ac18 is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus

Virology 367 (2007) 71 – 81 www.elsevier.com/locate/yviro

ac18 is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus Yanjie Wang, Wenbi Wu, Zhaofei Li 1 , Meijin Yuan, Guozhong Feng, Qian Yu, Kai Yang ⁎, Yi Pang State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China Received 14 February 2007; returned to author for revision 10 March 2007; accepted 9 May 2007 Available online 18 June 2007

Abstract orf18 (ac18) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, an ac18 knockout AcMNPV bacmid was generated to determine the role of ac18 in baculovirus life cycle. After transfection of Sf-9 cells, the ac18-null mutant showed similar infection pattern to the parent virus and the ac18 repair virus with respect to the production of infectious budded virus, occlusion bodies, or the formation of nucleocapsids as visualized by electron microscopy. The deletion mutant did not reduce AcMNPV infectivity for Trichoplusia ni in LD50 bioassay; however, it did take 24 h longer for deleted mutant to kill T. ni larvae than wild-type virus in LT50 bioassay. Our results demonstrate that ac18 is not essential for viral propagation both in vitro and in vivo, but it may play a role in efficient virus infection in T. ni larvae. © 2007 Elsevier Inc. All rights reserved. Keywords: Baculovirus; orf18; AcMNPV; Auxiliary gene

Introduction The family Baculoviridae includes a large group of DNA viruses that infect invertebrate species mainly belonging to the order Lepidoptera and contains two genera: Nucleopolyhedrovirus (NPV) and Granulovirus (GV) (Theilmann et al., 2005). Two types of baculoviral virions, budded virions (BVs) and occlusion-derived virions (ODVs), are produced during the life cycle. BVs can bud through the cell membrane to infect other cells, while ODVs remain in the nucleus and are embedded in a protein matrix, which is composed of polyhedrin during the late phase of infection (Theilmann et al., 2005). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most intensively studied member of baculoviruses. AcMNPV genome is about 134 kbp and

⁎ Corresponding author. Fax: +86 20 84037472. E-mail address: [email protected] (K. Yang). 1 Present address: Boyce Thompson Institute, Cornell University, Ithaca, NY 14850, USA. 0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2007.05.017

contains 154 potential methionine-initiated orfs of more than 150 nt (Ayres et al., 1994). To date, the functions of about 50% genes in AcMNPV genome have been determined. Some of the genes are essential genes, which are required for replication and cannot be deleted from the genome; some of the genes are auxiliary genes that are not essential for replication but may give the virus a certain selective advantage in nature (O'Reilly, 1997). orf18 (ac18) of AcMNPV (nt 14,398–15,459) encodes a putative protein of 353 amino acids with a predicted molecular mass of 40.865 kDa. Its homologs are present in almost all of the sequenced lepidopteran NPVs. The Ac18 protein is highly conserved in AcMNPV, Plutella xylostella NPV, Rachiplusia ou NPV, Bombyx mori NPV and Maruca vitrata NPV. The amino acid identities of Ac18 among these five baculoviruses are 87–99%, while the amino acid identities between Ac18 and its homologs of other lepidopteran NPVs are 21–52%. Homologs of Ac18 have not been identified in lepidopteran GVs, dipteran NPVs and hymenopteran NPVs. Sequence-based queries performed with InterProScan program showed that Ac18 was a protein of unknown function.

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Previous data indicated that the PstI-G region (7.6–13.1 m.u.) of AcMNPV, which includes ac18 (10.5–11.5 m.u.), was genetically unstable during serial passage of AcMNPV in a Trichoplusia ni cell line, and such deletion mutant became predominant in the serially passaged stocks (Kumar and Miller, 1987). Guarino and Summers (1988) reported that in a transientexpression assay system, a viral gene product, IE-1 and one of the cis-acting AcMNPV enhancers might be required late in infection. Expression of the late gene product was further increased by cotransfection of ac18 and orf16 (ac16) of AcMNPV. In this report, we generated an ac18 knockout AcMNPV mutant by homologous recombination in Escherichia coli (E. coli) and characterized it both in vitro and in vivo. We found that the ac18 deletion mutant had no striking phenotype in Sf-9 cells, including BV production, cytological changes and viral morphogenesis. However, the larval bioassays showed that the infectivity of the mutant virus did not reduce, but the mutant virus did take approximately 24 h longer to kill T. ni larvae than wild-type virus. We also examined ac18 transcription pattern and Ac18 localization in virus-infected Sf-9 cells. Our results suggest that Ac18 might play an important role in virus replication in vivo. Results RT-PCR analysis and mapping of the 5′ end of the ac18 transcript Baculovirus genes can be divided into early, late, and very late phases for transcription. To determine the temporal expression of ac18 transcript, RT-PCR was performed using total RNAs isolated from AcMNPV-infected Sf-9 cells at different time points as templates. We took advantage of AcMNPV ie-1 gene and vp39 gene as the controls for the early gene and late gene, respectively. As expected, a 1722-bp ie-1 transcript was amplified by ie-1-specific primers from 1 h p.i. (post-infection) to 72 h p.i. By contrast, a 1041-bp vp39 transcript was detectable from 12 h p.i. to 96 h p.i. when amplifying by vp39-specific primers (Fig. 1A). The earliest appearing ac18-specific transcript, which was indicative of a 1059-bp fragment amplified by ac18-specific primers, was detectable at 2 h p.i. and remained detectable up to 96 h p.i. (Fig. 1A). None of the ie-1, vp39 or ac18 transcripts was detected in the control experiments in which no reverse transcriptase was added prior to the PCR step (data not shown), indicating no possible contamination of AcMNPV DNA. The 5′ end of ac18 transcript was determined by 5′ RACE analysis with total RNAs isolated from AcMNPV-infected Sf-9 cells at 24 and 48 h p.i. With ac18-specific primers, a single cDNA was detected for ac18 transcript. The transcription initiation site of ac18 is 47 nt upstream of the translation initiation codon ATG and started at the first C (nt 15,502) of CGTGC motif (Fig. 1B). Thus, the ac18 transcript might belong to the early class of baculovirus transcripts and initiated within a rare baculovirus promoter motif as that of baculovirus DNA polymerase gene (Ohresser et al., 1994).

