Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

ABSTRACT

Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids.

IMPORTANCE To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132 deletion on the virus and multiple new findings on ac132.

INTRODUCTION

Baculoviruses in the family Baculoviridae are insect viruses that have rod-shaped, enveloped virions with single circular double-stranded DNA genomes. There are two types of structurally and functionally divergent virions: budded virus (BV) and occlusion-derived virus (ODV). ODVs are occluded in protein crystalline occlusion bodies (OBs).

Baculoviruses initiate infection in the midgut of the host insect. Upon ingestion, the viral OBs are dissolved under alkaline conditions in the midgut of larvae to release ODV virions, which penetrate the peritrophic matrix and infect epithelial cells. A group of ODV envelope-associated proteins, namely, per os infectivity factors (PIFs), including P74, PIF-1 to ∼6, AC83, and ODV-E66, are involved in the infection of midgut cells (1, 2). After cell entry, nucleocapsids are transported to the nuclear membrane in a process that involves actin polymerization (3), pass through nuclear pores, and enter the nucleus to initiate replication. A structural protein, P78/83, which is an activator of WASP-like protein and present in all alphabaculoviruses, is required for nucleation of G-actin to form F-actin filaments. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42, a capsid-associated protein, binds to PP78/83 and transports it into the nucleus (4).

Replication of baculoviruses proceeds through a series of stages mediated by an expression cascade of the viral genes. Genes encoding the proteins involved in DNA replication and other early events are transcribed early by the host RNA polymerase (5, 6). The later gene expression is catalyzed by a virus-encoded RNA polymerase (7, 8). Generally, genes encoding viral structural proteins and the proteins involved in viral assembly are transcribed by the viral RNA polymerase (9).

With the onset of viral DNA replication and gene expression, the virogenic stroma (VS), an electron-dense, chromatin-like structure surrounding multiple less dense spaces, forms near the center of nuclei of infected cells. It has been shown to be the site of viral genome replication and nucleocapsid assembly (9, 10). Part of the nucleocapsids bud out of the nucleus and traffic through the cytoplasm to the periphery. The major capsid proteins VP39 and EXON0 have been shown to interact with kinesin, a motor protein that is involved in anterograde transport in eukaryotic cells (11). The nucleocapsids finally bud through the cytoplasmic membrane that has been premodified by viral proteins to form BVs. BVs released from the cells spread to other susceptible tissues to cause systemic infection (12). The major BV envelope protein, GP64, in group I alphabaculoviruses is a low-pH-activated envelope fusion protein required for BV to exit from and enter cells (13–15). Another kind of envelope fusion protein, called F, present in group II alphabaculoviruses, betabaculoviruses, and the dipteran deltabaculovirus functions similarly to GP64 (16–18). The nucleocapsids remaining in the nucleus get enveloped by virus-induced membranes within the nucleoplasm and are occluded to form ODVs. Each ODV virion contains single or multiple nucleocapsids. When OBs released from the decayed cadavers of host larvae are consumed by other susceptible insects, ODV virions would initiate a new infection cycle in the midgut of the insects.

AcMNPV is the type species of the alphabaculoviruses. It contains a genome of 134 kbp, encoding about 150 protein-coding open reading frames (ORFs) (19, 20). Proteomics analyses revealed 44 and 34 proteins associated with AcMNPV ODV and BV, respectively (21, 22). However, the envelope and nucleocapsid components were not separated in proteomics analyses. Additional studies have localized some proteins onto the envelope, such as the PIF proteins on ODV envelope, GP64 and F-like protein on BV envelope, and ODV-E25 (E25), ODV-E18 (E18), and ODV/BV-E26 (E26) on both ODV and BV envelopes. Known important nucleocapsid-associated proteins include VP39, P6.9, P78/83, VP80, 38K, BV/ODV-C42, EC27, FP25, and EXON0. The roles of most structural proteins in infection and replication remain to be determined. Functional characterization of the individual viral structural proteins is crucial for understanding in detail all steps of viral infection, including entry, intracellular transport, morphogenesis, and egress.

