigs. The genome of AcMNPV-C6 was used as a reference to find a proper layout for the contigs. Gaps were filled through sequencing of PCR products by primer walking or specific oligonucleotide primers targeting contig ends between two adjacent contigs. The resulting sequence data were assembled into two circular dsDNA molecules. The results suggest that vAcRev contains two distinct circular dsDNA genomes. One genome was 118,582 bp in length and was designated as vAcRev-1 (GenBank accession no. KU697902), while the other was 138,991 bp and was designated as vAcRev-2 (GenBank accession no. KU697903).
Except for several ORFs which have a single nucleotide inserted or substituted, vAcRev-2 and vAc ∆P35 share over 99% nucleotide sequence or amino acid identities between the corresponding ORFs. Since vAc ∆P35 cannot replicate efficiently in Sf9, Se301 and SpLi-221 cell lines due to apoptosis, it is expected that vAcRev-2 would induce apoptosis and have aborted replication in these cell lines as well.
As single enzyme Bsu36I loci were found in both of the genome of vAcRev-1 and vAcRev-2, vAcRev DNA was linearized by Bsu36I digestion and then analyzed by pulsed field gel electrophoresis. The electrophoretogram exhibited two enzyme-digested fragments consistent with the expected sizes of vAcRev-1 and vAcRev-2 (Fig. 3B). This result confirmed that vAcRev is composed of a mixture of vAcRev-1 and vAcRev-2. The intensity of the two molar fragments is comparable, indicating that the molar ratio of vAcRev-1 and vAcRev-2 was approximately 1:1.
The above genomic analyses, together with the plaque morphology results, suggested that vAcRev-1 and vAcRev-2 cannot replicate individually in Sf9, Se301 and SpLi-221 cell lines due to the lack of essential genes and lack of ability to inhibit apoptosis, respectively. Thus, we argue that vAcRev-1 and vAcRev-2 exist in a mutualistic relationship where in vAcRev-1 provides an anti-apoptotic gene (the truncated Se-iap3 in vAcRev-1, which we have named vAcRev-iap3), while vAcRev-2 provides the essential genes which vAcRev-1 lacks. were digested with HindIII, BamHI or NcoI, and the digested fragments were separated on 0.7% agarose gel. EcoT14I-digested λ DNA was used as molecular size standards. (B) vAcRev genomic DNA was digested with Bsu36I and was analyzed by using pulsed field gel electrophoresis. λ DNA was used as a marker.  (Fig. 5A). Trypan blue staining assay showed that the relative survival rates (compared with gfp dsRNA-treated vAcRev-infected SpLi-221 cells) of vAcRev-iap3 and Se111 dsRNA-treated vAcRev-infected cells were 24.4% and 70.6%, respectively (Fig. 5B). DsRNA-treated cells without vAcRev infection did not undergo apoptosis (Fig. 5A). The silencing of vAcRev-IAP3 expression was confirmed by RT-PCR and western blot analysis (Fig. 5C). These results suggest that vAcRev-IAP3 acts as the primary helper to extend the host range of vAcRev, and Se111 does not appear to be necessary for replication of vAcRev but might increase the survival rate of infected cells.

vAcRev-iap3 is a truncated version of Se-iap3 and is expressed at early/late infection stages.
The iap3 gene cassette of vAcRev was subsequently analyzed and compared with its counterpart from SeMNPV. The vAcRev-iap3 ORF is 897 bp in length and encodes a polypeptide of 298 amino acids, with a predicted molecular mass of 34.2 kDa ( Figure S1 in the supplemental materials). Amino acid sequence alignment showed that vAcRev-IAP3 is a truncated version of Se-IAP3 that is lacking the first 16 amino acids at the N-terminus. vAcRev-IAP3 still contains the two BIR domains at the N-terminus and the RING finger motif at the C-terminus of Se-IAP3, which are both typical motifs for IAP proteins. The BIR motifs of baculovirus, vertebrate, and invertebrate exhibit several rigorously conserved residues, including three cysteines and a histidine that coordinate an atom of zinc in the center of a hydrophobic core. However, the second conserved cysteine residue of the SeMNPV-IAP3 BIR2 motif (CX 2 CX 16 HX 6 C) is replaced with a serine residue and this replacement is also found in vAcRev-IAP3 ( Figure S2 in the supplemental materials).
