Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

ABSTRACT

ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5′ rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment.

IMPORTANCE Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

INTRODUCTION

The Baculoviridae are a family of insect-specific double-stranded DNA (dsDNA) viruses. Viruses from this family are characterized by rod-shaped, enveloped nucleocapsids with circular, covalently closed, double-stranded DNA genomes of 80 to 180 kbp (1–3). The Baculoviridae family comprises four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus, and Deltabaculovirus. Alphabaculoviruses and betabaculoviruses infect lepidopteran larvae, whereas gammabaculoviruses and deltabaculoviruses infect hymenopteran and dipteran larvae, respectively (4). The alphabaculoviruses are further divided into group I and group II on the basis of phylogenetic analysis (5) and the type of envelope fusion protein (GP64 and F, respectively) (6).

The infection cycle of baculoviruses includes two distinct viral phenotypes: budded virus (BV) and occlusion-derived virus (ODV). Both BVs and ODVs are identical in terms of nucleocapsid structure and genetic information, but the composition of their envelopes is different to accommodate their respective functions in the infection cycle (7). ODVs, which become embedded in the nucleus into a protein matrix that forms polyhedra or occlusion bodies (OBs), are required for the horizontal transmission of the virus. The alkaline environment in the midgut lumen of the infected larvae releases ODVs from the OBs, enabling these viral particles to initiate primary infection of the mature columnar epithelial cells of the midgut (8). BVs, which are produced as nucleocapsids, egress from the nucleus, migrate through the cytoplasm, bud through a modified plasma membrane of infected cells (9), and initiate a secondary infection (10). However, there is limited knowledge regarding how the nucleocapsids are transported to the cell membrane and what determines the switch from BV to ODV.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species of the Alphabaculovirus genus and has a double-stranded DNA genome of ∼134 kbp that contains 154 predicted open reading frames (ORFs) based on the criterion that the ORF is a single, contiguous, nonoverlapping coding region (1), and the transcriptome of these AcMNPV genes over the course of infection in Trichoplusia ni cells has been analyzed (11). AcMNPV ORF11 (ac11) encodes a hypothetical protein of 340 amino acids with a putative molecular mass of 40.1 kDa (1). ac11 possesses a typical early promoter motif, CAGT. Although homologs of AC11 are found in all lepidopteran group I Alphabaculovirus genomes and in several group II genomes (12), its function in the life cycle remains unknown.

In this study, an ac11 knockout virus, Ac11KO, was constructed to investigate the functional role of ac11 in the AcMNPV life cycle. We identified the transcriptional phase of ac11 and the effects of an ac11 deletion on BV production and ODV assembly. In addition, the morphology of BVs and ODVs in Ac11KO-transfected cells was also examined by electron microscopy. The results indicated that ac11 is essential for BV production but that the deletion of ac11 does not affect viral DNA replication. The electron microscopy observations revealed that ac11 is required for ODV formation and the subsequent embedding of virions into OBs.

MATERIALS AND METHODS

Bacterial strains and bacmid DNA.

Escherichia coli strains TOP10 and DH10B (Invitrogen, USA) were used throughout the experiments. All of the restriction endonucleases and modifying enzymes used in this study were obtained from Roche Applied Science (Germany). All of the recombinant bacmids used in this study were propagated in E. coli strain DH10B.

Viruses, insect cells, and transfection.

Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9 (Sf9) insect cells were cultured at 27°C in TC-100 medium (WelGene, South Korea) supplemented with 10% fetal bovine serum (WelGene, South Korea) and subcultured every 3 to 4 days. The wild-type AcMNPV C6 strain and all recombinant AcMNPVs used in this study were propagated in Sf9 cells maintained in TC-100 medium. Transfections were performed by using Cellfectin reagent (Invitrogen, USA) according to the manufacturer's instructions.

Construction of ac11 knockout virus Ac11KO.

To construct the ac11 knockout virus (Ac11KO), the AcMNPV recombinant bacmid Ac-MK (13), in which the E. coli origin of replication (mini-F replicon) is coupled with a kanamycin resistance gene (Kan), was inserted into the locus between orf603 and the polyhedrin gene of the AcMNPV genome (Fig. 1A). Using Tn7-mediated transposition between Ac-MK and donor-S (13, 14) of the plasmid capture system, we generated the recombinant virus Ac11KO, in which the ac11 gene was disrupted with a pUC origin (pUC ori) and an ampicillin resistance gene (Amp) (Fig. 1A). The transposition procedure was conducted as described previously (14), with slight modifications. Briefly, for the transposition reaction, 12 ng of HindIII- and SphI-digested pPCS-S (donor-S) plasmid was combined with 200 ng of Ac-MK bacmid DNA. The TnsABC* transposase (New England BioLabs, United Kingdom) was added to the transposition reaction mixture, and the mixture was incubated at 37°C for 10 min. Next, 1 μl of the start solution was added to the mixture, and the mixture was then incubated at 30°C for at least 2 h. Finally, the transposition reaction was stopped by heating the mixture to 75°C for 10 min. The reacted DNA was transformed into competent E. coli DH10B cells (Invitrogen, USA), and the transformed cells were subsequently plated onto nutrient agar plates containing kanamycin (50 μg/ml) and ampicillin (50 μg/ml). The plates were incubated at 37°C for 2 days. Colonies resistant to both kanamycin and ampicillin were selected, and successful transposition was verified by PCR using primers specific to the ac11 gene and nucleotide sequence analysis.