Construction of ac18 knockout AcMNPV bacmid To determine whether ac18 is essential for viral replication, we generated a bacmid containing a knockout region in the ac18 gene via the λ Red recombination system in E. coli as described previously (Bideshi and Federici, 2000; Lin and Blissard, 2002). For this procedure, we constructed a transfer vector (pUC18-US-Cm-DS) in which the ac18 locus region was replaced with a chloramphenicol resistance gene (Cm) for antibiotic selection in E. coli. DH10Bac cells containing wildtype (wt) AcMNPV bacmid (bMON14272, ac18-positive control) (Luckow et al., 1993) were transformed with pBADgbaA and were induced by L-arabinose to allow expression of the λ Red system, and then the cultures were transformed with the linear DNA fragment containing Cm gene cassette and ac18 flanking regions. An 857-bp fragment of the ac18 coding region (nt 14,396–15,252) in AcMNPV bacmid was replaced by Cm gene cassette via homologous recombination (Fig. 2A). The resulting bacmid vAcac18KO was selected by growth on medium containing kanamycin and chloramphenicol. The absence of ac18 and the replacement with Cm gene in AcMNPV bacmid were confirmed by Southern blot hybridization analysis and PCR analysis. For Southern blot hybridization, an 857-bp fragment of AcMNPV, a deleted region of the ac18 gene, was PCR amplified using the primer pair 18P-U and 18P-D, purified and digoxigenin labeled, and was used as a probe (ac18 probe) to detect the ac18 gene (Fig. 2B). Using the primer pair CmU and CmD a 1039-bp fragment of the Cm gene was PCR amplified and also labeled with digoxigenin dUTP to be used as a probe (Cm probe) to detect the Cm gene (Fig. 2B) as previously described (Wu et al., 2006). wt AcMNPV bacmid and vAcac18KO were digested with PstI and hybridized with the ac18 probe or Cm probe, respectively. A 7.2-kbp PstI fragment, which the ac18 gene located in AcMNPV bacmid genome, hybridized strongly to the ac18 probe; however, there was no signal in vAcac18KO (Fig. 2C). Since an 857-bp fragment of ac18 gene was replaced by a 1039-bp Cm gene and a new PstI site was added in vAcac18KO, the AcMNPV bacmid 7.2-kbp PstI fragment had been separated to 5.4 kbp and 1.9 kbp. As expected, a 5.4-kbp PstI fragment of vAcac18KO hybridized strongly to Cm probe, but no signal in wt AcMNPV (Fig. 2C). The primers used in PCR analysis and the resulting products were shown in Figs. 2B and D. Primers FS11/18P-D produced no PCR product in vAcac18KO , but a 1399-bp fragment in wt. Similarly, primers 18P-U/FS22 produced no PCR product in vAcac18KO , but a 1338-bp fragment in wt. Primers FS11/CmD produced no PCR product in wt, but a 1581-bp fragment in vAcac18KO . Similarly, primers CmU/FS22 produced no PCR product in wt, but a 1520-bp fragment in vAcac18KO. Primers CmU/CmD produced no PCR product in wt, but a 1039-bp fragment in vAcac18KO . Primers 18P-U/18P-D produced no PCR product in vAcac18KO , but a 857-bp fragment in wt. These results demonstrated that the Cm gene successfully replaced ac18 and no intact ac18 gene cassette existed in vAcac18KO genome.

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Construction of knockout, repair, and wt AcMNPV bacmids containing polyhedrin and gfp (green fluorescent protein)

Fig. 1. (A) RT-PCR analysis of the transcription of ac18 performed on total RNA extracted from Sf-9 cells infected with AcMNPV at different time points post-infection (p.i.). Time points post-infection are indicated above the lanes. PCR products for different genes are indicated at the left. Sizes of different PCR products are indicated in bp. (B) 5′ RACE analysis of the ac18 transcription start site. The translation start codon is denoted in box. The arrowhead shows the location of the transcription initiation site of ac18. SP2-a and SP2-b are two second, nested primers located upstream of SP1 and were used together with the Oligo dT-anchor primer for the PCR amplification, respectively.