The canonical baculovirus promoter motifs for early genes (TATA box followed by CAGT) and late genes (TAAG) are present upstream of the predicted translation start codon of SeMNPV iap3. However, neither was found within 500 nt upstream from the predicted translation start codon of vAcRev-iap3. Similar to SeMNPV iap3, a consensus polyadenylation signal (AATAAA motif) is located 14 nucleotides downstream from the stop codon of vAcRev-iap3 (Fig. S1). Thus, the time-course of vAcRev-iap3 transcription and expression during vAcRev infection was investigated. RT-PCR showed that vAcRev-iap3 transcripts could be detected as early as 3 h p.i. in vAcRev-infected Hi5, Sf9, Se301 and SpLi-221 cells (Fig. 6A), suggesting that vAcRev-iap3 might be an early gene that is transcribed before viral DNA replication. The transcripts continued to be detectable up to 96 h p.i. By using a prepared anti-vAcRev-IAP3 monoclonal antibody, a major immunoreactive band of approximately 36 kDa was first detected at 6 h p.i. in vAcRev-infected Sf9 and SpLi-221 cells, 9 h p.i. in vAcRev-infected Hi5, and 24 h p.i. in Se301 cells, and the protein amount increased gradually through the late phase of infection (Fig. 6B). The size of the protein is consistent with the predicted 34.2 kDa of the vAcRev-iap3 gene, suggesting that no major post-translational modification occurs. Expression of SeIAP3 prevents apoptosis and rescues vAc ∆P35 replication. To further study the mechanism of the vAcRev host range expansion, four AcMNPV recombinants were constructed: vAc ∆P35-HSP-SeIAP3 (lacking P35 but bearing SeIAP3 which was under the control of hsp70 promoter), vAc ∆P35-SeIAP3-Se111 (lacking P35 but bearing SeIAP3 and Se111 expression cassettes), vAc ∆P35-SeIAP3 (lacking P35 but bearing SeIAP3 expression cassettes) and vAc ∆P35-Se111 (lacking P35 but bearing Se111 expression cassette) (Fig. 7A).
The three vAc ∆P35 non-permissive cell lines (Se301, Sf9 and SpLi-221 cells) infected with vAc ∆P35-SeIAP3 and vAc ∆P35-SeIAP3-Se111 exhibited a mixed phenotype, where some infected cells underwent apoptosis but other cells produced PIBs. Infection withvAc ∆P35-Se111 caused widespread apoptosis, which was comparable to vAc ∆P35 . However, apoptosis was totally inhibited and PIBs were formed in vAc ∆P35-HSP-SeIAP3 -infectedcells (Fig. 7B), in which Se-IAP3 was expressed from the strong constitutive hsp70 promoter 41 . These results suggested strongly that Se-IAP3 is a functional apoptosis inhibitor. Interestingly, our previous research showed that overexpression of Splt-P49 cannot support vAc ∆P35 replication in SpLi-221 cells, even though the overexpression blocks virus-induced apoptosis 27 . Our results suggested that Se-IAP3 might facilitate the vAc ∆P35 replication.

Discussion
In the present study, by co-transfecting Sf9 cells with an AcMNPV mutant which has a deletion of the anti-apoptotic gene p35 (vAc ∆P35 ) and cosmid 22 which represents a fragment of SeMNPV, a host range expanded recombinant virus strain (vAcRev) was generated. Host specificity of vAcRev was analyzed in four insect cell lines and three insect larvae. Substantial accumulation of BVs and PIBs indicates productive infection of vAcRev in Hi5, Sf9, SpLi-221 and Se301 cell lines. T. ni and S. exigua larvae are sensitive to vAcRev by oral inoculation. Moreover, vAcRev can kill S. litura larvae by hemocoelic injection of BV while the p35-null mutant cannot. These in vivo and in vitro assays indicate that vAcRev has a broader host range than its parental viruses vAc ∆P35 and SeMNPV.