FIG 1.

Construction of Ac11KO and Ac11Re. (A) Schematic diagram of the Ac-MK, Ac11KO, and Ac11Re viruses. The ac11 gene was knocked out by the insertion of pUC ori and Amp into amino acid 224 of ac11 via Tn7-mediated transposition. The deletion of ac11 was repaired by the replacement of Kan with ac11 and Cm via homologous recombination between pUC-19-MCP-ac11 and Ac11KO. The ac11 gene inserted into Ac11Re was driven by its own promoter. (B) RT-PCR analysis of ac11 and gp64 transcription. Total RNA from transfected Sf9 cells was extracted at 72 hpt. Lanes: M, 100-bp ladder; 1, mock-transfected Sf9 cells; 2, Ac-MK-transfected Sf9 cells; 3, Ac11KO-transfected Sf9 cells. (C) PCR verification of Ac11KO and Ac11Re. Lanes: M, 100-bp ladder; 1, Ac-MK; 2, Ac11KO; 3, Ac11Re. Primers Ac11-RealTime-Fw and Ac11-RealTime-Re are shown (arrows).

Construction of the repair virus Ac11Re.

To generate the ac11 knockout-repaired bacmid, denoted Ac11Re, the repair transfer vector pUC19-MCP-ac11 was constructed from pUC19-MCP (13) and the ac11 gene by using In-Fusion cloning (TaKaRa, Japan), as follows. First, the ac11 gene was PCR amplified with primers Ac11-In-Fusion-Fw (5′-CTTTTGCTGACTCGAAGCGAAGACGAAATGTTGGAC-3′) and Ac11-In-Fusion-Re (5′-AGCCCCTGTTCTCGAGCTTGTTATTTGCACGTCTGTC-3′), using Ac-MK DNA as the template. Ac11-In-Fusion-Fw and Ac11-In-Fusion-Re contain 15 bp homologous to the upstream and downstream regions of the pUC-19 XhoI digestion site (underlined), respectively. PCR amplifications were performed by using Phusion high-fidelity DNA polymerase (Finnzymes, Finland), and the In-Fusion cloning procedure was carried out by using an In-Fusion HD cloning kit (TaKaRa, Japan) according to the manufacturer's instructions. After 100 ng of the purified PCR product was mixed with 50 ng of XhoI-digested pUC19-MCP DNA (TaKaRa, Japan), 2 μl of 5× In-Fusion HD enzyme premix was added to the reaction mixture, and the mixture was preincubated at 50°C for 15 min. The resulting DNA was transformed into Stellar competent E. coli cells (Invitrogen, USA) according to the manufacturer's instructions, and the transformed cells were subsequently spread onto nutrient agar containing chloramphenicol (50 μg/ml) and ampicillin (50 μg/ml). The plates were incubated overnight at 37°C, and colonies resistant to chloramphenicol and ampicillin were selected and verified by restriction enzyme digestion and sequence analysis. The knocked-out ac11 repair bacmid Ac11Re was then generated via homologous recombination of the resulting pUC19-MCP-ac11 and Ac11KO genomic DNAs in cotransfected Sf9 cells and selected in E. coli cells plated onto a nutrient agar plate containing chloramphenicol (50 μg/ml) and ampicillin (50 μg/ml).

RNA and reverse transcription-PCR.

Sf9 cells (1 × 10⁶ cells/35-mm-diameter six-well plate) were transfected with viruses, and the total RNA from transfected Sf9 cells was isolated at 72 h posttransfection (hpt) by using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. After the RNA samples were treated with RNase-free DNase I (TaKaRa, Japan), reverse transcription-PCR (RT-PCR) was performed by using AccuPower RT/PCR premix (Bioneer, South Korea) in a 20-μl volume, according to the manufacturer's instructions. To amplify the ac11 gene, oligonucleotides Ac11-RealTime-Fw (5′-TACATTTCGGCGATTACACG-3′) and Ac11-RealTime-Re (5′-GAATTGGTGCCTCGTTTGT-3′) were used. The gp64 gene was amplified by using oligonucleotides AcGP64-F (5′-ATATGTGCTTTTGGCGG-3′) and AcGP64-R (5′-TTTGGCGCGTGGTGAAC-3′).

Phylogenetic analysis.

To investigate the relationship of ac11 and its homologous genes in other entomopathogenic dsDNA viruses, the ac11 homologs were aligned by using MUSCLE (15), and a phylogenetic tree was constructed by using MEGA6 (Molecular Evolutionary Genetics Analysis) software (16) with the neighbor-joining method and confirmed by a bootstrap analysis of 500 replicates.

Northern blot analysis.

Sf9 cells were infected with Ac-MK at a multiplicity of infection (MOI) of 10. Following gentle rocking for 1 h, the virus-containing culture medium was removed, and fresh medium was added after washing two times with incomplete TC-100 medium. For the Northern blot analysis, 10 μg of the total RNA isolated from infected Sf9 cells at various times postinfection (p.i.) was separated on a 1.5% agarose gel by using a formaldehyde-free RNA gel kit (Amresco, USA) according to the manufacturer's instructions. The separated RNAs were transferred onto a Hybond-N⁺ nylon hybridization transfer membrane (Amersham Pharmacia Biotech, USA). To generate a strand-specific probe, an ac11 gene fragment of ∼0.95 kb was amplified by using primers Ac11-Fw (5′-ATGTCTCTCGCTGCAAAGTT-3′) and Ac11-Re (5′-CCATAACACACAACAGGTCC-3′). A positive strand of the ac11 gene fragment was specifically amplified by using primer Ac11-Re and used as the template for labeling with digoxigenin (DIG) by using a DIG DNA labeling kit (Roche, Germany). The prehybridization, hybridization, washing, and detection procedures were performed by using the DIG luminescent detection kit for nucleic acids (Roche, Germany) according to the manufacturer's instructions.