To determine if ac18 deletion has any effect on occlusion body morphogenesis and to facilitate examination of virus infection, an ac18 knockout mutant vAcac18KO-ph-gfp containing polyhedrin and gfp genes was constructed by transposition of polyhedrin and gfp gene into the polyhedrin locus of vAcac18KO, using the BAC-to-BAC system (Invitrogen Life Technologies) (Fig. 3A). For rescue or confirmation of the phenotype resulting from the ac18 knockout, a repair bacmid vAcac18REP-ph-gfp was generated by inserting ac18 gene under the control of its native promoter as well as polyhedrin and gfp genes into the polyhedrin locus by transposition (Fig. 3A). Polyhedrin and gfp genes were also inserted into the polyhedrin locus of AcMNPV bacmid bMON14272 to generate a wt bacmid named vAcph-gfp , which was used as a wild-type control (Fig. 3A). Transposition events were confirmed later by GFP expression and occlusion body formation in bacmid DNA-transfected Sf-9 cells described as follows.

Fig. 2. Construction of ac18 knockout, repair, and wt AcMNPV bacmids. (A) The strategy for construction of an ac18 knockout bacmid containing a deletion of the ac18 gene by recombination in E. coli is shown in the diagram. An 857-bp fragment of the ac18 orf was deleted and replaced by chloramphenicol resistance gene (Cm). (B) Positions of primer pairs and probes used in the confirmation of disruption of ac18 and the correct insertion of Cm gene cassette. (C) Southern blot analysis of wt AcMNPV and vAcac18KO. ac18 probe and Cm probe were used to confirm the deletion of ac18 and the replacement by Cm. (D) PCR analysis of the presence or absence of sequence modifications in vAcac18KO or wt AcMNPV. The virus templates are shown above each lane, and the primer pairs used are shown below.

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Analysis of knockout, repair, and wt AcMNPV replication in infected Sf-9 cells To further assess whether ac18 is required for virus production, Sf-9 cells were transfected with vAcph-gfp, vAcac18KO-ph-gfp or vAcac18REP-ph-gfp bacmid, respectively. At 96 h post-transfection, supernatants were collected and the titers of BV were determined by TCID50 (50% Tissue Culture Infective Dose) end-point dilution assay on Sf-9 cells. We obtained high titer stocks of vAcph-gfp , vAcac18KO-ph-gfp and vAcac18REP-ph-gfp, respectively, and then these viruses were used to infect Sf-9 cells at an MOI (multiplicity of infection) of 5. Infected cells were monitored by fluorescence due to expression of GFP, which was under the control of the AcMNPV ie-1 promoter. No significant differences were observed among the three virus-infected cells at 12 h p.i. (Fig. 3B). Fluorescence was observed in almost all vAcph-gfp -, vAcac18KO-ph-gfp- or vAcac18REP-ph-gfp infected cells by 72 h p.i. (Fig. 3B), indicating that all these three viruses could generate infectious budded virions. Microscopic analysis showed that occlusion bodies formed in vAcph-gfp -, vAcac18KO-ph-gfp- or vAcac18REP-ph-gfp-infected cells (Fig. 3C). At 48 h p.i., almost similar proportion of the cells contained occlusion bodies. By 96 h p.i., no differences were observed in vAcph-gfp-, vAcac18KO-ph-gfp- or vAcac18REP-ph-gfp -infected cells. Almost every single cell infected with vAcph-gfp, vAcac18KO-ph-gfp or vAcac18REP-ph-gfp contained occlusion bodies (Fig. 3C). These above results indicated that the deletion of ac18 did not affect BV and polyhedra production. To further access the effect of ac18 deletion on virus replication and determine the replication kinetics of virus constructed, a virus growth curve analysis was performed. For this experiment, Sf-9 cells were infected with BVs at an MOI of 5, at selected time points the BV titers were determined by a TCID50 end-point dilution assay. Sf-9 cells infected with vAcph-gfp, vAcac18KO-ph-gfp or vAcac18REP-ph-gfp revealed steady increase in virus production and the slope of the growth curves of these three viruses had no significant difference (Fig. 3D). Electron microscopic analysis of ac18 wt, knockout and repair virus-infected cells To further analyze whether the deletion of ac18 has any effect on virus morphogenesis, electron microscopic analysis was performed with thin sections generated from virus-infected cells (Fig. 4). Observations of cells infected with wt virus (vAcph-gfp ), ac18 knockout virus (vAcac18KO-ph-gfp ) and ac18 repair virus (vAcac18REP-ph-gfp) showed typical cytological changes and viral morphogenesis of NPV infection and were morphologically