Restriction endonuclease patterns showed that the genetic material of vAcRev is mainly derived from vAc ∆P35 . Genomic sequencing and comparisons revealed that vAcRev is a mixture of two virus genotypes (vAcRev-1 and vAcRev-2). The main distinction between the vAcRev-1 and vAcRev-2 genomes is a region where 24.4 kb nucleotide sequence containing 26 ORFs (Ac43~Ac68) is replaced by a 4.0 kb nucleotide sequence containing three ORFs of SeMNPV (vAcRev-iap3, intact Se111 and truncated Se-lef8). Since the replaced region contains 10 essential genes, vAcRev-1 presumably cannot replicate on its own in cells, but it possesses a potential anti-apoptosis gene (vAcRev-iap3) which can resist the apoptosis response induced by vAcRev infection. In addition, as vAcRev-2 has a similar gene content and a similar gene arrangement to the genome of vAc ∆P35 , vAcRev-2 alone also presumably cannot replicate in Sf9, Se301 and SpLi-221 cells, but it provides the gene products of Ac43~Ac68, especially the 10 essential genes, to support vAcRev replication. Thus we propose that vAcRev-1 and vAcRev-2 are dependent on each other to replicate in Sf9, Se301 and SpLi-221 cells.
Recombination plays an important role in the generation of variability between virus strains and can produce recombinants with novel characteristics 42 . Recombination between two baculovirus genomes or between a baculovirus genome and cosmid/plasmid DNA representing a fragment of a baculovirus genome sometimes results in chimeric viruses with expanded host ranges 6,15,16,43 . However, to the best of our knowledge, the host range of the previously reported recombinant viruses is limited to insects that are hosts for one of the parental viruses. Thus, vAcRev is the first reported recombinant baculovirus that is derived from in vitro recombination in which the host range has expanded to a host that is non-permissive for both parental viruses, namely S. litura larvae and SpLi-221 cells.
Most baculovirus iap3 genes tested to date have anti-apoptotic ability 44 . When vAcRev-iap3 expression was knocked down by vAcRev-iap3 dsRNA, vAcRev infection induced apoptosis, indicating that vAcRev-iap3 is the responsible for suppressing apoptosis in vAcRev. To further investigate the role of iap3 in the vAcRev host range expansion to SpLi-221 cells, we constructed a series of recombinant viruses in which Se-iap3 was under the control of different promoters. Apoptosis was not suppressed completely in vAc ∆P35-SeIAP3 -infected cells in which Se-iap3 expression was under the control of its native promoter, while apoptosis could be totally inhibited in vAc ∆P35-HSP-SeIAP3 -infected cells in which Se-iap3 was expressed under the control of the constitutive hsp70 promoter. Moreover, occlusion bodies formed in the infected cells, indicating that infection could progress into the very late phase. Considering that both P35 and P49 are able to block apoptosis but fail to rescue viral replication in these cases, our results suggested that that either 1) apoptosis is the only block to AcMNPV infection in S. litura cells which is not consistent with our earlier results 27 , or 2) Se-IAP3 has another function that promotes viral replication other than inhibition of apoptosis.
Over-expression of SeIAP3 may have the same effects in vAcRev-infected cells. Although vAcRev-IAP3 is a truncated Se-IAP3 lacking the first 16 amino acid at the N-terminus, the typical IAP functional motifs, including two BIR motifs and a Ring-finger motif, remain. Thus we conclude that vAcRev-IAP3 acts similarly to SeIAP3. Interestingly, unlike the promoter region of Se-iap3 in which there are the consensus sequences of baculovirus promoter CAGT or TAAG motifs, a TATA box followed by a poly T fragment exists in the promoter region of vAcRev-iap3. We speculate that this region might contain a strong promoter to drive the expression of vAcRev-iap3.
There is a rapidly growing body of evidence revealing that IAPs are more than just inhibitors of apoptosis, but that they also play an important role in adaptive response to cellular stress, in cell proliferation, differentiation, signaling, motility and in immune response. Our findings that a viral IAP is involved in virus replication in a non-permissive host may open exciting perspectives for the future genetic engineering of baculoviruses.
The AcMNPV E2 strain, which was referred to as vAc WT in this paper, was propagated in T. ni larvae, Sf9 cells, or Hi5 cells. The SeMNPV US1 strain 48,49 was propagated in S. exigua larvae or Se301 cells. The SpltNPV G2 strain 50 was propagated in S. litura larvae or SpLi-221 cells. vAc P35-KO is a p35-null AcMNPV bacmid 27 . To facilitate the examination of viral infection, the AcMNPV polyhedrin (ph) and enhanced green fluorescence protein (gfp) genes were inserted into the ph locus of vAc ∆P35-KO via site-specific transposition as previously described 51 , and the resulting virus was designated vAc ∆P35 . PIBs were propagated and purified as previously described 42 . BVs were harvested from the infected larva hemolymph at 3 d p.i. 52 . The BV titers were determined by TCID 50 end point dilution assay in corresponding cells 52 .