5′ and 3′ rapid amplification of cDNA ends.

Total RNA from Ac-MK-infected Sf9 cells was isolated at 3, 6, and 12 hpt, and rapid amplification of cDNA ends (RACE)-ready first-strand cDNA was synthesized by using the SMARTer RACE cDNA amplification kit (Clontech, USA) according to the manufacturer's instructions. RACE PCR was carried out by using Advantage 2 polymerase mix (Clontech, USA) in a volume of 50 μl. PCR was performed under the following conditions: five cycles of 94°C for 30 s and 72°C for 3 min; five cycles of 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min; and 27 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min. Ac11GSP-R1 (5′-GGCCATACATTTCGGCGATTACACGC-3′) and Ac11GSP-F1 (5′-GTCAGCGTGCGCGTTCAAAACGG-3′) were used for 5′ and 3′ RACE PCR, respectively. The 5′ and 3′ RACE PCR products were subcloned into the pGEM-T Easy vector (Promega, USA) and sequenced in both directions. At least 10 colonies per time point were analyzed.

Titration of BV.

BV production was determined by quantitative PCR (qPCR) as previously described, with slight modifications (17, 18). Sf9 cells (1 × 10⁶ cells/35-mm-diameter six-well plate) were transfected with 1 μg of each bacmid DNA (Ac-MK, Ac11KO, and Ac11Re). At various times posttransfection, the supernatant containing BVs was harvested, and the cell debris was removed by centrifugation at 8,000 × g for 5 min. One milliliter of the above-described supernatant containing BVs was centrifuged at 80,000 × g for 2 h at 4°C, and the pellet was resuspended with 200 μl of lysis buffer (10 mM Tris-Cl [pH 7.5], 10 mM EDTA, 0.25% SDS, 20 μg/ml RNase A, and 80 μg/ml proteinase K). After incubation overnight at 65°C, the viral DNA was extracted by phenol extraction and alcohol precipitation. To perform qPCR, 2 μl of diluted DNA was used with a 2× DyNAmo HS SYBR green qPCR kit (Finnzymes, Finland) and primers IE1-RTF (5′-ACCATCGCCCAGTTCTGCTTATC-3′) and IE1-RTR (5′-GCTTCCGTTTAGTTCCAGTTGCC-3′), which amplify a 100-bp fragment of the ie-1 gene. A stock of wild-type Ac-MK (4.25 × 10⁸ PFU/ml) that was previously used to determine titers by endpoint dilution was serially diluted and used to develop a standard curve. The samples were analyzed by using a CFX96 real-time system (Bio-Rad, USA) under the following conditions: a preheating step for enzyme activation of 95°C for 15 min followed by 45 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 20 s.

The extracellular BV titers in the culture supernatants harvested at 24 and 72 hpt were also determined by using the endpoint dilution method in triplicate, as previously reported (19).

Quantification of viral DNA replication.

To assess viral DNA replication, a qPCR assay was performed as previously described (18), with slight modifications. Sf9 cells (1 × 10⁶ cells/35-mm-diameter well of a six-well plate) were transfected with 1 μg of bacmid DNA (Ac-MK, Ac11KO, repaired bacmid Ac11Re, and control GP64KO [13]). At the designated times posttransfection, the transfected cells were washed once with 1× phosphate-buffered saline (Sigma, USA) and centrifuged at 8,000 × g for 5 min. The harvested cell pellets were incubated for 30 min at 37°C in 250 μl of lysis buffer (10 mM Tris-Cl [pH 7.5], 10 mM EDTA, 0.25% SDS, 20 μg/ml RNase A) and then incubated overnight at 65°C after the addition of 80 μg/ml proteinase K. The viral DNA was extracted with 250 μl of phenol-chloroform and 250 μl of chloroform, and the aqueous layer containing the viral DNA was carefully harvested. Prior to PCR, 2 μl of total DNA from each time point was digested with 40 U of DpnI restriction enzyme (New England BioLabs, USA) overnight in a 20-μl total reaction mixture volume. For qPCR, 2 μl of the digested DNA was used with a 2× DyNAmo HS SYBR green qPCR kit (Finnzymes, Finland) and primers gp41-Fw (5′-CGTAGTGGTAGTAATCGCCGC-3′) and gp41-Re (5′-AGTCGAGTCGCGTCGCTTT-3′). The samples were analyzed by using a CFX96 real-time system (Bio-Rad, USA) under the following conditions: a preheating step for enzyme activation of 95°C for 15 min followed by 45 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 20 s.

BV partial purification and concentration.

