Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly
Entomology Department, Institute of Plant Protection, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israell(xm, 百拇医药
Received 5 July 2002/ Accepted 24 September 2002l(xm, 百拇医药
ABSTRACTl(xm, 百拇医药
Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.
INTRODUCTIONuf]v, 百拇医药
The arthropod virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Baculoviridae family, is able to infect 32 lepidopteran species (24). The complete genome of AcMNPV has been sequenced (3), and its replication in permissive cells has been studied extensively (7, 33). AcMNPV vectors are widely used as efficient tools for the expression of "foreign genes" in established cell lines and whole insects (7, 53). Recombinant AcMNPVs have been developed for use in pest control (29, 31, 48); moreover, baculovirus-based vectors have been considered for gene therapy purposes (1, 17). The ability of AcMNPV to interact with a wide range of hosts (50) makes it an attractive tool for elucidating which molecular factors are involved in determination of the host range of baculoviruses. Since AcMNPV can penetrate the nuclei of nonpermissive insect and mammalian cells, important mechanisms that determine the host range of AcMNPV are related to the expression of the viral genome (20, 44, 49-51).
AcMNPV gene expression is divided into early, late, and very late phases. Early-phase genes are transcribed by host RNA polymerase II before DNA replication, while late- and very-late-phase genes depend on viral DNA replication and are transcribed by a virus-specific RNA polymerase (4, 27). AcMNPV protein IE1, the product of immediate-early gene ie1, plays a central role in regulating viral infection (8, 15, 22, 37, 41, 61). IE1 is a potent transcriptional activator of early genes, such as 39K, p35, and he65 (9, 22, 25, 39), and is essential for both DNA replication and late gene transcription (35, 37, 39, 62, 67). IE1 negatively regulates the transcription of the pe38 and ie2 genes (22, 38, 39). IE0, the product of the ie0 gene, differs from IE1 only in that IE0 contains 54 additional amino acids at its amino terminus (resulting from splicing of the upstream ie0 exon and its subsequent fusion to the ie1 exon) (14). ie0 RNA is expressed only during the early phase of infection, and ie1 RNA is expressed during both early and late phases of infection (14, 36, 37). IE1 stimulates expression from the ie1 promoter but down-regulates expression from the ie0 promoter (37). The ie0 gene product also transactivates the ie1 promoter but does not affect expression from its own promoter (37).
The baculovirus genes p143 (helicase), hrf-1, hcf-1, p35, and ie2 were implicated in the determination of the host range of AcMNPV in various insect systems, including Bombyx mori, Lymantria dispar, Trichoplusia ni, and Spodoptera frugiperda (2, 13, 18, 44, 46, 56, 66).l&$4, 百拇医药
Recently, it was reported that AcMNPV infection of SL2 cells, a cell line derived from the Egyptian cotton worm Spodoptera littoralis, results in apoptosis and low yields of viral progeny (11, 19). The overexpression of p35, the apoptosis suppressor gene from AcMNPV (30), reduced apoptosis in AcMNPV-infected SL2 cells but did not improve the yield of AcMNPV budded viruses (BV) or viral infectivity for S. littoralis larvae (23). These results suggested that factors other than P35 were required by AcMNPV to replicate efficiently in SL2 cells. S. littoralis nucleopolyhedrovirus (SlNPV) replicates successfully in SL2 cells. To study the possibility that SlNPV could rescue AcMNPV infection, we cotransfected SL2 cells with AcMNPV DNA and a cosmid library representing the complete SlNPV genome. We isolated vAcSL2, a recombinant AcMNPV that yielded high titers of viral progeny in SL2 cells. vAcSL2 carried a 519-bp insert that disrupted the ie0 promoter, reducing the expression of this gene product and resulting in increased steady-state levels of IE1 relative to IE0. To confirm these observations, we mutagenized the ie0 promoter locus by inserting the chloramphenicol acetyltransferase (cat) gene and obtained a new recombinant AcMNPV with replication properties identical to those of vAcSL2. These data suggested that the overexpression of ie1 may enable efficient replication of AcMNPV in the SL2 host. Indeed, using various means to promote the overexpression of ie1, we were able to achieve productive AcMNPV infection of S. littoralis cells and larvae.
MATERIALS AND METHODS0, http://www.100md.com
Cell lines and viruses. S. frugiperda SF9 and S. littoralis SL2 cells were maintained and propagated in TNM-FH medium supplemented with 10% heat-inactivated fetal bovine serum (64). Wild-type AcMNPV strain E-2 (63) and SlNPV strain E-15 (32) were propagated on SF9 cells. SlNPV can replicate in both SF9 and SL2 cells. Viral growth curves were determined by plaque assays as described previously (23).0, http://www.100md.com
Plasmids and transfection. Plasmid pAcie1 contains an intact ie1 gene (26). pHspie1 contains the ie1 coding region under the control of the Drosophila melanogaster hsp70 promoter. The latter was restricted from phsp70PLVI+CAT (16) by using XbaI and BamHI and was subcloned into pBSK (Stratagene) bearing the ie1 coding region HincII-HindIII DNA fragment from pAcie1. pBgl-11.1 and pBgl-3.3 bear 11.1- and 3.3-kbp BglII fragments of vAcSL2, respectively, each cloned at the BglII site of pBSK (see below); the 11.1-kbp fragment contains the ie0 and ie1 genes. pie0PCAT bears the cat gene inserted at AcMNPV nucleotide 122,800 (3) to disrupt the ie0 promoter at position -31 upstream of the ATG start site. For this purpose, a 1.6-kbp SnaBI-SalI fragment bearing the ie0 promoter region was cloned into the corresponding site of pFastBac1 (Invitrogen Life Technologies). Then, a BamHI site was created by mutagenesis with overlap extension PCR and with complementary primers containing the desired restriction site (55). The primers used were 5'-GCCGGATCCAGTATAAGTAATTGA-3' and 5'-TTATACTGGATCCGGCGCGCGCA-3' (underlining indicates the BamHI site). The cat gene from pQ39CAT (25) was digested with BamHI and inserted into the newly created BamHI site to yield pie0PCAT.
Generation of a recombinant baculovirus by homologous recombination in Escherichia coli. The polyhedrin (polh) gene, which the manufacturer deleted from AcMNPV BACmid (Invitrogen Life Technologies), was first reintroduced by site-specific transposition with transfer vector pFastBac1 into E. coli DH10BAC cells (45). For this purpose, the complete polh gene, including its own promoter and termination sequence, was rescued from pI1 (12) by digestion with XhoI, filling in with Klenow DNA polymerase, and further restriction with EcoRI and was inserted into EcoRI- and Bst1107I-digested pFastBac1 to obtain pFastpolh. Site-specific transposition with pFastpolh and selection of DH10BAC cells harboring recombinant BACmid AcFastpolh were performed by following the manufacturer's (Invitrogen Life Technologies) instructions. Then, to disrupt the ie0 promoter in the viral (BACmid) genome, E. coli strain BJ5183 was transformed with recombinant BACmid AcFastpolh (5). The resulting strain, E. coli BJ-AcFP, was transformed with the 2.3-kb SnaBI-SalI fragment containing the cat gene-disrupted ie0 promoter sequence from pie0PCAT. Bacteria resistant to kanamycin and chloramphenicol were selected, and high-molecular-weight minipreparation DNA of recombinant BACmid AcFPCAT, with a cat gene insertion at the ie0 promoter (Acie0PCAT), was prepared as described in the Bac-to-Bac protocol (Invitrogen Life Technologies). The presence of the cat gene insertion at the ie0 promoter of BACmid AcFPCAT was confirmed by PCR with BACmid DNA template and primers CAT5' (5'-ATGGAGAAAAAAACACTG-3'), CAT3' (5'-TTACGCCCCGCCCT-3'), and IE0R (5'-GTGTCAACTTGCAACTGCTGAGCTTCTGC-3'). Acie0PCAT DNA was used to transfect insect cells to obtain recombinant virus vAcie0PCAT.
DNA transfection of insect cells was performed with Lipofectin as described previously (19)..ha[, 百拇医药
Rescue of AcMNPV replication in SL2 cells and isolation of AcMNPV recombinants. SL2 cells were cotransfected with either a complete cosmid library or individual cosmids of SlNPV (19) or AcMNPV, respectively. At 96 h after transfection, the culture supernatants were removed and recombinant viruses that replicated and formed polyhedra in SL2 cells were isolated by subsequent plaque purification in these cells..ha[, 百拇医药
Restriction endonuclease and Southern blot analyses. Viral DNA prepared from BV (19) was digested with various restriction endonucleases and subjected to agarose gel electrophoresis (0.8% agarose) for 16 h at 30 V. After the run, the gel was quickly blotted to a nylon membrane (47). Hybridization was performed by using a GeneScreen Plus membrane according to the manufacturer's instructions (NEN Life Science Products). Probe labeling was performed by using a Renaissance random primer fluorescein labeling kit (NEN Life Science Products). The 519-bp insert fragment (IF) from vAcSL2 and the 2.4-kb EcoRV-HindIII fragment of the AcMNPV ie1 gene (25) were used as hybridization probes.