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indistinguishable. Virogenic stroma, which was an electrondense and baculovirus-induced structure, could be found easily within the nucleus of the ac18 knockout virus-infected cells (Fig. 4A), but no such structure in mock-infected cells (data not shown). Nucleocapsids were observed at the electron-dense edges in the virogenic stroma (Fig. 4B). Enveloped virions containing multiple nucleocapsids prior to occlusion in the protein crystalline matrix of the developing occlusion bodies were observed within the ring zone (Fig. 4C). Ring zone is a significant and morphologically distinct peristromal compartment of nucleoplasm and yields along with maturation of the virogenic stroma. Polyhedra were observed in the ring zone of vAcac18KO-ph-gfp-infected cells (Fig. 4D). The sizes and shapes of the polyhedra in ac18 knockout virus-infected cells were similar to those in wt virus-infected cells (data not shown). These observations indicated that the deletion of ac18 did not affect the occlusion body morphogenesis. Effect of ac18 deletion of AcMNPV on infectivity for Trichoplusia ni larvae The infectivities of vAcph-gfp and vAcac18KO-ph-gfp were determined for newly molted third-instar T. ni larvae in 50% lethal dose (LD50) and 50% lethal time (LT50) bioassays. No significant difference in LD50 was observed between wt virus and ac18 knockout mutant (Table 1), but the LT50 of ac18 knockout mutant was 24 h longer than wt virus (Table 2), suggesting that the deletion of ac18 had no effect on virus oral infectivity, but it did affect the efficiency of virus infection in T. ni larvae. Localization of Ac18 in AcMNPV-infected insect cells To detect the subcellular localization of Ac18, two recombinant baculoviruses, vAcp18GFP and vAcac18GFP were constructed (Fig. 5A). The ac18-gfp chimera was inserted into the polyhedrin locus of AcMNPV bacmid by site-specific transposition and the native ac18 locus was left intact. GFP was fused to the C terminus of Ac18 and was expressed under the control of the ac18 native promoter in vAcac18GFP. As a control, GFP alone was expressed under the control of the ac18 promoter in vAcp18GFP (Fig. 5A). Sf-9 cells infected with vAcp18GFPor vAcac18GFP at an MOI of 10 were examined for GFP-specific fluorescence by confocal laser scanning (Fig. 5B). The observed localization of Ac18GFP changed over time in vAcac18GFP-infected cells. Fluorescence localized in cytoplasm along the periphery of the nucleus at 3 h p.i., moved into the nuclear at 16 h p.i. and accumulated at the intranuclear ring zone of infected cells until 72 h p.i., but could not be detected in the network of granular material of the nucleus which was possible to be the virogenic stroma. As a

Fig. 3. Analysis of virus replication in Sf-9 cells. (A) Schematic diagram of three viruses, vAcac18KO-ph-gfp, vAcac18REP-ph-gfp and vAcph-gfp, showing the polyhedrin (polh) and green fluorescent protein (gfp) genes inserted into the polyhedrin locus by Tn7-mediated transposition. (B) Sf-9 cells infected with vAc ph-gfp, vAcac18KO-ph-gfp, or vAcac18REP-ph-gfp at 12 and 72 h p.i., respectively. (C) Light microscopy of vAc ph-gfp-, vAcac18KO-ph-gfp-, or vAcac18REP-ph-gfp-infected cells at 48 and 96 h p.i. (D) Virus growth curves generated from an infection of virus in Sf-9 cells. Cells were infected at an MOI of 5 from each virus, and cells culture supernatants were harvested at the selected time points and assayed for the production of infectious virus by TCID50 assay. Each datum point represents the average from three independent infections. Error bars represent the standard errors.

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Fig. 4. Electron microscopic analysis of vAcac18KO-ph-gfp-infected Sf-9 cells to characterize ac18-null mutant. (A) Virogenic stroma appeared within the nucleus at 24 h p.i. (B) Normal nucleocapsids dispersed at the electron-dense edges of the stroma at 24 h p.i. (C) ODVs were forming in the ring zone by 48 h p.i. (D) Normal virions were embedded in the polyhedra. Scale bar, 250 nm.

contrast, fluorescence was first observed in the cytoplasm along the periphery of the nucleus at 3 h p.i., then throughout the cytoplasm and the nucleus in vAcp18GFP-infected cells from 16 h p.i. to 72 h p.i. (Fig. 5B). Discussion ac18 is a highly conserved gene in lepidopteran NPVs but sequence-based queries performed with InterProScan program shows that Ac18 homologues constitute a protein family (IPR010785) of unknown function. In this report, the role of ac18 in the context of AcMNPV infection in Sf-9 cells and T. ni larvae was examined with an ac18 knockout virus, which could propagate as a bacmid in E. coli. The ac18 knockout virus was able to propagate after transfection of Sf-9 cells and could generate infectious progeny viruses. The slope of virus growth curve of the ac18 knockout virus vAcac18KO-phgfp was similar to that of the wt virus vAcph-gfp and the repair virus vAcac18REP-ph-gfp . Subsequently, electron microscopy indicated that the rod-shaped nucleocapsids associating with Table 1 Dose–mortality of vAcph-gfp and vAcac18KO-ph-gfp for third-instar T. ni larvae Virus

LD50 (PIBs per larva)

ph-gfp

vAc vAcac18KO-ph-gfp

3

2.3 × 10 1.7 × 103

Table 2 Time–mortality of vAcph-gfp and vAcac18KO-ph-gfp for third-instar T. ni larvae

95% Fiducial limit (PIBs per larva) Lower

electron-dense edges of the virogenic stroma, ODVs and polyhedra containing ODVs were observed in the ac18 knockout virus-infected cells and showed no difference with those in wt virus-infected cells. These results, therefore, indicated that the ac18 gene was not essential for virus propagation and the ac18 deletion did not affect the occlusion body (OB) morphogenesis in AcMNPV-infected Sf-9 cells. Our results were consistent with the results obtained from a study performed by Kumar and Miller (1987), in which they found that mutants with deletion PstI-G (7.6–13.1 m.u.) region, which includes ac18 (10.5–11.5 m.u.) gene, were generated repeatedly during serial passage of AcMNPV in a T. ni cell line, indicating that ac18 is dispensable at the cellular level. Auxiliary genes are not essential for virus replication, but nonetheless provide it with some selective advantage. Auxiliary genes of baculovirus can be divided into categories: (1) genes likely to facilitate virus replication at the cellular level, e.g., protein kinase 2 (pk2) and protein tyrosine phosphatase (ptp) gene; (2) genes likely to function at the organismal level, e.g., cathepsin (cath) and chitinase (chiA) (O'Reilly, 1997). In a previous study, PstI-G deletion mutants of various sizes (ac18