Sf9 cells (1 × 10⁶ cells/35-mm-diameter well of a six-well plate) were transfected with 1 μg of bacmid DNA (Ac-MK, Ac11KO, and repaired bacmid Ac11Re). At 120 hpt, the supernatant containing BVs was harvested, and the BVs were purified as previously described (13). Briefly, the medium was harvested, and the cell debris was removed by centrifugation at 2,000 × g for 20 min. The supernatant (3 ml) was loaded onto a 25% sucrose cushion and centrifuged at 80,000 × g for 90 min at 4°C. The BV pellets were resuspended in 25 μl of 50 mM Tris-Cl (pH 7.5). An equal volume of 2× protein sample buffer (Sigma, USA) was added, and the samples were placed at 100°C for 10 min. Ten microliters of the Ac-MK and Ac11Re samples and the totality of the Ac11KO sample were analyzed by 12% SDS-PAGE and Western blotting.

Antibody preparation.

The preparation of the polyclonal antibody against AC11 was described previously (13). Briefly, the PCR-amplified ac11 gene was introduced into the E. coli expression vector pET30a(+) (Novagen, Germany) to obtain pET30a-ac11, which was transformed into E. coli BL21(DE3) cells. AC11 was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction and then purified by using Ni-nitrilotriacetic acid (NTA) Superflow cartridges (Qiagen, Germany) and fast protein liquid chromatography (FPLC) (GE Healthcare, USA). The purified protein was immunized into ICR female rabbits by intravenous injection. The first immunization of 200 μg/rabbit in incomplete Freund's adjuvant (Sigma Co., USA) was followed by a series of injections of 200 μg/rabbit in complete Freund's adjuvant (Sigma Co., USA) at 7-day intervals. The rabbits were bled 3 days after the last injection, and the antisera were separated after overnight storage of the total blood at 4°C.

Purification of BVs and ODVs.

Sf9 cells were infected with Ac-MK at an MOI of 0.1 and harvested at 5 days postinfection (dpi) by centrifugation at 1,800 × g for 10 min. The resulting supernatant was used for BV purification, and the pellet was used for ODV purification. The purification of BVs and ODVs and the fractionation of the envelope and nucleocapsid of BV and ODV were performed as previously described (7, 20).

Western blot analysis.

The protein samples were mixed with equal volumes of 2× protein sample buffer (Sigma, USA) and boiled at 100°C for 10 min. These samples were resolved by 12% SDS-PAGE, transferred onto a hydrophobic polyvinylidene difluoride (PVDF) membrane (GE Healthcare, USA), and probed with the following primary antibodies: rabbit polyclonal AC11 antiserum (1:5,000), rabbit polyclonal VP39 antiserum (1:5,000) (provided by Kai Yang, Sun Yat-Sen University), and mouse monoclonal GP64 AcV5 antibody (1:5,000) (eBioscience, USA). Peroxidase-conjugated goat anti-rabbit antibody (1:50,000) (ABM, Canada) or horseradish peroxidase-conjugated sheep anti-mouse antibody (1:10,000) (Amersham Biosciences, USA) was used as the secondary antibodies. The signals were detected with the Western blotting Luminol reagent (Santa Cruz Biotechnology, USA).

Electron microscopy.

For transmission electron microscopy (TEM), Sf9 cells (1 × 10⁶ cells/35-mm-diameter well of a six-well plate) were transfected with 1 μg of each bacmid DNA. At 24 and 72 hpt, the cells were harvested by centrifugation at 5,000 × g for 5 min and fixed for 4 h at 4°C with 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2). After postfixation with 1% OsO₄ in the same buffer, the samples were dehydrated in an ethanol-propylene oxide series and embedded in an Epon-Araldite mixture. Ultrathin sections were obtained with an RMC MT-X ultramicrotome and subsequently stained with a mixture of 2% uranyl acetate and Sato's lead (21). A JEM-1010 transmission electron microscope (JEOL, Japan) was used.

RESULTS

Generation of ac11 knockout and repair bacmids.

To study the function of ac11 in the viral life cycle, we generated an infectious bacmid, Ac11KO, in which the ac11 gene was knocked out. To achieve this, the Tn7-mediated transposition between Ac-MK and donor-S of the plasmid capture system was used to generate the recombinant bacmid Ac11KO, in which the ac11 gene was interrupted with a pUC origin (pUC ori) and an ampicillin resistance gene (Amp) 670 bp downstream of the predicted translational start site (Fig. 1A).

RT-PCR analysis was performed to confirm the lack of ac11 expression in Ac11KO-transfected Sf9 cells (Fig. 1B). RT-PCR using a gp64-specific primer set successfully amplified the corresponding gene from the cDNA of Sf9 cells transfected with Ac-MK or Ac11KO. A single RT-PCR product of the expected size was obtained from Sf9 cells transfected with Ac-MK, and no product was amplified from Ac11KO-transfected cells by using an ac11-specific primer set. These results demonstrated that the ac11 gene was successfully knocked out in Ac11KO.

To confirm the phenotypes resulting from the knockout of ac11, a repaired bacmid, Ac11Re, was also constructed. In this bacmid, ac11, expressed by its own promoter, is inserted upstream of the polyhedrin gene in Ac11KO (Fig. 1A). The internal genomic structure of the bacmids Ac-MK, Ac11KO, and Ac11Re was verified by PCR using specific primer sets (Fig. 1C) and nucleotide sequence analysis.

Phylogenetic analysis of ac11.