Western blot analysis. Virus-infected or plasmid-transfected cells were harvested and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with a pH 9 separating gel; this procedure allowed us to distinguish clearly the IE0 molecular species from the IE1 molecular species. Immunoblot analysis was performed with anti-IE0/IE1 antiserum as previously described (23, 54).(/wow7, 百拇医药
Metabolic labeling of viral polypeptides. SF9 or SL2 cells (105 cells) were infected with either wild-type or recombinant AcMNPV. At 45 h postinfection, the cells were washed once with Sf900 II medium depleted of methionine and cysteine (Invitrogen Life Technologies). The medium was subsequently replaced with 500 µl of the same medium supplemented with 15 µCi of a 35S-labeled methionine-cysteine mixture (Amersham, Little Chalfont, United Kingdom). After 2 h, cell extracts were prepared by adding SDS lysis buffer (53) and then subjected to PAGE. Autoradiography was performed as described before (53).
Bioassays. Carefully selected third-instar S. littoralis larvae (24 per dose, in triplicate) were injected with various doses of AcMNPV or vAcSL2 BV. Control larvae were injected with the same volume of TNM-FH complete medium. The percent mortality was calculated as the number of dead larvae (excluding larvae killed by the injection, normally one or two) divided by the number of larvae that survived the infection. No mortality was observed for mock-infected larvae (12, 23).(+#6p, http://www.100md.com
An oral bioassay of third-instar S. littoralis was performed by using a diet containing six concentrations of wild-type and recombinant viral polyhedra ranging from 5 x 102 to 2.5 x 105 polyhedral inclusion bodies (PIB)/mm2. After 24 h of exposure to the virus, the larvae were transferred to new containers containing a noncontaminated diet; larval death was monitored daily for 3 weeks. Thirty larvae were used for each dose, and the experiment was repeated twice.(+#6p, http://www.100md.com
RESULTS
Rescue of AcMNPV replication in S. littoralis SL2 cells. To study the potential of the SlNPV genome to sustain the replication of AcMNPV in SL2 cells, we cotransfected the cells with AcMNPV DNA and an overlapping cosmid library representing the entire SlNPV genome (19). After 6 days, we observed the appearance of polyhedra in the cell nuclei. By plaque purification, we were able to isolate a recombinant AcMNPV, vAcSL2, that replicated in SL2 and SF9 cells. At 96 h after infection of SL2 cells with vAcSL2, only 10 to 20% of the cells showed apoptosis, while 40 to 70% of the remaining cells showed clear polyhedra under direct microscopic observation (Fig. 1, upper left panel). In contrast, 90% of SL2 cells infected with AcMNPV at multiplicities of infection (MOIs) of 1 to 100 showed apoptosis and did not show PIB (Fig. 1, upper right panel). Compared to AcMNPV, vAcSL2 produced regular polyhedra in SF9 cells (Fig. 1, lower right and left panels, respectively). Since apoptosis has been implicated in the significant reduction of AcMNPV BV yields observed in SL2 cells (11), we compared the abilities of vAcSL2 and AcMNPV to replicate in these cells at MOIs of 0.5 and 5 (Fig. 2). BV were harvested from cell supernatants at 6, 9, 12, 28, and 48 h postinfection and titrated in SF9 cells. The titers of vAcSL2 obtained were significantly higher than those of AcMNPV: e.g., at 28 and 48 h after infection with an MOI of 0.5, the yield of vAcSL2 was 1,000-fold higher than that of AcMNPV (Fig. 2A).
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FIG. 1. vAcSL2 replicates in SL2 cells and forms polyhedra. SL2 and SF9 cells were infected with vAcSL2 or AcMNPV. Images are light micrographs (magnification, x400). Note the vAcSL2-infected cells producing polyhedra over the background of the intact cells, compared to AcMNPV-infected SL2 cell blebbing.yj\ge-, 百拇医药
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FIG. 2. BV yields from vAcSL2- and AcMNPV-infected SL2 cells. BV were collected from clarified cell supernatants at various times after infection with vAcSL2 or AcMNPV and titrated in SF9 cells (in triplicate). Error bars show standard deviations.yj\ge-, 百拇医药
Given that vAcSL2 replicated efficiently in SL2 cells, it was interesting to assess whether the vAcSL2 genome could rescue AcMNPV replication. For this purpose, we cotransfected SL2 cells with intact AcMNPV DNA and vAcSL2 DNA digested with various restriction enzymes. At about 72 to 96 h after transfection, the cells were examined microscopically for the presence of polyhedra. vAcSL2 DNA digested with either SmaI, XhoI, HindIII, or BglII rescued production of polyhedra, while EcoRV-digested DNA did not rescue it (Table 1). Remarkably, only EcoRV treatment is supposed to disrupt the ie1 open reading frame. BglII-digested vAcSL2 genomic DNA showed two new fragments of about 11.1 and 3.3 kbp (designated and ß, respectively), not present in AcMNPV (Fig. 3A), due to the presence of a new BglII site in fragment D of BglII-digested AcMNPV (see below). To determine whether either of these two fragments was able to rescue AcMNPV amplification in SL2 cells, they were subcloned in pBSK to obtain plasmids pBgl-11.1 and pBgl-3.3 (see Materials and Methods). Transfection of SL2 cells with each of these plasmids and AcMNPV DNA showed that only pBgl-11.1 could rescue formation of polyhedra (Table 1).
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TABLE 1. Rescue of wild-type AcMNPV replication in SL2 cells by vAcSL2 DNA fragmentsay3y+, 百拇医药
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FIG. 3. vAcSL2 contains a 519-bp insert downstream of the ie0 TATA element that disrupts the basic transcriptional unit. (A) vAcSL2 DNA (lane 1) and AcMNPV DNA (lane 2) were digested with BglII, and the restricted DNA fragments were analyzed in a 0.7% agarose gel. New BglII fragments in vAcSL2 are indicated by arrows ( and ß). Lane M, molecular size markers DNA digested with BstEII) (in kilobase pairs). (B) Partial physical map and sequence of fragment downstream of the new BglII site. Indicated are the ie0 TATA element (bold), the transcriptional initiation motif CAGT (bold and underlined), and the downstream IF sequence that increases the distance between the motifs (repeated sequence is underlined). AcMNPV map units are indicated as well. The boxes show the locations of the ie0 5'-terminal coding region and the me53 gene, which are transcribed in opposite directions (arrows). Restriction sites: H, HindIII; B, BglII; S, SalI.
Sequencing of the and ß fragments showed that the new BglII site corresponded to a point mutation in nucleotide 125 (A to G) in the coding region of viral gene me53 which resulted in a conservative change (Lys 42 to Arg). The vAcSL2 11.1-kbp fragment that was able to rescue formation of polyhedra contained the whole ie0-ie1 region of AcMNPV and included a new insert of 519 bp (named IF) located upstream of the ie0 gene at position -88 from the ATG start site (Fig. 3B). The sequence 5'-TCGTAAATCAG-3' was repeated and flanked both ends of the IF at positions -78 to -88 and positions -586 to -597 from the ie0 ATG start site (Fig. 3B, underlining). The IF increased the distance between the ie0 CAGT transcriptional start site and the TATA element by 519 bp (Fig. 3B, bold underlining).o/i83i@, 百拇医药
Expression of ie0 in vAcSL2-infected cells. We observed that in AcMNPV-infected SL2 cells, the steady-state levels of IE1 and IE0 were almost identical throughout the infection (Fig. 4A; see also Fig. 6C); in contrast, in AcMNPV-infected permissive SF9 cells (Fig. 4B), the steady-state levels of IE1 early in infection were low relative to those of IE0 and later increased progressively. It should be noted that the double bands of IE1 and IE0 represented the phosphorylated and dephosphorylated main electrophoretic species reported (15) (see also below). The presence of the IF upstream of the transcriptional start site of ie0 suggested that it might result in a lower level of expression of this gene in vAcSL2-infected cells. Indeed, IE0 was synthesized at very low steady-state levels in vAcSL2-infected SL2 and SF9 cells compared to AcMNPV-infected cells (Fig. 4A and B), and the steady-state levels of IE1 were higher than those of IE0.