Virus

LT50 (h)

Upper 3

1.7 × 10 1.2 × 103

3

3.3 × 10 2.5 × 103

ph-gfp

vAc vAcac18KO-ph-gfp

72.0 96.0

95% Fiducial limit (h) Lower

Upper

63.5 90.5

80.5 101.5

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Fig. 5. Subcellular localization of Ac18-GFP fusion protein in Sf-9 cells infected with GFP-tagged viruses. (A) Schematic map of construction of GFP-tagged recombinant bacmids. None-fused GFP under the control of ac18 promoter was transposed into the polyhedrin locus of AcMNPV bacmid to generate vAcp18GFP. Ac18 fused with GFP tag under the control of its native promoter was transposed into the polyhedrin locus of AcMNPV bacmid, the resulting recombinant bacmid was named vAcac18GFP. (B) Confocal images of Sf-9 cells. Cells were infected with vAcac18GFP at an MOI of 10. At 3, 16 and 72 h p.i., cells were examined for fluorescence by confocal laser scanning microscopy. Cells infected with vAcp18GFP were used as a control. For each time point, GFP-specific fluorescence micrographs are shown to the left of the merged micrographs. Scale bar, 10 μm.

was deleted in some mutants) became predominant in the stock during serial passage of AcMNPV in a T. ni cell line or successive passage of AcMNPV in larvae of Manduca sexta by feeding the occluded form of the virus (Kumar and Miller, 1987). These results implied that ac18 was an auxiliary gene with a possible function at the organismal level. Recently, a novel baculovirus auxiliary gene, fibroblast growth factor (vfgf), has been characterized in detail. No obvious differences in AcMNPV mutants which have or do not have vfgf with respect to BV production, viral proteins synthesis and shut-off of viral or host proteins, or genome replication were found in Sf21 cells (Detvisitsakun et al., 2006). Coinfection of insect cells with AcMNPV mutants which have or do not have vfgf indicated that vfgf did not confer a consistent advantage for virus replication after several passages (Detvisitsakun et al., 2006). Deletion of vfgf had no effect on the infectivity of AcMNPV. However, lack of vfgf delayed the time of death in two host species, Spodoptera frugiperda and T. ni, when the virus was delivered by feeding but not by intrahemocoelic

injection (Detvisitsakun et al., 2007). By contrast, B. mori nucleopolyhedrovirus (BmNPV) BV production was reduced in BmN cells and B. mori larvae infected with a Bm-vfgf deletion mutant. The larval bioassays also revealed that deletion of vfgf did not reduce the infectivity, but the mutant virus did take 20 h longer to kill B. mori larvae than wild-type BmNPV, when tested either by BV injection or by polyhedrin-inclusion body ingestion (Katsuma et al., 2006). These results suggest that vfgf is involved in efficient infection to the permissive hosts and also imply some auxiliary genes could function at the organismal level. In present study, bioassays performed with T. ni larvae did not reveal significant difference between the LD50 of ac18 knockout virus and that of wt virus, but the LT50 of ac18 knockout virus was 24 h longer than wt virus in T ni. larvae, indicating that Ac18 might have an important role in virus replication in T. ni larvae. Using a recombinant virus that expresses Ac18 fused to an enhanced green fluorescent protein as a visual marker to follow protein transport and localization within the nucleus during

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infection, we found that in the late phase of virus infection, Ac18-GFP mainly localized into the intranuclear ring zone. Previous studies showed that occlusion-derived virions (ODVs) formation occurs in this region (Williams and Faulkner, 1997). Some important structural proteins, such as P74 which was essential for oral infectivity of ODV and had been proposed to play a role in midgut attachment and/or fusion, showed similar localization (Slack et al., 2001). Wu et al. (2006) found that 38K of AcMNPV localized to the virogenic stroma of nucleus of Sf-9 cells and 38K might play a role in nucleocapsid assembly and was essential for viral replication in the AcMNPV life cycle. The virogenic stroma was thought to be the active site for viral DNA replication, condensation, and packaging into capsids (Fraser, 1986; Young et al., 1993) and ODV assembly and polyhedra forming occur within the ring zone (Williams and Faulkner, 1997). The location of Ac18 in the ring zone but not in the virogenic stroma suggested that this protein could be associated with ODV formation and/or regulation of late viral genes or the virus–host interaction, but might not involved in the viral DNA replication and nucleocapsids assembly. In summary, we examined the transcription of ac18 gene in Sf-9 cells and found that it was an early gene. AcMNPV mutant lacking a functional ac18 gene replicated similarly to wt virus in Sf-9 cells and T. ni larvae, but the LT50 of the mutant lacking ac18 gene was 24 h longer than wt virus, demonstrating that ac18 is not an essential gene for viral invasion and replication but affects the efficiency of virus infection in some degree in T. ni larvae. Electron microscopic analysis of ac18 knockout virus-infected cells did not find any obvious difference with wt virus-infected cells. Detection of the localization of Ac18 in AcMNPV-infected Sf-9 cells detected that it is localized to the intranuclear ring zone. All these results suggested that ac18 is not necessary for AcMNPV propagation but affects the efficiency of virus infection in vivo.