ac132 is located within a cluster of five late-expressed ORFs (orf129 to -133), which are oriented in the same direction on the genome of AcMNPV, transcribed from the promoters recognized by the viral RNA polymerase, and the transcripts of which have same 3′ termini (19, 23–26). In a recent transcriptomics analysis of AcMNPV in a Trichoplusia ni cell line, ac132 was revealed as one of the 20 viral genes with the highest transcript levels detected at 0, 12, 18, 24, 36, and 48 h postinfection (hpi), and it was classified into the G4 gene cluster, in which the transcripts increased dramatically between 6 and 12 hpi and peaked between 18 and 24 hpi (26). Homologs of ac132 have been found in all sequenced group I alphabaculovirus genomes (20, 27). Proteomics analysis showed that AC132 protein was associated with both ODV and BV (21, 22). However, it has not been functionally characterized yet. A homolog of ac132 in Bombyx mori NPV (BmNPV), bm109, was shown to be required for virus replication (28), suggesting ac132 may play an important role in virus replication.

To investigate the role of ac132 in replication cycle of AcMNPV, the expression time course, location, and subcellular location of AC132 protein in the virions and in infected cells were analyzed in this study. AcMNPV mutants lacking ac132 were constructed and characterized to evaluate the effects of ac132 deletion on BV reproduction, viral DNA replication, and late gene expression and morphogenesis in transfected cells. It was found that ac132 encodes a nucleocapsid-associated protein that is required for production of BV and multiply enveloped ODV.

MATERIALS AND METHODS

Virus, cell line, and primers.

bMON14272 is the original bacmid derived from the AcMNPV strain E2. It was maintained in DH10B cells as described previously (29). The Sf9 cell line is a clonal isolate of the parental cell line IPLB-Sf21-AE derived from the fall armyworm, Spodoptera frugiperda (30). The cells were cultured at 26°C in Grace's medium (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum, penicillin (100 μg/ml), and streptomycin (100 μg/ml). The DNA primers used in the experiments are listed in Table 1.

TABLE 1.

Oligonucleotides used to generate knockout and repair bacmid constructs

Oligonucleotide	Sequencea
130koFP	5′-TCAAAAATATACTCACGGGAACGCTGACAAAAAAATAAGCCTTCGAATAAATACCTGTG-3′
130koRP	5′-AGTTTTAATTTGTAATCGGTATTGACAAAATAGGCCATGGAACCAGCAATAGACATAAG-3′
gfpUP1	5′-TATCTCGAGATGGTGAGCAAGGG-3′
gfpDP1	5′-ATAGCATGCTTACTTGTACAGCTCGTC-3′
SV40UP	5′-TACGAATTCGATCATAATCAGCC-3′
SV40DP	5′-CGGACTAGTGATCCAGACATG-3′
ac132UP	5′-GTCGTATACCATCAACACGGTGCAGC-3′
ac132DP	5′-TTGGAATTCGATGTCGACGTGCTAG-3′
polhUP1	5′-GCGACTAGTGTTCACCTCCCTTTTCT-3′
polhUP	5′-GCTGTCGACATGCCGGATTATTC-3′
polhDP	5′-TAGCTGCAGTTAATACGCCGGACC-3′
polhUP2	5′-CCGGTCGACATCATGGAGATAATTAAAAT-3′

^aSequences of restriction sites are underlined.

Antisera and antiserum preparation.