Three replicates were performed for each trial. Larval death was monitored daily until larvae died or pupated. Data was conducted using the SPSS data processing software.
Restriction endonuclease analysis and genome sequencing. Viral DNAs were extracted from PIBs as described previously 42 . The DNAs were analyzed by pulsed-field gel electrophoresis or digested with restriction endonucleases as described elsewhere 54 .
The vAcRev genome was sequenced by using 454 pyrosequencing 55 . Bioinformatics pipelines were used to assemble contigs from the sequence data production 56 . The draft assembly was followed by a labor-intensive finishing phase where the assembled sequences were improved using targeted sequencing to resolve misassembled regions, close sequence gaps, and improve coverage and accuracy in sparsely covered regions of the genome.
Generation of dsRNAs and RNA interference (RNAi). dsRNAs were prepared as described previously 57,58 . Briefly, a 286-bp fragment of vAcRev-iap3 and a 477-bp fragment of g fp were PCR-amplified by using vAcRev DNA as template. The PCR primers were designed to contain a 5′ T7 RNA polymerase binding site (TAATACGACTCACTATAGG) followed by sequences specific for target genes. The sequences of the primer pairs are as follows. For iap3: T7-vAcRev-iap3-U (CGGGATCCTAATACGACTCACTATAGGAATTGGAGAGAGGGCGACGATC, BamHI site is underlined) and T7-vAcRev-iap3-D (GGAATTCTAATACGACTCACTATAGGAATCACATGACCGCACGGCA, EcoRI site is underlined). For gfp: T7-gfp-U (CGGGATCCTAATACGACTCACTATAGGGTGTTCAATG CTTTTCAAGATAC, BamHI site is underlined) and T7-gfp-D (GGAATTCTAATACGACTCACTATAGG CTGTTACAAACTCAAGAAGGACC, EcoRI site is underlined). After purification with a High Pure PCR Purification Kit (Roche Molecular Biochemicals), the PCR products were used as templates to produce dsRNA using an Ampliscribe TM T7-Flash TM Transcription Kit (EPICENTER). RNase-free DNase was added to digest the DNA templates. After incubation at 95 °C for 2 min and slow cooling to room temperature, the annealed dsRNA were purified by using an RNeasy Mini Kit (QIAGEN). The concentrations of dsRNA were determined with a spectrophotometer. The resulting dsRNAs were stored at − 70 °C until use.
SpLi-221, Sf9 or Se301 cells were mock-transfected or transfected with 2 μ g of vAcRev-iap3 or gfp dsRNA using Trans Messenger Transfection Reagent (Qiagen). Twenty-four hour later, the transfected cells were infected with vAcRev at an MOI of 10 TCID 50 /cell. The cells were observed for the occurrence of apoptosis with a light microscope.
Cell viability assay. Viable cell numbers were assessed by using trypan blue staining assay as previously described 59 .
Transcriptional analysis of vAcRev-iap3. Hi5, Sf9, Se301, and SpLi-221 cells were infected with vAcRev at an MOI of 20 and were harvested at different time points (p.i.), respectively. Time zero was defined as the time when the inoculum was added to the cells. Total cellular RNA was extracted as mentioned above. RT-PCR was conducted using a RNA PCR Kit (TaKaRa, Ver.3.0) employing 1 μ g of total RNA as template. The PCR products were analyzed using a 1% agarose gel.
Scientific RepoRts | 6:28072 | DOI: 10.1038/srep28072 Antibody preparation and immunoblot analysis. Two peptides corresponding to amino acids 79 to 275 and 1 to 313 of SeMNPV IAP3 were synthesized by Abmart and were used to generate polyclonal antibodies in three mice. The monoclonal antibodies of SeMNPV IAP3 were screened by bone marrow hybridoma fusion technology, and the spleen cells of mice which had the best immune response were fused with myeloma cells (SP2/0). Western blotting was performed by standard procedures as previously described 60 . Briefly, the infected-cells were harvested and lysed at the indicated time points. The mouse monoclonal anti-vAcRev-IAP3 antibody (1:200) or a mouse monoclonal anti-actin antibody (1:2000; Abmart) were used as the primary antibodies, and horseradish peroxidase-conjugated anti-mouse IgG (1:5000; GE Healthcare) as a secondary antibody. The blots were visualized using enhanced chemiluminescence reagent (ECL Advance Western Blotting Detection Kit; GE Healthcare).