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FIG. 4. IE1 and IE0 synthesis in vAcSL2- and AcMNPV-infected cells. (A and B) SL2 and SF9 cells (105) were infected with vAcSL2 and AcMNPV at an MOI of 5. At various times postinfection, the cells were harvested, and cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-IE0/IE1 antiserum. mi, mock-infected cells. (C) Expression of ie1 in transfected cells. SF9 cells (105) were transfected with 1 µg of pBgl-11.1 (bearing the 、 fragment) or pHspie1 (bearing ie1 under the control of the hsp70 promoter). At 24 h after transfection, cell extracts were subjected to SDS-PAGE and immunoblot analysis. m-transf., mock-transfected cells. Molecular mass markers are indicated.g, 百拇医药
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FIG. 6. vAcie0PCAT structure and analysis and synthesis of IE1 and IE0. (A) Genomic organization. The dashed lines indicate the site of insertion of the cat gene (box). The other boxes show the locations of the ie0 coding region (exon 1 and exon 2) and the ie1 and me53 genes and their corresponding promoters (upper arrows). IE0R, CAT5', and CAT3' were primers used for PCR analysis. The dotted lines indicate the expected sizes of amplified fragments. (B) PCR analysis. (Left panel) cat gene amplification. Lane 1, no template; lane 2, AcMNPV; lane 3, vAcie0PCAT. (Right panel) Lane 1, cat gene marker (same as lane 3 in the left panel); lanes 2 to 4, cat gene plus exon 1 (primers CAT3' and IE0R), with no template in lane 2, AcMNPV in lane 3, and vAcie0PCAT in lane 4. (C) SL2 (105 cells) were infected with AcMNPV and vAcie0PCAT at an MOI of 5. pHspie1, SF9-transfected cell control. At various times postinfection, cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-IE0/IE1 antiserum as described in the legend to Fig. 4. Molecular mass markers are indicated.
A comparison of SF9 cells transfected with pBgl-11.1 and with pHspie1 (bearing only the ie1 gene under the control of the D. melanogaster hsp70 promoter) confirmed that IE1 was the main electrophoretic species synthesized; IE1 was clearly distinguished from IE0 (15) (see also Materials and Methods) (Fig. 4C). IE0 was undetectable in pBgl-11.1-transfected cells; in comparison, AcMNPV-infected SF9 cells synthesized IE0 and IE1 (Fig. 4C).d\, http://www.100md.com
To find out whether the IF of vAcSL2 was derived from the SlNPV genome, we performed Southern blot analysis. SlNPV, AcMNPV, and vAcSL2 genomic DNAs were digested with HindIII (Fig. 5A, lanes 2, 3, and 4, respectively) and PstI (Fig. 5A, lanes 7, 8, and 9, respectively). Hybridization with labeled IF at 60°C showed that vAcSL2 bore the IF, as expected, but did not show fragments homologous to IF in the genomes of SlNPV and AcMNPV (Fig. 5B, lanes 4 and 9, lanes 2 and 7, and lanes 3 and 8, respectively). Performing the hybridization at a lower stringency revealed a weak signal corresponding to the HindIII G and PstI B fragments of AcMNPV (8.15 and 21.6 kbp, respectively) that bear the IE0 and IE1 coding sequences but not to SlNPV. Also, Southern blot analysis with the same IF probe and cellular DNA did not indicate the presence of a homologous sequence in the SL2 cell genome (data not shown). A GenBank search did not reveal homology between the IF and any reported sequences. Thus, we concluded that the IF was probably the result of a recombinational event between AcMNPV and viral or cellular DNA, but we could not establish its exact genomic source.
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FIG. 5. Southern blot analysis of AcMNPV recombinants. (A) HindIII- or PstI-digested viral DNAs. (B) Hybridization with fluorescein-labeled IF (PCR amplified from pBgl-11.1) at 60°C. (C) Hybridization with fluorescein-labeled EcoRV-HindIII ie1 fragment. Lanes 2 and 7, SlNPV; lanes 3 and 8, AcMNPV; lanes 4 and 9, vAcSL2; lanes 5 and 10, vBgl3; lanes 6 and 11, vHsp-1; lane 1, undigested pBgl-11.1; lane M, molecular size markers (in kilobase pairs).h?44, http://www.100md.com
To avoid the possibility that the phenotype of vAcSL2 was due to some unexpected mutation in the viral genome, we constructed a recombinant AcMNPV in which the ie0 promoter region was disrupted by insertion of the cat gene. For this purpose, we used the method described by Bideshi and Federici (5). Plasmid pie0PCAT was used to transpose the mutated ie0 promoter region into BACmid AcFastpolh, and the resultant BACmid construct was transfected into SF9 cells to further isolate recombinant baculovirus vAcie0PCAT (see Materials and Methods) (Fig. 6A). The constructs were confirmed by PCR analysis with a primer complementary to the cat gene and a primer 159 bp downstream of the transposition site (CAT3' and IE0R, respectively) (Fig. 6B). The effect of disruption of the ie0 promoter on the synthesis of IE0 and IE1 during the infectious cycle of vAcie0PCAT was studied as described above by using immunoblot analysis (Fig. 6C). vAcie0PCAT infection of SL2 cells reproduced the patterns of expression of ie0 and ie1 observed with vAcSL2 expressing mainly ie1 (compare to AcMNPV-infected cells and to pHspie1-transfected cells in Fig. 6C and Fig. 4A). Direct microscopic observation of vAcie0PCAT-infected SL2 cells revealed polyhedra (data not shown). Moreover, vAcie0PCAT showed a significant improvement in BV yields in SL2 cells compared to wild-type AcMNPV (Fig. 7). Identical results were obtained when the cat gene was inserted in the opposite orientation (data not shown).
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FIG. 7. BV yields from vAcie0PCAT- and AcMNPV-infected SL2 cells. BV were collected from clarified cell supernatants at various times after infection (p.i.) with an MOI of 0.5 and titrated in SF9 cells (in triplicate). Error bars indicate standard deviations.*:r^%, 百拇医药
Formation of AcMNPV polyhedra can be rescued in SL2 cells by increasing the level of expression of ie1. The above results suggested that enhancing ie1 expression might rescue the replication of AcMNPV in SL2 cells. To test this hypothesis, we cotransfected SL2 cells with either pBgl-11.1 or pHspie1 and AcMNPV DNA. At 72 to 96 h after transfection, formation of polyhedra was detected in the transfected cells, while control cells cotransfected with pBSK and AcMNPV DNA did not show polyhedra (data not shown). Moreover, by subsequent plaque purification, we were able to isolate from pHspie1 and pBgl-11.1 cotransfection supernatants new recombinant viruses (vHsp-1 and vBgl3, respectively) that replicated in SL2 cells, forming polyhedra. Southern blot analysis with an ie1 probe (see Materials and Methods) showed that vHsp-1 bore two copies of the ie1 gene (Fig. 5C, lanes 6 and 11). vBgl3 bore the IF in the viral genome (Fig. 5C, lanes 5 and 10). Lighter bands observed in all of the lanes corresponded to the common background usually observed with this chemiluminescence detection method under these exposure conditions (Fig. 5C). Thus, in the first case, cotransfection with pHspie1 appeared to have resulted in the insertion of a second ie1 gene in the viral genome. In the second case, the region upstream of the ie0 gene appeared to have been replaced by an IF copy.
Infection of SF9 cells with vAcSL2, vHsp-1, vBgl3, and AcMNPV followed by metabolic labeling with 35S-labeled amino acids (see Materials and Methods) resulted in similar polypeptide profiles (Fig. 8, lanes 3, 4, 5, and 2, respectively). The most prominent polypeptide detected at 45 h postinfection was polyhedrin. This marker, which reflected the succesful completion of the viral replication cycle, was clearly detected in SL2 cells infected with vAcSL2, vBgl3, and vHsp-1 but not AcMNPV (Fig. 8, lanes 8, 9, 10, and 7, respectively). As expected, polyhedrin was not observed in mock-infected SF9 and SL2 cells (Fig. 8, lanes 1 and 6, respectively).}, 百拇医药
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FIG. 8. Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (105) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with AcMNPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of AcMNPV polyhedrin.