time point. First-strand DNA complementary to RNA (cDNA) synthesis was carried out using Avian Myeloblastosis virus (AMV) reverse transcriptase and the oligo-dT primers according to the manufacturer's instruction. Then the cDNA mixtures were amplified by PCR using the ac18-specific primers 18RTP1: 5′-GAATTCGAACGTTTGTTGAATCAACTA-3′ (EcoRI site was underlined) and 18RT-P2: 5′-CTCGAGTTAGTATAACGAAAAAATACAT-3′ (XhoI site was underlined). AcMNPV ie1 gene and vp39 gene were used as the control for the early gene and late gene, respectively. The primer pair: ie-1U: 5′-ATGACGCAAATTAATTTTAACGC-3′ and ie-1D: 5-ACAATTTAGTTTTTGTTCCG-3′ was designed for ie-1; the primer pair: vp39U: 5′-GCGCTAGTGCCCGTGGGTAT-3′ and vp39D: 5′-TTAGACGGCTATTCCTCCACCTGC-3′ were designed for vp39. The obtained PCR products were analyzed in 1.0% agarose gel. Control experiments in which no reverse transcriptase was added prior to the PCR step were performed to detect any possible viral DNA contamination. The 5′ end of ac18 transcript was determined using the 5′/3′ RACE Kit, 2nd Generation (Roche) employing 2 μg total RNAs isolated from 24 and 48 h p.i. as template, respectively. According to the manufacturer's recommendations, the first strand cDNA was synthesized with an ac18-specific primer SP1: 5′-GTCAATATGGCATGTCACGGCTG-3′ (nt 15,262–15,284). The cDNA was purified with High PCR Purification Kit (Roche) and was added with a homopolymeric A-tail to the 3′ end using Terminal Transferase and dATP. The tailed cDNAs were amplified by PCR using the oligo-dT anchor primer and the nested ac18-specific primer SP-2a: 5′-ATGCCAACTTTGCTTTAG-3′ (nt 15,351–15,368) or SP-2b: 5′-CAAACTACGGCTTCGAAACAATCT-3′ (nt 15,320–15,343). All PCR products were gel purified and cloned into pMD18-T (TaKaRa) and sequenced with M13F (− 47) or M13R (− 48) primers. Deletion of ac18

Materials and methods Virus, cell line and insects Sf-9 insect cell line, the clonal isolate 9 from IPLB-Sf21-AE cells which derived from the fall armyworm S. frugiperda, was cultured at 27 °C in Grace's medium (Invitrogen Life Technologies) containing 10% fetal bovine serum, penicillin (100 μg/ml) and streptomycin (30 μg/ml). Larvae of T. ni were reared on an artificial diet at 28 °C. AcMNPV BV stocks were prepared by infecting third instar T. ni larvae with polyhedra and extracting hemolymph from infected insects 3 days p.i. as previously described (O'Reilly et al., 1992). Total RNA isolation, RT-PCR and 5′ RACE analysis Total RNAs were isolated from 1 × 106 mock-infected and AcMNPV-infected Sf-9 cells at an MOI of 5 with the RNeasy Mini Kit (Qiagen) at 0, 1, 2, 6, 12, 24, 48, 72 and 96 h p.i. After the RNA samples were treated with RNase-Free DNase Set (Qiagen), RT-PCR was performed using the RNA PCR Kit Ver. 3.0 (TaKaRa) employing 2 μg total RNA as the template per

An AcMNPV bacmid with deletion of ac18 gene was made by ET recombination as previously described (Wu et al., 2006). We first generated a transfer vector in which the ac18 locus region was replaced with Cm for antibiotic selection in E. coli. A 481-bp 5′ flank of ac18 was PCR amplified from the AcMNPV bacmid using the primers 18FS21: 5′-CTGCAGTCTGTCGTCGTCAATATGGC-3′ (PstI site was underlined) and 18FS22: 5′-AAGCTTAATCGTAACCCCGTCGCTGT-3′ (HindIII site was underlined). The PCR product was digested with PstI and HindIII and then ligated into vector pUC18-Cm (Wu et al., 2006) to generate the recombinant plasmid named pUC18-US-Cm. With primers 18FS11: 5′-CAGCTCTGTCAGATGCCGTTCTCCTT-3′ (SacI site was underlined) and 18FS12: 5′-GGATCCAAACAACACAATTTTCTAACAA-3′ (BamHI site was underlined), a 541-bp 3′ flank of ac18 was PCR amplified from the AcMNPV bacmid. The PCR product was digested with SacI and BamHI and cloned into plasmid pUC18-US-Cm that was digested with SacI/BamHI to generate a final ac18 knockout transfer vector named pUC18-US-Cm-DS. This transfer vector was digested with SacI and HindIII, and the resulting linear 2.1-kbp fragment containing