The NCBI BLAST search result with the Ac11 peptide sequence against the nr database demonstrated that 20 of the alphabaculoviruses with complete viral genome sequences available in GenBank have an ac11 homolog, whereas the other genera of baculoviruses and other large dsDNA viruses do not contain an ac11 homolog (Fig. 2). Among the 20 alphabaculoviruses, 17 and 3 belonged to group I and II alphabaculoviruses, respectively. All 17 group I alphabaculoviruses were clustered in the same clade exclusively, and the 3 group II alphabaculoviruses were Lymantriidae-specific alphabaculoviruses. It was shown previously that Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) is the only group II alphabaculovirus that has an ac11 homolog (12); however, it is expected that more group II alphabaculoviruses containing ac11 may be found as more alphabaculovirus genomes are sequenced.

FIG 2.

Neighbor-joining phylogenetic tree of ac11 and its homologs in baculoviruses. The following viruses were included: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (GenBank accession no. NP_054040), Plutella xylostella multiple nucleopolyhedrovirus (PxMNPV) (accession no. YP_758477), Bombyx mori nucleopolyhedrovirus (BmNPV) (accession no. AGX01103), Bombyx mandarina nucleopolyhedrovirus (BomaNPV) (accession no. YP_002884243), Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) (accession no. NP_703001), Maruca vitrata nucleopolyhedrovirus (MaviNPV) (accession no. YP_950734), Thysanoplusia orichalcea nucleopolyhedrovirus (ThorNPV) (accession no. YP_007250422), Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (accession no. YP_803404), Choristoneura murinana nucleopolyhedrovirus (ChmuNPV) (accession no. YP_008992232), Hyphantria cunea nucleopolyhedrovirus (HcNPV) (accession no. YP_473329), Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) (accession no. NP_046167), Choristoneura rosaceana multiple nucleopolyhedrovirus (ChroMNPV) (accession no. YP_008378495), Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) (accession no. YP_611103), Philosamia cynthia nucleopolyhedrovirus (PhcyNPV) (accession no. AFY62937), Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) (accession no. NP_848322), Choristoneura occidentalis nucleopolyhedrovirus (ChocNPV) (accession no. YP_008378642), Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) (accession no. NP_203177), Lymantria xylina nucleopolyhedrovirus (LyxyNPV) (accession no. YP_003517771), Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) (AHC69531) and Euproctis pseudoconspersa nucleopolyhedrovirus (EupsNPV) (accession no. YP_002854732). The robustness of the tree was tested by a bootstrap analysis (500 replicates). The numbers on the nodes indicate the bootstrap values.

Transcriptional analysis of ac11.

The temporal transcription of ac11 during viral replication was investigated by using Northern blotting (Fig. 3A). The transcript of ac11 was detected as early as 2 h postinfection (hpi) in Ac-MK-infected Sf9 cells, accumulated to its maximum level at 3 hpi, and markedly declined after 12 hpi. Additionally, 5′ RACE analysis using the total RNA isolated from infected Sf9 cells mapped the transcription start site of ac11 to 45 nucleotides (nt) upstream of the translation initiation ATG codon and ∼30 nt downstream of two TATA sequences, at an A residue inside a typical baculovirus early promoter motif, CAGT (Fig. 3B). These results suggested that ac11 is an early gene.

FIG 3.

Transcription of ac11 in Ac-MK-infected Sf9 cells (A) and structural organization of core promoter elements and the termination signal in the ac11 gene (B). (A) Total RNA from Sf9 cells infected with Ac-MK was extracted at 0.5, 1, 2, 3, 6, 12, 24, 48, and 72 hpi and separated on a 1.5% agarose gel. The RNAs were hybridized with a labeled ac11 gene. (B) Typical core promoter elements, including the TATA box (TATA) motif, the CAGT initiator (INR) motif, and the downstream activating region (DAR) motif, were located upstream of the ac11 ORF. The putative polyadenylation signal was located within the ac11 ORF. The RNA start (position +1) and stop (position +1059) sites are indicated by arrows.

BV production is abolished by deletion of ac11.

To determine the effect of ac11 deletion on virus replication, Sf9 cells were separately transfected and infected with Ac-MK, Ac11KO, or Ac11Re. Microscopy analysis revealed no difference among the three viruses at 24 hpt. At 48 hpt, only a small proportion of the cells contained OBs. By 72 hpt, significant differences were observed between the Ac-MK- and Ac11Re-transfected cells and the Ac11KO-transfected cells. Although a large proportion of the Ac-MK- and Ac11Re-transfected Sf9 cells contained OBs, the number of Ac11KO-transfected cells containing OBs did not increase. Furthermore, at 72 hpi, none of the Ac11KO-infected cells contained OBs (Fig. 4). These results suggested that the deletion of ac11 leads to a defect in the production of infectious BV progeny in Sf9 cells.

FIG 4.

Light microscopy of Sf9 cells transfected or infected with Ac-MK, Ac11KO, or Ac11Re. Sf9 cells (1 × 10⁶) were transfected with 1 μg of Ac-MK, Ac11KO, or Ac11Re bacmid DNA. Additionally, 500 μl of culture medium was harvested from Ac-MK-, Ac11KO-, or Ac11Re-transfected cells at 96 hpt and used to infect Sf9 cells (1 × 10⁶). At the designated time points, transfected and infected Sf9 cells were observed a under a light microscope.