The expression of ie1 was implicated in the induction of apoptosis in S. frugiperda cells (57). Also, it was reported that AcMNPV-infected SL2 cells underwent apoptosis and expressed the antiapoptotic gene p35 at very low levels (23). Thus, we hypothesized that the overexpression of ie1 would enhance p35 expression as well, neutralizing the potential increase in the apoptotic response of the cell. Indeed, P35 steady-state levels were higher in vAcSL2-infected cells than in AcMNPV-infected SL2 cells (Fig. 9, lanes 1 to 3 and lanes 4 to 6, respectively).h'[m', http://www.100md.com
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FIG. 9. Synthesis of the apoptosis suppressor P35 in vAcSL2-infected SL2 cells. SL2 cells were infected at an MOI of 5 with vAcSL2 (lanes 1 to 3) or AcMNPV (lanes 4 to 6). Cell extracts (105 cell equivalents) were harvested at various times postinfection and subjected to SDS-PAGE and immunoblot analysis with anti-P35 antiserum (23). A molecular mass marker is indicated.h'[m', http://www.100md.com
Infectivity of vAcSL2 for S. littoralis larvae. To determine whether the ability of vAcSL2 to replicate efficiently in SL2 cells is a cell line phenomenon or has organismic implications, we compared the infectivity of vAcSL2 with that of AcMNPV for S. littoralis larvae. For this purpose, S. littoralis third-instar larvae were infected with increasing doses of viral polyhedra orally (see Materials and Methods) or via the intrahemocoelic route (by injection of increasing doses of BV). No differences in AcMNPV and vAcSL2 infectivities were measured for the oral route which, under these conditions, resulted in only 10 to 14% larval death at the higher PIB doses (data not shown). However, significant differences in larval mortality were observed for intrahemocoelic inoculation of BV: a 10-fold increase in the AcMNPV dose was required to obtain 50 and 70% mortality of the larval population (at doses of 100 and 1,000 PFU, respectively) (Fig. 10).
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FIG. 10. Mortality of S. littoralis larvae infected with vAcSL2 and AcMNPV. S. littoralis larvae (third instar, 24 larvae per dose) were injected with increasing concentrations of AcMNPV or vAcSL2 BV. The mortality rate was calculated as the number of larvae that died from the infection divided by the number of survivors. The results are the averages of three independent experiments. Error bars indicate standard deviations.;7, 百拇医药
DISCUSSION;7, 百拇医药
Expression of ie1 and replication of AcMNPV in S. littoralis. IE1 displayed steady-state levels equivalent to or lower than those of IE0 during the early phase of AcMNPV infection of permissive SF9 cells (Fig. 4B and 6C). Late in infection, IE1 steady-state levels increased significantly over those of IE0 in SF9 cells (54 and ibid), but in poorly permissive SL2 cells, the steady-state levels of IE1 and IE0 remained equivalent throughout the infection (Fig. 4A and 6C). The IF disrupted the continuity of the ie0 transcriptional unit by increasing the distance between the TATA element and the CAGT transcriptional start site (6, 22, 58), suggesting that indeed the transcription of ie0 would be diminished in vAcSL2-infected cells; however, this aspect was not directly analyzed in this study (e.g., by transcriptional mapping). However, the steady-state levels of IE0 were reduced consistently in vAcSL2-infected cells (Fig. 4), resulting in higher steady-state levels of IE1 than of IE0 in vAcSL2-infected SL2 cells compared to AcMNPV-infected cells (Fig. 4A). vAcSL2 showed a significant increase in infectivity toward S. littoralis SL2 cells (Fig. 1) and larvae (Fig. 10, BV injection). However, no change in infectivity was measured when infection was performed via the oral route. Thus, it appears that viral factors besides the overexpression of IE1 relative to IE0 were required to overcome the initial response of S. littoralis larvae (probably of immune origin) (68) to viral infection.
The IF displayed the same repeat sequence, 5'-TCGTAAATCAG-3', at its 5' and 3' termini, suggesting that it originated by some recombinational event between viral (AcMNPV or SlNPV) and cellular (SL2) sequences. However, we could not determine the exact origin of the IF by Southern blot analysis. Thus, we evaluated whether the insertion of another arbitrary DNA fragment (e.g., the cat gene) (Fig. 6) instead of the IF into the ie0 promoter locus might reproduce the vAcSL2 phenotype. Indeed, cat gene-mediated disruption of the ie0 promoter region resulted in enhanced replication of recombinant vAcie0PCAT in SL2 cells (Fig. 7) that correlated with lower steady-state levels of IE0 than of IE1 (Fig. 6C).jx, http://www.100md.com
The above data suggested the following. (i) There was a block to ie1 expression in AcMNPV-infected SL2 cells (probably late expression, although it was not rigorously measured by comparing it with ie1 expression in aphidicolin-treated infected cells) (compare Fig. 4A and B). (ii) An increase in the steady-state levels of IE1 relative to IE0 may enable the replication of AcMNPV in SL2 cells (see below).
We confirmed the latter hypothesis by cotransfecting AcMNPV and plasmid pHspie1 or the ie0-ie1 genetic unit present in the vAcSL2 genome (pBgl-11.1) and subsequently isolating new recombinant viruses that overexpressed ie1 (Table 1 and Fig. 8). Our results provide the first evidence that AcMNPV can replicate in a poorly permissive cell line (and insect) through an increase in the level of expression of the ie1 gene (see below).w, 百拇医药
IE0 and IE1 functions. IE1 is a multifunctional protein that plays a crucial role in the life cycle of AcMNPV: (i) transactivation of the delayed early and late classes of genes (37, 39), (ii) its own continued expression during infection, and (iii) repression of other immediate-early genes.w, 百拇医药
The finding that lower steady-state levels of IE0 and higher steady-state levels of IE1 enabled AcMNPV replication in SL2 cells suggested that IE0 may be involved in the regulation of IE1 function. Thus, elevated IE1 levels may be required in SL2 cells to stimulate genes involved in AcMNPV DNA replication, such as DNA polymerase, p35, and helicase genes (10, 43, 52, 54, 67), to become directly involved in binding to a replication origin and catalyzing early steps that lead to the assembly of a replication complex (33, 42). Also, the reduction of apoptosis in vAcSL2-infected SL2 cells may be the result of enhanced p35 gene expression (Fig. 9) (23).
IE1 has separate domains for transactivation and DNA binding that are essential for the modulation of baculovirus gene expression (35, 60, 61). Little is known about the role of IE0 as a transregulator, although it possesses an additional N-terminal 54 amino acids compared to IE1. Transient expression assays revealed that IE0 could transactivate the ie1 promoter but did not affect expression from the ie0 promoter, while IE1 stimulated expression from the ie1 promoter but down-regulated expression from the ie0 promoter (37). IE1 was shown to bind, probably as a dimer (60), to specific DNA sequences present in hrs that have been demonstrated to function as cis-acting enhancers of viral transcription and origins of replication (34, 40, 41, 59) and to sequences present in promoters of genes that it down-regulates (41). The motif 5'-ACBYGTAA-3' is present in all DNA fragments that have been shown to bind IE1, including ie0 (41). Thus, it was proposed that transcriptional repression by IE1 occurs when such a single binding motif is present in the DNA sequence and that IE1 may have a lower affinity for these single motifs than for the complete palindromic sequences present in the hrs (42).
The same IE1 domains that are involved in DNA binding are present in IE0. Moreover, it was suggested that the putative helix-loop-helix domain present at C-terminal residues 543 to 568 mediates oligomerization and DNA binding of IE1 (61). Taken together, these and the above data suggest that IE0 and IE1 can form heterodimers (39). It is conceivable that the IE0-IE1 heterodimer has a function different from that of the IE1-IE1 homodimer: IE1 or the IE1-IE1 dimer could up-regulate the ie1 promoter and down-regulate the ie0 promoter (41), but the formation of an IE0-IE1 complex could compete for free IE1 molecules, preventing the formation of IE1-IE1 dimers, and/or could compete for DNA binding of the IE1 homodimer, which is required to enhance the expression of ie1 and other IE1-transactivatable promoters.{dmoy, http://www.100md.com
In addition, it was postulated that a host factor(s) which transactivates the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) ie1 promoter is present in SF9 cells but absent in Ld652Y cells (21), and a 38-kDa protein with hr gene binding capacity was isolated from SF9 cells (28). Similarly, such a factor could be missing or not very abundant in SL2 cells, resulting in a low level of ie1 expression late in infection and consequently in an insufficient amount of functional IE1 to compete with the IE1-IE0 complex or to displace IE0 from it and transactivate essential AcMNPV replication genes. Either the reduction of ie0 expression or the overexpression of ie1 could overcome the IE1 deficiency. A variation of this model could include cellular proteins in the IE0-IE1 complex.
In support of the IE0-IE1 dimer hypothesis, a recent study suggested that IE0 and IE1 interact in regulating the expression of the AcMNPV he65 gene during the infectious cycle (39). Moreover, IE0 was postulated to be part of a tripartite protein-DNA-IE1 complex (39, 60). Recently, it was observed that a gradation of gene activation occurs in transfected Ld652Y cells, depending on the presence of IE1 alone, IE1 and IE0, or IE0 alone from OpMNPV (65). It was proposed that maintaining a low level of IE0 is a priority during a normal OpMNPV infection (65). Further studies will be required to prove directly the IE1-IE0 protein-protein interaction hypothesis and its biological role.w9h$, 百拇医药
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Received 5 July 2002/ Accepted 24 September 2002l(xm, 百拇医药
ABSTRACTl(xm, 百拇医药
Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.