The anti-AC132 polyclonal antiserum was prepared as described below. The AcMNPV orf132 was amplified as an EcoRI-SalI fragment and inserted into the corresponding sites of pET28a (Invitrogen) to construct pET-Ac132. Escherichia coli BL21(DE3)/pLysS cells transformed with pET-Ac132 were induced with isopropyl-β-d-thiogalactopyranoside (IPTG), and the His-tagged AC132 protein was purified with Ni-nitrilotriacetic acid (NTA) resin (Qiagen). The eluate was subjected to SDS-PAGE, and the band of the His-tagged AC132 protein was cut from the gel, homogenized, and used to immunize mice. Two weeks after the first inoculation, the animal was subjected to three boosts at 2-week intervals. Ten days after the final boost, the animal was bled, and the serum was prepared for use in this study. Similarly, the anti-E25, anti-VP39, and anti-BV/ODV-C42 rabbit polyclonal antisera and the anti-PP31 mouse polyclonal antiserum were separately generated with His-tagged AcMNPV E25 (31), VP39, BV/ODV-C42, and PP31, which were expressed in E. coli.

The anti-GP64 and anti-E18 rabbit polyclonal antisera were provided by Zhi-Hong Hu. The anti-IE1 and anti-P6.9 rabbit polyclonal antisera were provided by Kai Yang.

Western blot analysis.

Whole-cell extracts of transfected or infected Sf9 cells or other designated forms of protein samples were resolved by SDS-PAGE, followed by electroblotting to BioTace polyvinylidene difluoride (PVDF) membrane with a liquid transfer apparatus. The blots were probed with the AC132-specific mouse antiserum (1:3,000) or other designated antisera. IRDye 800CW-conjugated goat anti-mouse (or anti-rabbit) antibody (1:5,000) (LI-COR) was used as the secondary antibody. Fluorescence was detected by LI-COR Odyssey. SDS-PAGE and immunohybridizations to Western blots were performed in accordance with standard protocols and the manufacturer's instructions (32).

Virion purification and fractionation.

Sf9 cells were infected with AcMNPV at a multiplicity of infection (MOI) of 0.1 and harvested at 5 days postinfection. Two hundred milliliters of the harvested medium was centrifuged at 9,000 rpm for 20 min. The supernatant and the pellet were used for BV purification and ODV purification, respectively. Purification of BV and ODV was performed as previously described (33). For BV purification, 80 ml of supernatant was centrifuged at 9,000 rpm (Beckman JA-30.50 rotor) for 20 min to remove cell debris. The supernatant was decanted and centrifuged at 4°C at 100,000 × g (Beckman JS-24 rotor) for 90 min. The pellet was resuspended in 500 μl of 0.1× Tris-EDTA (TE) buffer, overlaid onto a 20, 30, 40, 50 and 60% (wt/vol) discontinuous sucrose gradient, and centrifuged at 100,000 × g (MLS50 rotor) for 90 min at 4°C. The virus fraction was collected, 1:4 diluted with 0.1× TE, and centrifuged at 100,000 × g for 90 min at 4°C. The BV pellet was resuspended in 200 μl of 0.1× TE and stored at −20°C.

For ODV purification, the cell pellet was resuspended in 15 ml of 0.2% Triton X-100 and subjected to sonication on ice. The cell lysates were diluted with 0.2% Triton X-100 to a 60-ml total volume and then layered onto a 30% sucrose–0.2% Triton X-100 cushion and centrifuged at 9,000 rpm (JS-24 rotor) for 20 min. The pellet was resuspended in 5 ml of 0.2% Triton X-100, layered onto a 33.5-ml 35 to 60% (wt/vol) sucrose gradient in H₂O, and centrifuged at 100,000 × g for 30 min at 4°C. The OB fraction was collected and washed twice by suspension in H₂O and centrifugation. The OB pellet was resuspended in 2 ml of H₂O, mixed with 3 ml of 3× OB lysis buffer (0.3 M Na₂CO₃, 0.51 M NaCl, 0.03 M EDTA [pH 11]), incubated at 37°C for 40 min, and then centrifuged at 500 × g for 5 min. The supernatant was layered onto a 20, 30, 40, 50, and 60% (wt/vol) discontinuous sucrose gradient and centrifuged at 50,000 × g (Beckman JS-24 rotor) for 30 min at 4°C. The virus fraction was collected, washed by dilution in 3 volumes of 0.1× TE, and centrifuged at 50,000 × g (Beckman JM-24 rotor) for 60 min at 4°C. The ODV pellet was resuspended in 200 μl of 0.1× TE and stored at −20°C.