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Cm gene cassette and ac18 flank regions was gel purified, suspended in distilled water to a final concentration of 200 ng/μl. To facilitate homologous recombination between the Cm gene and the bacmid target sequence, DH10Bac cells (DH10B contains AcMNPV bacmid bMON14272) were transformed with pBAD-gbaA (Muyrers et al., 1999). pBAD-gbaA contains the λ Red recombinant genes gamma, beta, and alpha that encode a RecBC inhibitor, an ssDNA annealing protein, and a 5′→3′ dsDNA exonuclease, respectively (Muyrers et al., 1999). The resulting clone cells were induced by the addition of L-arabinose to allow expression of the λ Red system, made competent and electro-transformed with 1 μg of the purified linear 2.1-kbp fragment as previously described (Pijlman et al., 2002). The eletroporated cells containing altered genotype of the recombinant bacmid were incubated at 37 °C for 1 h in 1 ml SOC (Sambrook and Russell, 2001) medium and subsequently spread onto agar medium containing 20 μg of chloramphenicol per ml, 50 μg of kanamycin per ml and 7 μg of tetracycline per ml. Plates were incubated at 37 °C for 2 days and the chloramphenicol- and kanamycin-resistant colonies were selected and verified by PCR analysis and Southern blot analysis. The resulting ac18 knockout bacmid was named vAcac18KO. Southern blot analysis and PCR analysis Southern blot hybridization analysis was used to confirm the absence of ac18 gene in AcMNPV bacmid and its replacement by the Cm gene. The deletion fragment of ac18 gene was PCR amplified from AcMNPV bacmid w i t h p r i m e r s 1 8 P - U : 5 ′ - A A G C T TATA C AT G A C A TAAACCGCTTC-3′ (HindIII site was underlined) and 18P-D: 5′-GAGCTCAGATTTTGACTATTACGGC-3′ (SacI site was underlined). The PCR product was purified and digoxigenin (DIG High Prime Labeling and Detection Starter Kit I; Roche Biochemicals) labeled overnight to be used as a probe for Southern blot hybridization to detect the deletion of ac18 gene. Meanwhile, the fragment containing the Cm gene was PCR amplified with primers CmU and CmD, and this PCR product was also labeled with digoxigenin dUTP and was used as a probe to detect the replacement of Cm gene (Wu et al., 2006). AcMNPV bacmid DNA and vAc ac18KO were isolated from E. coli cells according to the Bac-to-Bac protocol (Invitrogen Life Technologies). PstI digested bacmid DNA was run overnight in ethidium bromide-stained 0.8% agarose gels and DNA was transferred onto NYTRAN N nylon transfer membrane (Scheicher and Schuell). Hybridization and colorimetric detection with nitroblue tetrazolium-5-bromo4-chloro-3-indolylphosphate were performed according to the manufacturer's recommendations. The disruption of ac18 gene and the insertion of Cm gene were further confirmed by PCR analysis. Primers CmU and CmD were used to detect the correct insertion of the Cm gene cassette. Primers 18P-U and 18P-D, which are just the knockout part of ac18, were used to confirm the deletion region. Primer pairs FS11/18P-D, 18P-U/FS22, FS11/CmD

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and CmU/FS22 were used to examine the recombination junctions of the upstream and downstream flanking region. Construction of knockout, repair, and wt AcMNPV bacmids containing polyhedrin and gfp To construct repair bacmid, a 1623-bp fragment containing ac18 gene with its native promoter and poly (A) tail was PCR amplified using primers Ac18RP-U: 5′-GAATTCTGAATCGTAACCCCGTCGCT-3′ (EcoRI site was underlined) and Ac18RP-D: 5′-TCTAGACAGACAATGTTAAAAGTAGG-3′ (XbaI site was underlined). This PCR product was digested with EcoRI/XbaI and ligated with pFB1-ph-gfp (Wu et al., 2006), which was digested with the same enzymes previously to give pFB1-ac18-ph-gfp. Electrocompetent DH10B cells containing helper plasmid pMON7124 and the bacmid bMON14272 were transformed with pFB1-ph-gfp to generate a control virus named vAcph-gfp. Electrocompetent DH10B cells containing pMON7124 helper plasmid and vAc ac18KO were transformed with donor plasmids pFB1-ph-gfp and pFB1-ac18-ph-gfp, to generate ac18-null bacmid vAcac18KO-ph-gfp and ac18 repair bacmid vAc ac18REP-ph-gfp , respectively. The correct recombinant bacmids were electroporated into E. coli DH10B cells and screened for tetracycline sensitivity to ensure that the isolated bacmids were free of helper plasmids. Bacmid DNAs were extracted and purified with QIAGEN Large-Construct Kit and quantified by optical density. Analysis of virus growth curve To assess whether ac18 is required for virus production and determine the replication kinetics of virus constructed, a virus growth curve analysis was performed as described previously (Wu et al., 2006). For this experiment, 2 × 106 Sf-9 cells were transfected with 2.0 μg of the bacmid DNA using appropriate Cellfectin liposome reagent (Invitrogen Life Technologies), virus supernatant was collected at 96 h posttransfection. The titers of BV were determined by TCID50 end-point dilution assay on Sf-9 cells (O'Reilly et al., 1992), high titer stocks of virions were obtained. Then Sf-9 cells were infected in triplicate with BV (vAcph-gfp , vAcac18KO-ph-gfp , or vAcac18REP-ph-gfp) at an MOI of 5 and at selected time points the titers were determined by a TCID50 end-point dilution assay on Sf-9 cells. Electron microscopy For electron microscopy, 1 × 106 Sf-9 cells were infected with BV (vAcph-gfp , vAcac18KO-ph-gfp , or vAcac18REP-ph-gfp) at an MOI of 5. Mock-infected cells were treated similarly but without the addition of BV. At 24, 48 and 72 h p.i., cells were dislodged and centrifuged at 3000×g for 10 min. Cells were fixed, dehydrated, embedded, sectioned, and stained as described previously (Li et al., 2005). Samples were viewed with a JEM-100CX/II transmission electron microscope at an accelerating voltage of 80 kV.