To better define the effect of the absence of ac11 on virus replication and investigate the replication kinetics of the virus constructs, BV levels were analyzed by using a 50% tissue culture infective dose (TCID₅₀) and qPCR. To achieve this, Sf9 cells were transfected with Ac-MK, Ac11KO, or Ac11Re bacmid DNA, and BV titers were determined by TCID₅₀ endpoint dilution at the selected time points. Although Sf9 cells transfected with Ac-MK or Ac11Re displayed a normal increase in BV production that reached equivalent titers, no BV was detected in Ac11KO-transfected cells at any time point tested, indicating that no infectious virus was produced (Fig. 5A). These results suggested that ac11 is required for infectious-BV production in Sf9 cells.

FIG 5.

Analysis of BV production. (A) Extracellular BV titers from Sf9 cells separately transfected with Ac-MK, Ac11KO, and Ac11Re were determined at 24 and 72 hpt by TCID₅₀ endpoint dilution. Each point represents the average titer derived from three independent TCID₅₀ assays. The error bars represent standard deviations. (B) The virus titer, regardless of virion infectivity, of the supernatants of Sf9 cells transfected with Ac-MK, Ac11KO, or Ac11Re was determined by qPCR analysis at the designated time points. A stock of wild-type Ac-MK (4.25 × 10⁸ PFU/ml) whose titer was previously determined by endpoint dilution was serially diluted and used to develop a standard curve (shown in the inset). (C) Western blot analysis of BV isolated from Sf9 cells transfected with Ac-MK, Ac11KO, or Ac11Re at 72 hpt. The blots were probed with a monoclonal antibody specific for the VP39 nucleocapsid protein. (D) Detection of viral DNA in Sf9 cells infected with serially passaged Ac11KO. The passage numbers (P2 to P5) are indicated for Sf9 cells infected with serially passaged Ac11KO.

The TCID₅₀ endpoint dilution assay determines only the production of infectious BVs and cannot detect if any noninfectious BVs are produced. To address this concern, the BV titers were also assayed by qPCR, which determines the BV titers by detecting the viral genomes regardless of infectivity (Fig. 5B). The Ac-MK- and Ac11Re-transfected cells revealed a steady increase in BV production, whereas only a background level of viral genomes resulting from the initially transfected bacmid DNA was detected up to 96 hpt in the Ac11KO-transfected cells.

An additional assay was performed to confirm the absence of BV production in Ac11KO-transfected cells. Western blotting using BVs purified from Ac-MK-, Ac11KO-, and Ac11Re-transfected-cell supernatants was performed to compare the levels of the nucleocapsid protein VP39 (Fig. 5C). VP39 was detected in the Ac-MK- and Ac11Re-transfected-cell supernatants, but this nucleocapsid protein was not detected in the Ac11KO-transfected-cell supernatants. Moreover, when the Ac11KO-transfected medium was serially passaged (up to five passages) in Sf9 cells and viral replication was explored by PCR analysis using gp64-specific primers, no viral replication was observed at any of the passages (Fig. 5D). These results suggested that the deletion of ac11 results in a viral phenotype incapable of producing BVs.

Viral DNA replication is not affected by deletion of ac11.

To determine whether ac11 is required for viral DNA replication, a qPCR analysis was performed to investigate the initiation and levels of viral DNA replication in Ac-MK- and Ac11KO-transfected cells up to 72 hpt (Fig. 6). Equal amounts of transfected cells were collected at the designated time points, and the total DNA from the cell lysates was extracted and subjected to qPCR. The results showed that both Ac11KO and GP64KO synthesized similar levels of nascent DNA compared with Ac-MK, suggesting that the onset and level of viral DNA replication in the initially transfected cells are not affected by the deletion of ac11. However, although the levels of DNA replication for Ac-MK increased after 24 hpt, correlating with the spread of infection due to the production of BVs, the DNA replication levels of Ac11KO did not increase up to 72 hpt, which correlates with the absence of BV production in transfected cells.

FIG 6.

Time course of viral replication in transfected cells. In total, Sf9 cells (1 × 10⁶) were transfected with 1 μg of Ac-MK, Ac11KO, Ac11Re, or GP64KO bacmids. At the designated time points, total cellular DNA from Sf9 cells transfected with each bacmid DNA was isolated, digested with the restriction enzyme DpnI to eliminate the input bacmid, and analyzed by qPCR. The results are from three separate transfections, and the error bars represent standard deviations.

Substructural localization of AC11 in virions.

To determine whether AC11 is associated with virions, BV and ODV were purified from Sf9 cells infected with Ac-MK and analyzed by Western blotting. Furthermore, the biochemically fractionated nucleocapsid and envelope fractions of BV and ODV were also analyzed. As a control, to confirm the efficiency of BV fractionation, the nucleocapsid protein VP39 and the BV envelope-specific protein GP64 were analyzed by Western blotting. The results showed that both VP39 and GP64 were observed in the expected fractions; however, AC11 was not detected in either the BV or the ODV fraction (Fig. 7), which was in accordance with data from previous proteomic studies of AcMNPV ODV (22) and BV (23), suggesting that AC11 is not a virus structural protein.

FIG 7.

Western blot analysis of AC11 in purified and fractionated virions. BVs and ODVs were purified by using a sucrose gradient and analyzed by SDS-PAGE and Western blotting. The blots were probed individually with an anti-AC11 antibody to detect AC11, an anti-AcV5 monoclonal antibody to detect the BV envelope protein GP64, and an anti-VP39 antibody to detect the nucleocapsid protein VP39. GP64 and VP39 were analyzed to confirm the correct fractionation of the BV particle in the envelope (E) and nucleocapsid (NC) fractions, respectively.