BVs and ODVs were fractionated into envelope and nucleocapsid preparations as previously described (34) with modification. Briefly, 170 μl of BV or ODV was mixed with 800 μl of 2.0% NP-40–10 mM Tris (pH 8.5) and incubated at room temperature for 60 min with gentle agitation. The solution was layered onto a 4-ml 30% (vol/vol) glycerol–10 mM Tris (pH 8.5) cushion and centrifuged at 150,000 × g for 60 min at 4°C (MLS 50 rotor). The envelope proteins were recovered from the top of the gradient and mixed with 4 volumes of ice-cooled acetone and precipitated by centrifugation (150,000 × g for 45 min). Following one wash with ice-cooled acetone, the pellet was dissolved in 10 mM Tris (pH 7.4). The nucleocapsid pellet was resuspended in 0.1× TE buffer.

Immunoblotting was performed as described above with polyclonal antibodies against AC132, GP64, E25 (1:5,000) (31), or VP39 (1:10,000) (31).

Immunofluorescence assays and confocal microscopy.

Sf9 cells seeded on the surface of coverslips placed in 35-mm-dimater dishes were inoculated with infectious supernatant of AcMNPV at an MOI of 5. At designated time points after infection, the cells on the coverslips were fixed with Immunol staining fixing solution (Beyotime), incubated with AC132-specific antibodies, and then incubated with rhodamine (tetramethyl rhodamine isocyanate [TRITC])-conjugated goat anti-mouse IgG (PTG Lab) (1:60) and stained with Hoechst 33258 (Beyotime), as previously described (31). Finally, the cells were sealed on microscope slides with antifade mounting medium (Beyotime) and subjected to a confocal microscopic assay with a Zeiss LSM510 Meta confocal laser scanning microscope for fluorescence using wavelengths of 550 nm for rhodamine and 352 nm for Hoechst 33258. All images were digitally recorded and merged by the use of Zeiss software.

Bacmid construction.

The sequences of the oligonucleotides used to generate the knockout and repair bacmid constructs used in this study are shown in Table 1. The deletion of ac132 from the AcMNPV genome was done by using the λ Red system, as previously described (35). Briefly, the chloramphenicol acetyltransferase gene (cat) cassette was amplified from a pKS-cat template plasmid by PCR with primers ac132koFP and ac132koRP, which contained a 40-nucleotide (nt)-long homologous arm corresponding to the sequence (nt 111833 to 111872) immediately upstream of the start codon ATG and the 3′ end (nt 112183 to 112222) of AcMNPV ac132 (nt 111873 to 112532), respectively. The PCR product was electrotransformed into arabinose-induced BW25113 competent cells harboring bMON14272 and plasmid pKD46 encoding λ Red recombinase. The resultant bacmid was named vAc^Ac132ko (see Fig. 4B).

FIG 4.

Construction of ac132 knockout, repair, and wt AcMNPV bacmids. (A) Map of vAc^PH-gfp1 showing the ac132 and polh loci. A copy of the egfp ORF under the control of AcMNPV p10 promoter and a copy of polh with the native promoter were inserted back into the polh locus in the opposite orientation. (B) Maps showing the modification of the ac132 and polh loci in ac132 knockout and repair AcMNPV bacmids. At the ac132 locus, a 310-bp sequence of the ac132 ORF was replaced with the cat gene. In the polh locus, a copy of egfp under the control of the p10 promoter and a copy of polh with its native promoter (vAc^{ac132ko-PH-gfp}), a copy of ac132 with the native promoter and a copy of egfp under the control of the p10 promoter and a copy of polh with its native promoter (vAc^{ac132ko-rep-PH-gfp}), or a copy of ac132 with the native promoter and a copy of polh with the native promoter (vAc^{ac132ko-rep-PH}) were inserted by transposition. (C) Western blot analysis for the presence or absence of AC132 in extracts of the cells infected by vAc^{ac132ko-PH-gfp} (ko) or wild-type AcMNPV (wt) or vAc^{ac132ko-rep-PH} (Rep) at 48 hpt.