INTRODUCTIONuf]v, 百拇医药
The arthropod virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Baculoviridae family, is able to infect 32 lepidopteran species (24). The complete genome of AcMNPV has been sequenced (3), and its replication in permissive cells has been studied extensively (7, 33). AcMNPV vectors are widely used as efficient tools for the expression of "foreign genes" in established cell lines and whole insects (7, 53). Recombinant AcMNPVs have been developed for use in pest control (29, 31, 48); moreover, baculovirus-based vectors have been considered for gene therapy purposes (1, 17). The ability of AcMNPV to interact with a wide range of hosts (50) makes it an attractive tool for elucidating which molecular factors are involved in determination of the host range of baculoviruses. Since AcMNPV can penetrate the nuclei of nonpermissive insect and mammalian cells, important mechanisms that determine the host range of AcMNPV are related to the expression of the viral genome (20, 44, 49-51).
AcMNPV gene expression is divided into early, late, and very late phases. Early-phase genes are transcribed by host RNA polymerase II before DNA replication, while late- and very-late-phase genes depend on viral DNA replication and are transcribed by a virus-specific RNA polymerase (4, 27). AcMNPV protein IE1, the product of immediate-early gene ie1, plays a central role in regulating viral infection (8, 15, 22, 37, 41, 61). IE1 is a potent transcriptional activator of early genes, such as 39K, p35, and he65 (9, 22, 25, 39), and is essential for both DNA replication and late gene transcription (35, 37, 39, 62, 67). IE1 negatively regulates the transcription of the pe38 and ie2 genes (22, 38, 39). IE0, the product of the ie0 gene, differs from IE1 only in that IE0 contains 54 additional amino acids at its amino terminus (resulting from splicing of the upstream ie0 exon and its subsequent fusion to the ie1 exon) (14). ie0 RNA is expressed only during the early phase of infection, and ie1 RNA is expressed during both early and late phases of infection (14, 36, 37). IE1 stimulates expression from the ie1 promoter but down-regulates expression from the ie0 promoter (37). The ie0 gene product also transactivates the ie1 promoter but does not affect expression from its own promoter (37).
The baculovirus genes p143 (helicase), hrf-1, hcf-1, p35, and ie2 were implicated in the determination of the host range of AcMNPV in various insect systems, including Bombyx mori, Lymantria dispar, Trichoplusia ni, and Spodoptera frugiperda (2, 13, 18, 44, 46, 56, 66).l&$4, 百拇医药
Recently, it was reported that AcMNPV infection of SL2 cells, a cell line derived from the Egyptian cotton worm Spodoptera littoralis, results in apoptosis and low yields of viral progeny (11, 19). The overexpression of p35, the apoptosis suppressor gene from AcMNPV (30), reduced apoptosis in AcMNPV-infected SL2 cells but did not improve the yield of AcMNPV budded viruses (BV) or viral infectivity for S. littoralis larvae (23). These results suggested that factors other than P35 were required by AcMNPV to replicate efficiently in SL2 cells. S. littoralis nucleopolyhedrovirus (SlNPV) replicates successfully in SL2 cells. To study the possibility that SlNPV could rescue AcMNPV infection, we cotransfected SL2 cells with AcMNPV DNA and a cosmid library representing the complete SlNPV genome. We isolated vAcSL2, a recombinant AcMNPV that yielded high titers of viral progeny in SL2 cells. vAcSL2 carried a 519-bp insert that disrupted the ie0 promoter, reducing the expression of this gene product and resulting in increased steady-state levels of IE1 relative to IE0. To confirm these observations, we mutagenized the ie0 promoter locus by inserting the chloramphenicol acetyltransferase (cat) gene and obtained a new recombinant AcMNPV with replication properties identical to those of vAcSL2. These data suggested that the overexpression of ie1 may enable efficient replication of AcMNPV in the SL2 host. Indeed, using various means to promote the overexpression of ie1, we were able to achieve productive AcMNPV infection of S. littoralis cells and larvae.
MATERIALS AND METHODS0, http://www.100md.com
Cell lines and viruses. S. frugiperda SF9 and S. littoralis SL2 cells were maintained and propagated in TNM-FH medium supplemented with 10% heat-inactivated fetal bovine serum (64). Wild-type AcMNPV strain E-2 (63) and SlNPV strain E-15 (32) were propagated on SF9 cells. SlNPV can replicate in both SF9 and SL2 cells. Viral growth curves were determined by plaque assays as described previously (23).0, http://www.100md.com
Plasmids and transfection. Plasmid pAcie1 contains an intact ie1 gene (26). pHspie1 contains the ie1 coding region under the control of the Drosophila melanogaster hsp70 promoter. The latter was restricted from phsp70PLVI+CAT (16) by using XbaI and BamHI and was subcloned into pBSK (Stratagene) bearing the ie1 coding region HincII-HindIII DNA fragment from pAcie1. pBgl-11.1 and pBgl-3.3 bear 11.1- and 3.3-kbp BglII fragments of vAcSL2, respectively, each cloned at the BglII site of pBSK (see below); the 11.1-kbp fragment contains the ie0 and ie1 genes. pie0PCAT bears the cat gene inserted at AcMNPV nucleotide 122,800 (3) to disrupt the ie0 promoter at position -31 upstream of the ATG start site. For this purpose, a 1.6-kbp SnaBI-SalI fragment bearing the ie0 promoter region was cloned into the corresponding site of pFastBac1 (Invitrogen Life Technologies). Then, a BamHI site was created by mutagenesis with overlap extension PCR and with complementary primers containing the desired restriction site (55). The primers used were 5'-GCCGGATCCAGTATAAGTAATTGA-3' and 5'-TTATACTGGATCCGGCGCGCGCA-3' (underlining indicates the BamHI site). The cat gene from pQ39CAT (25) was digested with BamHI and inserted into the newly created BamHI site to yield pie0PCAT.
Generation of a recombinant baculovirus by homologous recombination in Escherichia coli. The polyhedrin (polh) gene, which the manufacturer deleted from AcMNPV BACmid (Invitrogen Life Technologies), was first reintroduced by site-specific transposition with transfer vector pFastBac1 into E. coli DH10BAC cells (45). For this purpose, the complete polh gene, including its own promoter and termination sequence, was rescued from pI1 (12) by digestion with XhoI, filling in with Klenow DNA polymerase, and further restriction with EcoRI and was inserted into EcoRI- and Bst1107I-digested pFastBac1 to obtain pFastpolh. Site-specific transposition with pFastpolh and selection of DH10BAC cells harboring recombinant BACmid AcFastpolh were performed by following the manufacturer's (Invitrogen Life Technologies) instructions. Then, to disrupt the ie0 promoter in the viral (BACmid) genome, E. coli strain BJ5183 was transformed with recombinant BACmid AcFastpolh (5). The resulting strain, E. coli BJ-AcFP, was transformed with the 2.3-kb SnaBI-SalI fragment containing the cat gene-disrupted ie0 promoter sequence from pie0PCAT. Bacteria resistant to kanamycin and chloramphenicol were selected, and high-molecular-weight minipreparation DNA of recombinant BACmid AcFPCAT, with a cat gene insertion at the ie0 promoter (Acie0PCAT), was prepared as described in the Bac-to-Bac protocol (Invitrogen Life Technologies). The presence of the cat gene insertion at the ie0 promoter of BACmid AcFPCAT was confirmed by PCR with BACmid DNA template and primers CAT5' (5'-ATGGAGAAAAAAACACTG-3'), CAT3' (5'-TTACGCCCCGCCCT-3'), and IE0R (5'-GTGTCAACTTGCAACTGCTGAGCTTCTGC-3'). Acie0PCAT DNA was used to transfect insect cells to obtain recombinant virus vAcie0PCAT.
DNA transfection of insect cells was performed with Lipofectin as described previously (19)..ha[, 百拇医药
Rescue of AcMNPV replication in SL2 cells and isolation of AcMNPV recombinants. SL2 cells were cotransfected with either a complete cosmid library or individual cosmids of SlNPV (19) or AcMNPV, respectively. At 96 h after transfection, the culture supernatants were removed and recombinant viruses that replicated and formed polyhedra in SL2 cells were isolated by subsequent plaque purification in these cells..ha[, 百拇医药
Restriction endonuclease and Southern blot analyses. Viral DNA prepared from BV (19) was digested with various restriction endonucleases and subjected to agarose gel electrophoresis (0.8% agarose) for 16 h at 30 V. After the run, the gel was quickly blotted to a nylon membrane (47). Hybridization was performed by using a GeneScreen Plus membrane according to the manufacturer's instructions (NEN Life Science Products). Probe labeling was performed by using a Renaissance random primer fluorescein labeling kit (NEN Life Science Products). The 519-bp insert fragment (IF) from vAcSL2 and the 2.4-kb EcoRV-HindIII fragment of the AcMNPV ie1 gene (25) were used as hybridization probes.