Electron microscopy analysis of Ac-MK-, Ac11KO-, and Ac11Re-transfected cells.

To further analyze whether the deletion of ac11 has any effect on virus morphogenesis, an electron microscopic analysis was performed with thin sections generated from cells transfected with Ac-MK, Ac11KO, or Ac11Re (Fig. 8 and 9). At 24 hpt, cells transfected with Ac-MK, Ac11KO, or Ac11Re exhibited the typical baculovirus infection symptoms, including enlarged nuclei, a typically reorganized electron-dense virogenic stroma (Fig. 8A to C), and rod-shaped nucleocapsids associating with the electron-dense edges of the virogenic stroma. The virogenic stroma is the active site for viral DNA replication, condensation, and packaging into capsids (24, 25). In Ac-MK- and Ac11Re-transfected cells, a significant number of nucleocapsids was observed in the cytoplasm and budding at the plasma membrane (Fig. 8D and F). In contrast, in Ac11KO-transfected cells, the nucleocapsids were consistently observed only in the nucleus up to 72 hpt (Fig. 8E). In addition, no nucleocapsid was observed in the cytoplasm or budding at the plasma membrane (Fig. 8E). At 72 hpt, nucleocapsids associated with edges of the virogenic stroma, forming bundles, aligning with de novo-developed nuclear envelopes, and acquiring envelopes, were observed in both Ac-MK- and Ac11Re-transfected cells. These enveloped virions contain multiple nucleocapsids prior to occlusion in the protein crystalline matrix of the developing OBs (Fig. 9A and C), and OBs containing numerous enveloped virions were observed in the ring zone (Fig. 9D and F). In Ac11KO-transfected cells, although bundles of nucleocapsids and masses of electron-lucent tubular structures also appeared at the electron-dense edges of the stroma, none of them were enveloped to form ODVs (Fig. 9B).

FIG 8.

Transmission electron microscopy analysis of Sf9 cells transfected with Ac-MK (A and D), Ac11KO (B and E), or Ac11Re (C and F) at 24 hpt. (A to C) Enlarged nucleus (Nu) and virogenic stroma (VS) in Ac-MK-, Ac11KO-, and Ac11Re-transfected cells. c, cytoplasm; nm, nuclear membrane. (D and F) Higher-magnification micrographs of Ac-MK- and Ac11Re-transfected cells displaying normal nucleocapsids (arrows) residing in the cytoplasm (c) and budding from the plasma membrane (pm). (E) In Ac11KO-transfected cells, nucleocapsids and masses of electron-lucent tubular structures were observed in the nucleus, but no nucleocapsids were observed in the cytoplasm.

FIG 9.

Nucleocapsid envelopment and OB morphogenesis. (A, C, D, and F) In Ac-MK- or Ac11Re-transfected cells, normally enveloped virions containing multiple nucleocapsids (A and C, arrows) were embedded within the OBs (P) (D and F, arrows). (B and E) In Ac11KO-transfected cells, bundles of nucleocapsids (arrows) and masses of electron-lucent tubular structures (white triangle) appeared at the electron-dense edges of the stroma (B), and no normal virions were embedded in the OBs (E).

In addition, Ac11KO ODVs were not embedded in the OBs, although their shape and size were similar to those of Ac-MK and Ac11Re ODVs (Fig. 9E). These observations indicated that the deletion of ac11 did not abolish nucleocapsid or OB morphogenesis but affected the formation of normal BV and ODV and the subsequent embedding of ODVs into OBs.

Expression of BV/ODV-associated genes is not affected by deletion of ac11.

Because, as an early gene, ac11 may also regulate the expression of late genes, the disruption of ac11 might affect the expression of some genes associated with BVs or ODVs, consequently inhibiting BV production, ODV envelopment, and the formation of normal OBs. To address this possibility, we investigated the expression of 21 viral genes that have been reported to play an important role in the viral life cycle in Ac11KO-transfected Sf9 cells at 48 and 72 hpt using RT-PCR. These 21 genes included the BV/ODV envelope protein-encoding genes ac16 (bv/odv-e26), ac23 (f-like), ac66, ac78, ac94 (odv-e25), and ac143 (odv-e18); the BV/ODV nucleocapsid-associated genes ac9 (p78/83), ac54 (vp1054), ac77 (vlf-1), ac89 (vp39), ac98 (38k), ac100 (p6.9), ac101 (bv/odv-c42), ac104 (vp80), ac109, ac141 (exon0), ac142, and ac146; the ODV envelope-related genes ac46 (odv-e66) and ac80; and the BV envelope protein-encoding gene ac128 (gp64). However, the expression of these genes in Ac11KO-transfected Sf9 cells showed no differences from that observed in Ac-MK-transfected cells, demonstrating that the deletion of the ac11 gene did not affect the expression of these 21 BV/ODV-associated genes (data not shown). It is possible that AC11 could affect the expression of other BV/ODV-associated genes that have not yet been reported. Otherwise, AC11 could be indirectly involved in the process of ODV envelopment and nucleocapsid egress from the nucleus.