A DNA fragment containing the egfp ORF, coding for enhanced green fluorescent protein (EGFP), was PCR amplified with primers gfpUP1 and gfpDP1 and inserted between the XhoI and SphI sites of pFastBacDual 1 to make an intermediate vector, pFBD-gfp. Another fragment containing the AcMNPV polh ORF was amplified using the primers polhUP and polhDP and inserted between the SalI and PastI sites of pFBD-gfp, producing pFBD-PH-gfp. pFBD-PH-gfp was electroporated into E. coli DH10B containing bMON14272 and a helper plasmid, pMON7124 (29), which encodes a transposase, to generate a polh- and gfp-containing wild-type (wt) bacmid vAc^PH-gfp1. pFBD-PH-gfp was electroporated into E. coli DH10B containing ac132 knockout bacmid vAc^ac132ko and pMON7124 to generate polh- and gfp-containing and ac132 knockout bacmid vAc^{ac132ko-PH-gfp}.

A fragment containing ac132 with native promoter (248 bp upstream of the start codon ATG) was amplified using primers ac132UP and ac132DP and inserted between the BstZ17I and EcoRI sites of pFBD-gfp to make pFBD-gfp-ac132. A copy of simian virus 40 (SV40) poly(A) signal sequence (241 bp) was amplified using primers SV40UP and SV40DP and inserted between the EcoRI and SpeI sites of pFBD-gfp-ac132 to generate pFBD-gfp-ac132-SV40PA. A copy of AcMNPV polh ORF was PCR amplified with the primers polhUP1 and polhDP, digested with SpeI and PstI, and ligated with pFBD-gfp-ac132-SV40PA cut with the same enzymes to make pFBD-ac132-PH-gfp. The transfer vector pFBD-ac132-PH-gfp was electroporated into E. coli DH10B containing vAc^ac132ko and pMON7124 to generate ac132-repaired bacmid vAc^{ac132ko-rep-PH-gfp}.

A fragment containing AcMNPV polh with its native promoter was PCR amplified using primers polhUP2 and polhDP and inserted between the SalI and PstI sites of pFastBac1 to make pFB-PH1. pFB-PH1 was cut with SnaBI and SalI to remove the original polh promoter, the end was filled, and the fragment was religated to produce pFB-PH2. pFBD-ac132-PH-gfp was cut withBstz17 I and XhoI to remove the Pp10-gfp part, end filled, and religated to make pFBD-ac132-PH. pFB-PH2 and pFBD-ac132-PH were separately electroporated into E. coli DH10B containing vAc^ac132ko and pMON7124 to generate vAc^ac132ko-PH and vAc^{ac132ko-rep-PH}.

Titration of BV.

Sf9 cells were infected with infectious cell culture supernatants at an MOI of 0.1 for the wt and the ac132 knockout repair viruses or inoculated with 1 ml of the supernatant from the transfection with the ac132 knockout mutant. The cell culture supernatants were collected at the designated time points, and the infectious budded-virus titers were determined using a 50% tissue culture infectious dose (TCID₅₀) endpoint dilution assay (36). Virus infection was determined by monitoring green fluorescent protein (GFP) expression by fluorescence microscopy.