Western blot analysis. Virus-infected or plasmid-transfected cells were harvested and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with a pH 9 separating gel; this procedure allowed us to distinguish clearly the IE0 molecular species from the IE1 molecular species. Immunoblot analysis was performed with anti-IE0/IE1 antiserum as previously described (23, 54).(/wow7, 百拇医药
Metabolic labeling of viral polypeptides. SF9 or SL2 cells (105 cells) were infected with either wild-type or recombinant AcMNPV. At 45 h postinfection, the cells were washed once with Sf900 II medium depleted of methionine and cysteine (Invitrogen Life Technologies). The medium was subsequently replaced with 500 µl of the same medium supplemented with 15 µCi of a 35S-labeled methionine-cysteine mixture (Amersham, Little Chalfont, United Kingdom). After 2 h, cell extracts were prepared by adding SDS lysis buffer (53) and then subjected to PAGE. Autoradiography was performed as described before (53).
Bioassays. Carefully selected third-instar S. littoralis larvae (24 per dose, in triplicate) were injected with various doses of AcMNPV or vAcSL2 BV. Control larvae were injected with the same volume of TNM-FH complete medium. The percent mortality was calculated as the number of dead larvae (excluding larvae killed by the injection, normally one or two) divided by the number of larvae that survived the infection. No mortality was observed for mock-infected larvae (12, 23).(+#6p, http://www.100md.com
An oral bioassay of third-instar S. littoralis was performed by using a diet containing six concentrations of wild-type and recombinant viral polyhedra ranging from 5 x 102 to 2.5 x 105 polyhedral inclusion bodies (PIB)/mm2. After 24 h of exposure to the virus, the larvae were transferred to new containers containing a noncontaminated diet; larval death was monitored daily for 3 weeks. Thirty larvae were used for each dose, and the experiment was repeated twice.(+#6p, http://www.100md.com
RESULTS
Rescue of AcMNPV replication in S. littoralis SL2 cells. To study the potential of the SlNPV genome to sustain the replication of AcMNPV in SL2 cells, we cotransfected the cells with AcMNPV DNA and an overlapping cosmid library representing the entire SlNPV genome (19). After 6 days, we observed the appearance of polyhedra in the cell nuclei. By plaque purification, we were able to isolate a recombinant AcMNPV, vAcSL2, that replicated in SL2 and SF9 cells. At 96 h after infection of SL2 cells with vAcSL2, only 10 to 20% of the cells showed apoptosis, while 40 to 70% of the remaining cells showed clear polyhedra under direct microscopic observation (Fig. 1, upper left panel). In contrast, 90% of SL2 cells infected with AcMNPV at multiplicities of infection (MOIs) of 1 to 100 showed apoptosis and did not show PIB (Fig. 1, upper right panel). Compared to AcMNPV, vAcSL2 produced regular polyhedra in SF9 cells (Fig. 1, lower right and left panels, respectively). Since apoptosis has been implicated in the significant reduction of AcMNPV BV yields observed in SL2 cells (11), we compared the abilities of vAcSL2 and AcMNPV to replicate in these cells at MOIs of 0.5 and 5 (Fig. 2). BV were harvested from cell supernatants at 6, 9, 12, 28, and 48 h postinfection and titrated in SF9 cells. The titers of vAcSL2 obtained were significantly higher than those of AcMNPV: e.g., at 28 and 48 h after infection with an MOI of 0.5, the yield of vAcSL2 was 1,000-fold higher than that of AcMNPV (Fig. 2A).
fig.ommitteedyj\ge-, 百拇医药
FIG. 1. vAcSL2 replicates in SL2 cells and forms polyhedra. SL2 and SF9 cells were infected with vAcSL2 or AcMNPV. Images are light micrographs (magnification, x400). Note the vAcSL2-infected cells producing polyhedra over the background of the intact cells, compared to AcMNPV-infected SL2 cell blebbing.yj\ge-, 百拇医药
fig.ommitteedyj\ge-, 百拇医药
FIG. 2. BV yields from vAcSL2- and AcMNPV-infected SL2 cells. BV were collected from clarified cell supernatants at various times after infection with vAcSL2 or AcMNPV and titrated in SF9 cells (in triplicate). Error bars show standard deviations.yj\ge-, 百拇医药
Given that vAcSL2 replicated efficiently in SL2 cells, it was interesting to assess whether the vAcSL2 genome could rescue AcMNPV replication. For this purpose, we cotransfected SL2 cells with intact AcMNPV DNA and vAcSL2 DNA digested with various restriction enzymes. At about 72 to 96 h after transfection, the cells were examined microscopically for the presence of polyhedra. vAcSL2 DNA digested with either SmaI, XhoI, HindIII, or BglII rescued production of polyhedra, while EcoRV-digested DNA did not rescue it (Table 1). Remarkably, only EcoRV treatment is supposed to disrupt the ie1 open reading frame. BglII-digested vAcSL2 genomic DNA showed two new fragments of about 11.1 and 3.3 kbp (designated and ß, respectively), not present in AcMNPV (Fig. 3A), due to the presence of a new BglII site in fragment D of BglII-digested AcMNPV (see below). To determine whether either of these two fragments was able to rescue AcMNPV amplification in SL2 cells, they were subcloned in pBSK to obtain plasmids pBgl-11.1 and pBgl-3.3 (see Materials and Methods). Transfection of SL2 cells with each of these plasmids and AcMNPV DNA showed that only pBgl-11.1 could rescue formation of polyhedra (Table 1).
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TABLE 1. Rescue of wild-type AcMNPV replication in SL2 cells by vAcSL2 DNA fragmentsay3y+, 百拇医药
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FIG. 3. vAcSL2 contains a 519-bp insert downstream of the ie0 TATA element that disrupts the basic transcriptional unit. (A) vAcSL2 DNA (lane 1) and AcMNPV DNA (lane 2) were digested with BglII, and the restricted DNA fragments were analyzed in a 0.7% agarose gel. New BglII fragments in vAcSL2 are indicated by arrows ( and ß). Lane M, molecular size markers DNA digested with BstEII) (in kilobase pairs). (B) Partial physical map and sequence of fragment downstream of the new BglII site. Indicated are the ie0 TATA element (bold), the transcriptional initiation motif CAGT (bold and underlined), and the downstream IF sequence that increases the distance between the motifs (repeated sequence is underlined). AcMNPV map units are indicated as well. The boxes show the locations of the ie0 5'-terminal coding region and the me53 gene, which are transcribed in opposite directions (arrows). Restriction sites: H, HindIII; B, BglII; S, SalI.
Sequencing of the and ß fragments showed that the new BglII site corresponded to a point mutation in nucleotide 125 (A to G) in the coding region of viral gene me53 which resulted in a conservative change (Lys 42 to Arg). The vAcSL2 11.1-kbp fragment that was able to rescue formation of polyhedra contained the whole ie0-ie1 region of AcMNPV and included a new insert of 519 bp (named IF) located upstream of the ie0 gene at position -88 from the ATG start site (Fig. 3B). The sequence 5'-TCGTAAATCAG-3' was repeated and flanked both ends of the IF at positions -78 to -88 and positions -586 to -597 from the ie0 ATG start site (Fig. 3B, underlining). The IF increased the distance between the ie0 CAGT transcriptional start site and the TATA element by 519 bp (Fig. 3B, bold underlining).o/i83i@, 百拇医药
Expression of ie0 in vAcSL2-infected cells. We observed that in AcMNPV-infected SL2 cells, the steady-state levels of IE1 and IE0 were almost identical throughout the infection (Fig. 4A; see also Fig. 6C); in contrast, in AcMNPV-infected permissive SF9 cells (Fig. 4B), the steady-state levels of IE1 early in infection were low relative to those of IE0 and later increased progressively. It should be noted that the double bands of IE1 and IE0 represented the phosphorylated and dephosphorylated main electrophoretic species reported (15) (see also below). The presence of the IF upstream of the transcriptional start site of ie0 suggested that it might result in a lower level of expression of this gene in vAcSL2-infected cells. Indeed, IE0 was synthesized at very low steady-state levels in vAcSL2-infected SL2 and SF9 cells compared to AcMNPV-infected cells (Fig. 4A and B), and the steady-state levels of IE1 were higher than those of IE0.