DISCUSSION

Baculovirus ac11 is a highly conserved gene because homologs of ac11 have been found in all lepidopteran group I alphabaculovirus genomes and in three group II genomes, implying that it might perform a common key function in the baculovirus life cycle. However, the function of the ac11 gene remains unknown. In this study, we investigated the role of AcMNPV ac11 in viral replication using an ac11 knockout virus, Ac11KO, and determined that it is an essential gene in the life cycle.

Although it has been reported that Bm4, an ac11 homolog of the Bombyx mori nucleopolyhedrovirus (BmNPV), is not essential (26), our results revealed that BV production is blocked by the deletion of ac11, suggesting that these homologs play somewhat different roles in the infection cycle of the corresponding virus. Infection can be initiated without ac11, as evidenced by the presence of OBs in Ac11KO-transfected Sf9 cells, but infection was restricted to the initially transfected cells. This phenotype was further confirmed by BV titration and Western blot assays. In addition, the qPCR analysis results revealed that ac11 is not essential for DNA replication. Previously, many AcMNPV genes have also been deleted to study their specific function in the viral life cycle. Among them, the following group of genes has been shown to be essential for BV production. ac25 (dbp) encodes a single DNA binding protein that is essential for the production of nucleocapsids and virogenic stroma (27). ac76 expresses a type II integral membrane protein localized to the ring zone late in infection (28, 29). Both ac78, which encodes an envelope protein located in both BVs and ODVs (13), and ac80 (gp41), which encodes a tegument protein located between the virion envelope and capsid (30–32), are required for the egress of nucleocapsids from the nucleus. ac93 encodes a BV- and ODV-associated nucleocapsid protein that is present in the BV envelope fraction (33). In addition, ac9 (p78/83) (34), ac54 (vp1054) (35), ac77 (vlf-1) (36), ac98 (38k) (20), ac142 (37), and ac146 (38), which are essential for BV production, have been shown to encode nucleocapsid proteins located in both BV and ODV. Interestingly, all of these genes are not essential for DNA replication as is the case for ac11, which was investigated in this study.

The electron microscopy observations indicated that abundant nucleocapsids with a normal appearance were observed in the intrastromal space of the virogenic stroma of Ac11KO-transfected cells and were morphologically indistinguishable from those in cells transfected with either the Ac-MK or Ac11Re bacmid, indicating that nucleocapsid assembly is not affected by the deletion of ac11. The normal nucleocapsids also revealed that new viral DNA was incorporated into the capsids. This is consistent with data from qPCR analysis, which demonstrated that ac11 is not involved in viral DNA synthesis. However, no nucleocapsid budding through the nuclear membrane into the cytoplasm was observed in Ac11KO-transfected cells, indicating that inefficient BV production from Ac11KO-transfected cells was due to inefficient egress of nucleocapsids from the nucleus to the cytoplasm. These results demonstrate that ac11 is not required for nucleocapsid formation but is essential for nucleocapsid egress from the nucleus to form the BV.

Several morphogenetic processes, such as nucleocapsid bundling, envelopment of ODVs, and embedding of ODVs into OBs, have been reported to occur in the ring zone (8). Our results revealed that although the nucleocapsids could normally bundle together in the nuclear ring zone in Ac11KO-transfected cells, none were enveloped to form ODVs, and the nonenveloped bundles were never incorporated into the developing OBs. Therefore, the results suggest that AC11 is required for nucleocapsid envelopment and the subsequent embedding of ODVs into OBs. Recently, several other genes, including ac53, ac76, ac77 (vlf-1), ac78, ac93, ac94, ac98 (38k), ac103 (p48), ac109, and ac142, have been shown to be essential for ODV formation (13, 28, 33, 36, 37, 39–42). Deletion of ac53 (39) or ac98 (38k) (41) leads to defects in nucleocapsid assembly, and deletion of ac76 (28) or ac93 (33) affects intranuclear microvesicle formation, resulting in subsequent nucleocapsid envelopment. The knockout of ac78 (13), ac103 (p48) (43), ac109 (42), or ac142 (37) does not affect nucleocapsid assembly but interferes with nucleocapsid envelopment, which is similar to what was observed with the knockout of ac11 in this study. Western blot analysis demonstrated that AC11 is not a structural component of BVs or ODVs, suggesting that the perturbation in ODV envelopment did not result from a lack of structural integrity due to the absence of AC11.

Previously, Chen et al. (11) reported that ac11 is a late gene using transcriptome analysis of AcMNPV. However, they analyzed the RNA extracted at late viral infection phases (6, 12, 18, 24, 36, and 48 hpi). Because RNA extracted at early viral infection phases (for example, 2 to 3 hpi) was not analyzed, those authors were unable to classify ac11 as either an early gene or a late gene. In this study, Northern blotting and 5′ RACE results for the ac11 transcript revealed that ac11 is an early transcriptional gene that is transcribed from 2 hpi and peaks at 3 hpi. Additionally, the transcription started from the “A” residue in the early promoter motif CAGT, which is located 36 bp upstream of the translation start codon. These results clearly demonstrate that ac11 is an early gene.

In conclusion, this study suggests that ac11 is an essential gene for BV production and ODV envelopment. The deletion of ac11 does not affect virus DNA replication, and ac11 is an early gene that is not a structural component of the BV or ODV. Although the exact role of ac11 in the process of ODV envelopment and nucleocapsid egress from the nucleus is remains unclear, the results of this study will lead to a better understanding of baculovirus infection processes.