To determine the titers of infectious and noninfectious BVs released from transfected cells, the genome DNAs contained in BVs in the supernatants from the transfected cell cultures were purified and measured by real-time quantitative PCR (qPCR), as previously described (68). Briefly, 400 μl of the supernatant collected from transfected cell culture was mixed with an equal volume of the lysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM EDTA, 0.5% SDS, 20 μg/ml RNase A, 80 μg/ml protease K) and incubated at 50°C overnight. DNA was extracted with 800 μl of phenol-chloroform-isoamyl alcohol (25:24:1). The aqueous layer was removed and mixed with 0.8 volume of isopropanol. DNA was precipitated by centrifugation. The pellet was dissolved in 10 μl of double-distilled H₂O (ddH₂O). One microliter of each purified DNA sample was used as the template for each qPCR.

Quantitative real-time PCR.

Viral DNA replication was assayed by real-time PCR, as previously described (37). Briefly, Sf9 cells transfected with vAc^{ac132ko-PH-gfp}, vAc^{ac132ko-rep-PH-gfp}, or vAc^gp64ko were harvested at designated time points posttransfection, and the DNA was extracted by phenol-chloroform-isoamyl alcohol extraction. Each DNA pellet was dissolved in 100 μl of ddH₂O. Prior to the PCR, 8 μl of total DNA from each time point was digested with DpnI in a 20-μl total reaction volume. Two microliters of the digested DNA was mixed with the SYBR green I real-time PCR master mix kit (Toyobo) and the qPCR primers Q-65972F and Q-66072R (37) in a 20-μl reaction mixture. The samples were analyzed in a Bio-Rad CFX96 qPCR cycler. The results were analyzed using CFX Manager 2.1 (Bio-Rad) software. The qPCR product corresponded to a 100-bp region of the AcMNPV gp41 gene. The number of viral DNA genome copies within each sample was calculated by using a standard curve generated from a dilution series of a plasmid containing AcMNPV gp41.

Electron microscopy.

A total of 1 × 10⁶ Sf9 cells per dish (35 mm) were transfected with 1.0 μg of vAc^{ac132ko-res-PH} or vAc^ac132ko-PH. At 48, 72, 80, and 96 h posttransfection (hpt), the cells were fixed and then dislodged and precipitated at 3,000 rpm for 5 min. The cell pellet was dehydrated, embedded, sectioned, and stained as described previously (38). The samples were examined with a FEI Tecnai G²20 TWIN transmission electron microscope at an accelerating voltage of 200 kV.

Immunoprecipitation.

Sf9 cells (8 × 10⁶ in total) infected with AcMNPV were harvested at 18, 24, and 36 hpi, respectively. The cell pellets were washed three times with phosphate-buffered saline (PBS) and then mixed and resuspended in 800 μl of lysis buffer (20 mM Tris-HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 100 μM phenylmethylsulfonyl fluoride [PMSF]), incubated on ice with shaking for 1 h. The lysates were clarified by centrifugation at 11,000 rpm for 25 min. The supernatant was mixed with the AC132-specific antiserum (1:600), rocked at 4°C for 2 h, and then mixed with 40 μl of protein A/G Plus beads (Santa Cruz Biotechnology) and rocked at 4°C for 6 h. The beads were precipitated and washed three times with cold lysis buffer and then mixed with 40 μl of 2× Laemmli buffer, denatured at 100°C, and precipitated. The supernatant was subjected to 15% (for E18 and P6.9) or 12% (for AC132 and other proteins) SDS-PAGE and Western blotting. The blots were probed with the individual antisera to AC132 (1:3,000), VP39 (1:10,000), P6.9 (1:2,000), E25 (1:5,000), E18 (1:8000), IE1 (1:2,000), PP31 (1:10,000), or BV/ODV-C42 (1:3,000).

RESULTS

AC132 is associated with the nucleocapsids of both BV and ODV.