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FIG. 4. IE1 and IE0 synthesis in vAcSL2- and AcMNPV-infected cells. (A and B) SL2 and SF9 cells (105) were infected with vAcSL2 and AcMNPV at an MOI of 5. At various times postinfection, the cells were harvested, and cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-IE0/IE1 antiserum. mi, mock-infected cells. (C) Expression of ie1 in transfected cells. SF9 cells (105) were transfected with 1 µg of pBgl-11.1 (bearing the 、 fragment) or pHspie1 (bearing ie1 under the control of the hsp70 promoter). At 24 h after transfection, cell extracts were subjected to SDS-PAGE and immunoblot analysis. m-transf., mock-transfected cells. Molecular mass markers are indicated.g, 百拇医药
fig.ommitteedg, 百拇医药
FIG. 6. vAcie0PCAT structure and analysis and synthesis of IE1 and IE0. (A) Genomic organization. The dashed lines indicate the site of insertion of the cat gene (box). The other boxes show the locations of the ie0 coding region (exon 1 and exon 2) and the ie1 and me53 genes and their corresponding promoters (upper arrows). IE0R, CAT5', and CAT3' were primers used for PCR analysis. The dotted lines indicate the expected sizes of amplified fragments. (B) PCR analysis. (Left panel) cat gene amplification. Lane 1, no template; lane 2, AcMNPV; lane 3, vAcie0PCAT. (Right panel) Lane 1, cat gene marker (same as lane 3 in the left panel); lanes 2 to 4, cat gene plus exon 1 (primers CAT3' and IE0R), with no template in lane 2, AcMNPV in lane 3, and vAcie0PCAT in lane 4. (C) SL2 (105 cells) were infected with AcMNPV and vAcie0PCAT at an MOI of 5. pHspie1, SF9-transfected cell control. At various times postinfection, cell extracts were subjected to SDS-PAGE and immunoblot analysis with anti-IE0/IE1 antiserum as described in the legend to Fig. 4. Molecular mass markers are indicated.
A comparison of SF9 cells transfected with pBgl-11.1 and with pHspie1 (bearing only the ie1 gene under the control of the D. melanogaster hsp70 promoter) confirmed that IE1 was the main electrophoretic species synthesized; IE1 was clearly distinguished from IE0 (15) (see also Materials and Methods) (Fig. 4C). IE0 was undetectable in pBgl-11.1-transfected cells; in comparison, AcMNPV-infected SF9 cells synthesized IE0 and IE1 (Fig. 4C).d\, http://www.100md.com
To find out whether the IF of vAcSL2 was derived from the SlNPV genome, we performed Southern blot analysis. SlNPV, AcMNPV, and vAcSL2 genomic DNAs were digested with HindIII (Fig. 5A, lanes 2, 3, and 4, respectively) and PstI (Fig. 5A, lanes 7, 8, and 9, respectively). Hybridization with labeled IF at 60°C showed that vAcSL2 bore the IF, as expected, but did not show fragments homologous to IF in the genomes of SlNPV and AcMNPV (Fig. 5B, lanes 4 and 9, lanes 2 and 7, and lanes 3 and 8, respectively). Performing the hybridization at a lower stringency revealed a weak signal corresponding to the HindIII G and PstI B fragments of AcMNPV (8.15 and 21.6 kbp, respectively) that bear the IE0 and IE1 coding sequences but not to SlNPV. Also, Southern blot analysis with the same IF probe and cellular DNA did not indicate the presence of a homologous sequence in the SL2 cell genome (data not shown). A GenBank search did not reveal homology between the IF and any reported sequences. Thus, we concluded that the IF was probably the result of a recombinational event between AcMNPV and viral or cellular DNA, but we could not establish its exact genomic source.
fig.ommitteedh?44, http://www.100md.com
FIG. 5. Southern blot analysis of AcMNPV recombinants. (A) HindIII- or PstI-digested viral DNAs. (B) Hybridization with fluorescein-labeled IF (PCR amplified from pBgl-11.1) at 60°C. (C) Hybridization with fluorescein-labeled EcoRV-HindIII ie1 fragment. Lanes 2 and 7, SlNPV; lanes 3 and 8, AcMNPV; lanes 4 and 9, vAcSL2; lanes 5 and 10, vBgl3; lanes 6 and 11, vHsp-1; lane 1, undigested pBgl-11.1; lane M, molecular size markers (in kilobase pairs).h?44, http://www.100md.com
To avoid the possibility that the phenotype of vAcSL2 was due to some unexpected mutation in the viral genome, we constructed a recombinant AcMNPV in which the ie0 promoter region was disrupted by insertion of the cat gene. For this purpose, we used the method described by Bideshi and Federici (5). Plasmid pie0PCAT was used to transpose the mutated ie0 promoter region into BACmid AcFastpolh, and the resultant BACmid construct was transfected into SF9 cells to further isolate recombinant baculovirus vAcie0PCAT (see Materials and Methods) (Fig. 6A). The constructs were confirmed by PCR analysis with a primer complementary to the cat gene and a primer 159 bp downstream of the transposition site (CAT3' and IE0R, respectively) (Fig. 6B). The effect of disruption of the ie0 promoter on the synthesis of IE0 and IE1 during the infectious cycle of vAcie0PCAT was studied as described above by using immunoblot analysis (Fig. 6C). vAcie0PCAT infection of SL2 cells reproduced the patterns of expression of ie0 and ie1 observed with vAcSL2 expressing mainly ie1 (compare to AcMNPV-infected cells and to pHspie1-transfected cells in Fig. 6C and Fig. 4A). Direct microscopic observation of vAcie0PCAT-infected SL2 cells revealed polyhedra (data not shown). Moreover, vAcie0PCAT showed a significant improvement in BV yields in SL2 cells compared to wild-type AcMNPV (Fig. 7). Identical results were obtained when the cat gene was inserted in the opposite orientation (data not shown).
fig.ommitteed*:r^%, 百拇医药
FIG. 7. BV yields from vAcie0PCAT- and AcMNPV-infected SL2 cells. BV were collected from clarified cell supernatants at various times after infection (p.i.) with an MOI of 0.5 and titrated in SF9 cells (in triplicate). Error bars indicate standard deviations.*:r^%, 百拇医药
Formation of AcMNPV polyhedra can be rescued in SL2 cells by increasing the level of expression of ie1. The above results suggested that enhancing ie1 expression might rescue the replication of AcMNPV in SL2 cells. To test this hypothesis, we cotransfected SL2 cells with either pBgl-11.1 or pHspie1 and AcMNPV DNA. At 72 to 96 h after transfection, formation of polyhedra was detected in the transfected cells, while control cells cotransfected with pBSK and AcMNPV DNA did not show polyhedra (data not shown). Moreover, by subsequent plaque purification, we were able to isolate from pHspie1 and pBgl-11.1 cotransfection supernatants new recombinant viruses (vHsp-1 and vBgl3, respectively) that replicated in SL2 cells, forming polyhedra. Southern blot analysis with an ie1 probe (see Materials and Methods) showed that vHsp-1 bore two copies of the ie1 gene (Fig. 5C, lanes 6 and 11). vBgl3 bore the IF in the viral genome (Fig. 5C, lanes 5 and 10). Lighter bands observed in all of the lanes corresponded to the common background usually observed with this chemiluminescence detection method under these exposure conditions (Fig. 5C). Thus, in the first case, cotransfection with pHspie1 appeared to have resulted in the insertion of a second ie1 gene in the viral genome. In the second case, the region upstream of the ie0 gene appeared to have been replaced by an IF copy.
Infection of SF9 cells with vAcSL2, vHsp-1, vBgl3, and AcMNPV followed by metabolic labeling with 35S-labeled amino acids (see Materials and Methods) resulted in similar polypeptide profiles (Fig. 8, lanes 3, 4, 5, and 2, respectively). The most prominent polypeptide detected at 45 h postinfection was polyhedrin. This marker, which reflected the succesful completion of the viral replication cycle, was clearly detected in SL2 cells infected with vAcSL2, vBgl3, and vHsp-1 but not AcMNPV (Fig. 8, lanes 8, 9, 10, and 7, respectively). As expected, polyhedrin was not observed in mock-infected SF9 and SL2 cells (Fig. 8, lanes 1 and 6, respectively).}, 百拇医药
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FIG. 8. Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (105) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with AcMNPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of AcMNPV polyhedrin.