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Bioassays In order to determine if ac18 deletion has any effect on the infectivity of AcMNPV, bioassays were performed as previously described (Eldridge et al., 1992). Fourth-instar T. ni larvae were hemocoelic injected with 10 μl inoculum which BVs diluted in Grace's medium at the concentration of 2 × 106 TCID50/ml. PIBs were harvested from dead larvae and purified by centrifugation and resuspended in double-distilled water as described previously (O'Reilly et al., 1992). To determine the LD50, 10 μl of samples, containing 0 (control), 2 × 102, 1 × 103, 2 × 103, 1 × 104 and 2 × 104 PIBs, respectively, was applied onto a small piece of artificial diet in a 1-OZ containers. Newly molted third-instar T. ni larvae were allowed to feed individually until all the diet was consumed and then added fresh diet. Only the larvae that completely ingested the diet with virus within 12 h were further reared. Mortality was recorded daily after infection until larvae died or pupated. 45 larvae per dosage were used in the infection experiments and the experiments were repeated for three times. The LT50 was determined by the droplet feeding method as previously described (Hughes and Wood, 1981). Briefly, newly molted third-instar T. ni were starved for 6 h and then inoculated with a solution containing 1% sucrose (w/v), 0.01% blue food coloring (FD and C Blue No.1), as well as PIBs (1 × 108 PIBs/ml). Inoculated larvae were maintained individually in 12-well plates provided with artificial diet; each well was added 1.5 ml 1.5% agar previously to keep the humidity. Mortality was recorded every 12 h after infection until larvae died or pupated. 36 larvae per virus were used in the infection experiments and the experiments were repeated three times. The LD50 value was determined using the probit analysis and LT50 value was determined using the Kaplan–Meier estimator in the bioassays.

with a GFP tag under the control of the ac18 promoter in the resulting bacmid which was referred to as vAcac18GFP. Primers Ac18CF-D2: 5′-TCTAGAAATGAATAGCGGCGACG-3′ (XbaI site was underlined) and Ac18RP-U were used to amplify ac18 promoter from AcMNPV bacmid. The EcoRI/XbaI digested PCR product was inserted into pFB1-ph- to generated pFB1ph--p18. The orf of gfp was digested with XbaI and PstI from pUC19-gfp and the resulting fragment was cloned into XbaI/ PstI site of pFB1-ph--p18 to generate a donor plasmid pFB1p18-gfp. The control bacmid vAcp18GFP, which only GFP was expressed under the control of ac18 promoter, was generated in a procedure similar to that for vAcac18GFP. Sf-9 cells (2 × 106) were transfected with 2 μg vAcp18GFP or vAcac18GFP bacmid DNA, respectively. At 96 h p.i., supernatants were collected and the titers of BV were determined by TCID50 end-point dilution assay on Sf-9 cells. For confocal microscopy, Sf-9 cells (1 × 105 ) were seeded onto glass coverslips and left to stand for several hours to allow cell attachment. Cells were infected with vAcp18GFP or vAcac18GFP virus at an MOI of 10, respectively. At 3, 16, 24, 48 and 72 h p.i., cells were visualized with a Leica TCS SP2 confocal laser scanning microscope for fluorescence using a wavelength of 488 nm laser line for GFP. All images were digitally recorded and merged by the use of Leica software. Acknowledgments We thank Dr. Wenjun Zhang and Dr. Guanghong Li for helping us with the statistical analysis. We are also grateful to Ms Miner He for the insect cell culture. This research was supported by the National Major Basic Research Project (‘973’) of China (Nos: 2003CB114202, 2006CB102005) and the National Nature Science Foundation of China (Nos: 30530540, 30470069). References

Generation of GFP fusion recombinant bacmids and confocal microscopy To monitor the localization of Ac18 in AcMNPV-infected insect cells, Ac18 was expressed in frame with GFP to create an Ac18-GFP chimera. ac18 orf (without stop codon TAA) with its native promoter was amplified from AcMNPV bacmid using primers Ac18CF-D: 5′-TCTAGATTAGTATAACGAAAAAATACAT-3′ (XbaI site was underlined) and Ac18RP-U. The EcoRI/ XbaI digested PCR product was inserted into pFB1-ph- to generated pFB1-ph - -ac18. The polyhedrin promoter was removed from pFastBac1 to create pFB1-ph- as previously described (Dai et al., 2004). The orf of gfp was digested with XbaI and PstI from pUC19-gfp and the resulting fragment was cloned into XbaI/PstI site of pFB1-ph--ac18 to generate a donor plasmid pFB1-ac18-gfp. According to the Bac-to-Bac instruction manual (Invitrogen Life Technologies), DH10Bac cells which contain AcMNPV bacmid bMON14272 and helper plasmid pMON7124 were transformed with pFB1-ac18-gfp and the ac18-gfp chimera was site-specific transposed into the AcMNPV bacmid polyhedrin locus. Thus Ac18 was expressed

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