The expression of ie1 was implicated in the induction of apoptosis in S. frugiperda cells (57). Also, it was reported that AcMNPV-infected SL2 cells underwent apoptosis and expressed the antiapoptotic gene p35 at very low levels (23). Thus, we hypothesized that the overexpression of ie1 would enhance p35 expression as well, neutralizing the potential increase in the apoptotic response of the cell. Indeed, P35 steady-state levels were higher in vAcSL2-infected cells than in AcMNPV-infected SL2 cells (Fig. 9, lanes 1 to 3 and lanes 4 to 6, respectively).h'[m', http://www.100md.com
fig.ommitteedh'[m', http://www.100md.com
FIG. 9. Synthesis of the apoptosis suppressor P35 in vAcSL2-infected SL2 cells. SL2 cells were infected at an MOI of 5 with vAcSL2 (lanes 1 to 3) or AcMNPV (lanes 4 to 6). Cell extracts (105 cell equivalents) were harvested at various times postinfection and subjected to SDS-PAGE and immunoblot analysis with anti-P35 antiserum (23). A molecular mass marker is indicated.h'[m', http://www.100md.com
Infectivity of vAcSL2 for S. littoralis larvae. To determine whether the ability of vAcSL2 to replicate efficiently in SL2 cells is a cell line phenomenon or has organismic implications, we compared the infectivity of vAcSL2 with that of AcMNPV for S. littoralis larvae. For this purpose, S. littoralis third-instar larvae were infected with increasing doses of viral polyhedra orally (see Materials and Methods) or via the intrahemocoelic route (by injection of increasing doses of BV). No differences in AcMNPV and vAcSL2 infectivities were measured for the oral route which, under these conditions, resulted in only 10 to 14% larval death at the higher PIB doses (data not shown). However, significant differences in larval mortality were observed for intrahemocoelic inoculation of BV: a 10-fold increase in the AcMNPV dose was required to obtain 50 and 70% mortality of the larval population (at doses of 100 and 1,000 PFU, respectively) (Fig. 10).
fig.ommitteed;7, 百拇医药
FIG. 10. Mortality of S. littoralis larvae infected with vAcSL2 and AcMNPV. S. littoralis larvae (third instar, 24 larvae per dose) were injected with increasing concentrations of AcMNPV or vAcSL2 BV. The mortality rate was calculated as the number of larvae that died from the infection divided by the number of survivors. The results are the averages of three independent experiments. Error bars indicate standard deviations.;7, 百拇医药
DISCUSSION;7, 百拇医药
Expression of ie1 and replication of AcMNPV in S. littoralis. IE1 displayed steady-state levels equivalent to or lower than those of IE0 during the early phase of AcMNPV infection of permissive SF9 cells (Fig. 4B and 6C). Late in infection, IE1 steady-state levels increased significantly over those of IE0 in SF9 cells (54 and ibid), but in poorly permissive SL2 cells, the steady-state levels of IE1 and IE0 remained equivalent throughout the infection (Fig. 4A and 6C). The IF disrupted the continuity of the ie0 transcriptional unit by increasing the distance between the TATA element and the CAGT transcriptional start site (6, 22, 58), suggesting that indeed the transcription of ie0 would be diminished in vAcSL2-infected cells; however, this aspect was not directly analyzed in this study (e.g., by transcriptional mapping). However, the steady-state levels of IE0 were reduced consistently in vAcSL2-infected cells (Fig. 4), resulting in higher steady-state levels of IE1 than of IE0 in vAcSL2-infected SL2 cells compared to AcMNPV-infected cells (Fig. 4A). vAcSL2 showed a significant increase in infectivity toward S. littoralis SL2 cells (Fig. 1) and larvae (Fig. 10, BV injection). However, no change in infectivity was measured when infection was performed via the oral route. Thus, it appears that viral factors besides the overexpression of IE1 relative to IE0 were required to overcome the initial response of S. littoralis larvae (probably of immune origin) (68) to viral infection.
The IF displayed the same repeat sequence, 5'-TCGTAAATCAG-3', at its 5' and 3' termini, suggesting that it originated by some recombinational event between viral (AcMNPV or SlNPV) and cellular (SL2) sequences. However, we could not determine the exact origin of the IF by Southern blot analysis. Thus, we evaluated whether the insertion of another arbitrary DNA fragment (e.g., the cat gene) (Fig. 6) instead of the IF into the ie0 promoter locus might reproduce the vAcSL2 phenotype. Indeed, cat gene-mediated disruption of the ie0 promoter region resulted in enhanced replication of recombinant vAcie0PCAT in SL2 cells (Fig. 7) that correlated with lower steady-state levels of IE0 than of IE1 (Fig. 6C).jx, http://www.100md.com
The above data suggested the following. (i) There was a block to ie1 expression in AcMNPV-infected SL2 cells (probably late expression, although it was not rigorously measured by comparing it with ie1 expression in aphidicolin-treated infected cells) (compare Fig. 4A and B). (ii) An increase in the steady-state levels of IE1 relative to IE0 may enable the replication of AcMNPV in SL2 cells (see below).
We confirmed the latter hypothesis by cotransfecting AcMNPV and plasmid pHspie1 or the ie0-ie1 genetic unit present in the vAcSL2 genome (pBgl-11.1) and subsequently isolating new recombinant viruses that overexpressed ie1 (Table 1 and Fig. 8). Our results provide the first evidence that AcMNPV can replicate in a poorly permissive cell line (and insect) through an increase in the level of expression of the ie1 gene (see below).w, 百拇医药
IE0 and IE1 functions. IE1 is a multifunctional protein that plays a crucial role in the life cycle of AcMNPV: (i) transactivation of the delayed early and late classes of genes (37, 39), (ii) its own continued expression during infection, and (iii) repression of other immediate-early genes.w, 百拇医药
The finding that lower steady-state levels of IE0 and higher steady-state levels of IE1 enabled AcMNPV replication in SL2 cells suggested that IE0 may be involved in the regulation of IE1 function. Thus, elevated IE1 levels may be required in SL2 cells to stimulate genes involved in AcMNPV DNA replication, such as DNA polymerase, p35, and helicase genes (10, 43, 52, 54, 67), to become directly involved in binding to a replication origin and catalyzing early steps that lead to the assembly of a replication complex (33, 42). Also, the reduction of apoptosis in vAcSL2-infected SL2 cells may be the result of enhanced p35 gene expression (Fig. 9) (23).
IE1 has separate domains for transactivation and DNA binding that are essential for the modulation of baculovirus gene expression (35, 60, 61). Little is known about the role of IE0 as a transregulator, although it possesses an additional N-terminal 54 amino acids compared to IE1. Transient expression assays revealed that IE0 could transactivate the ie1 promoter but did not affect expression from the ie0 promoter, while IE1 stimulated expression from the ie1 promoter but down-regulated expression from the ie0 promoter (37). IE1 was shown to bind, probably as a dimer (60), to specific DNA sequences present in hrs that have been demonstrated to function as cis-acting enhancers of viral transcription and origins of replication (34, 40, 41, 59) and to sequences present in promoters of genes that it down-regulates (41). The motif 5'-ACBYGTAA-3' is present in all DNA fragments that have been shown to bind IE1, including ie0 (41). Thus, it was proposed that transcriptional repression by IE1 occurs when such a single binding motif is present in the DNA sequence and that IE1 may have a lower affinity for these single motifs than for the complete palindromic sequences present in the hrs (42).
The same IE1 domains that are involved in DNA binding are present in IE0. Moreover, it was suggested that the putative helix-loop-helix domain present at C-terminal residues 543 to 568 mediates oligomerization and DNA binding of IE1 (61). Taken together, these and the above data suggest that IE0 and IE1 can form heterodimers (39). It is conceivable that the IE0-IE1 heterodimer has a function different from that of the IE1-IE1 homodimer: IE1 or the IE1-IE1 dimer could up-regulate the ie1 promoter and down-regulate the ie0 promoter (41), but the formation of an IE0-IE1 complex could compete for free IE1 molecules, preventing the formation of IE1-IE1 dimers, and/or could compete for DNA binding of the IE1 homodimer, which is required to enhance the expression of ie1 and other IE1-transactivatable promoters.{dmoy, http://www.100md.com
In addition, it was postulated that a host factor(s) which transactivates the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) ie1 promoter is present in SF9 cells but absent in Ld652Y cells (21), and a 38-kDa protein with hr gene binding capacity was isolated from SF9 cells (28). Similarly, such a factor could be missing or not very abundant in SL2 cells, resulting in a low level of ie1 expression late in infection and consequently in an insufficient amount of functional IE1 to compete with the IE1-IE0 complex or to displace IE0 from it and transactivate essential AcMNPV replication genes. Either the reduction of ie0 expression or the overexpression of ie1 could overcome the IE1 deficiency. A variation of this model could include cellular proteins in the IE0-IE1 complex.
In support of the IE0-IE1 dimer hypothesis, a recent study suggested that IE0 and IE1 interact in regulating the expression of the AcMNPV he65 gene during the infectious cycle (39). Moreover, IE0 was postulated to be part of a tripartite protein-DNA-IE1 complex (39, 60). Recently, it was observed that a gradation of gene activation occurs in transfected Ld652Y cells, depending on the presence of IE1 alone, IE1 and IE0, or IE0 alone from OpMNPV (65). It was proposed that maintaining a low level of IE0 is a priority during a normal OpMNPV infection (65). Further studies will be required to prove directly the IE1-IE0 protein-protein interaction hypothesis and its biological role.w9h$, 百拇医药
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