The ac132 ORF comprises 660 nt and encodes a polypeptide with a predicted size of 25.1 kDa. Bioinformatic analysis of the amino acid sequence of AC132 with the Quick2D and COILS programs identified a putative coiled coil domain containing three heptad repeats (ESAVFQELERLENAVVVLENE; amino acids [aa] 59 to 79; the residues at the a and d positions of each repeat are in boldface type) in the N terminus. Coiled-coil-containing proteins exhibit a broad range of different functions. To determine whether AC132 was an envelope protein or a nucleocapsid-associated protein, purified BVs and ODVs were fractionated into envelope and nucleocapsid fractions separately and analyzed by SDS-PAGE and Western blotting. The capsid protein VP39 (39), envelope protein E25 (40), and the BV envelope protein GP64 (13) in the same samples were also probed, as controls. As a result, a polypeptide was detected by AC132-specific antibodies in the nucleocapsid fractions of both BV and ODV but not detected in the envelope fractions. VP39, E25, and GP64 were detected only in the expected fractions (Fig. 1A). These results indicated that AC132 was localized to the nucleocapsids of both BV and ODV. In an SDS-PAGE gel, the band of AC132 migrated between the bands of E25 (25 kDa) and His-tagged AC132, which was expressed in E. coli (29.6 kDa) and loaded for comparison (Fig. 1B). The estimated molecular mass of AC132 was 28 kDa. It was somewhat larger than predicted, suggesting that AC132 was modified posttranslation.

FIG 1.

Analysis of AC132 in purified and fractionated virions. (A) BV and ODV of AcMNPV were purified by using a sucrose gradient and fractionated into envelope and nucleocapsid fractions, which were analyzed by SDS-PAGE and Western blotting. AC132 was probed with AC132-specific polyclonal antibodies. The major capsid protein VP39, envelope protein E25, and BV envelope protein GP64 were detected with the corresponding specific polyclonal antibodies. NC, nucleocapsid fraction; E, envelope fraction. (B) Purified ODV of AcMNPV (lanes 1 and 2) and His-tagged AC132 expressed in E. coli (lane 3) were subjected to SDS-PAGE and Western blotting. AC132 (lane 2) and His-tagged AC132 (lane 3) and E25 (lane 1) were probed with the corresponding antibodies.

Expression time course and subcellular localization of AC132 in infected Sf9 cells.

ac132 was previously shown to be a late-transcribed gene (25). In this study, the time course of accumulation of AC132 in AcMNPV-infected cells was analyzed by SDS-PAGE and Western blotting. AC132 was first detected in the extracts of infected cells at 12 hpi, and the peak level of accumulation occurred at 24 hpi (Fig. 2A). In comparison, the level of VP39 was much lower than that of AC132 at 12 hpi, but it rose and remained higher than that of AC132 after 18 hpi. The size of the AC132 protein detected in the infected cells was the same as those from AcMNPV BV and ODV (Fig. 2B).

FIG 2.

Time course analysis of AcMNPV AC132 expressed in infected Sf9 cells. (A) The cells inoculated with infectious cell culture supernatant containing AcMNPV were harvested at the designated time points postinfection, and the cell extracts were subjected to SDS-PAGE and immunoblot analysis with AC132-specific antibodies. AcMNPV VP39 and cellular β-actin were also probed as controls. (B) The extracts of Sf9 cells infected 24 hpi and purified AcMNPV BV and ODV were subjected to SDS-PAGE and Western blot assays with AC132-specific polyclonal antibodies. CL, cytolysates; M, protein molecular mass ladders.

Subcellular localization of AC132 in infected Sf9 cells was analyzed by immunofluorescence microscopy. Sf9 cells infected with AcMNPV, at an MOI of 5, were collected at 0, 12, 18, 24, 36, 48, and 72 hpi, blotted with AC132-specific antibodies and rhodamine-conjugated goat anti-mouse IgG, stained with Hoechst 33258, and subjected to confocal microscopy (Fig. 3). The AC132 labeled by rhodamine with red fluorescence was first observed predominantly in the cytoplasm at 12 hpi. A few interspersed fluorescent dots were present at the center of the nucleus (blue color). At 18 hpi, red fluorescence still remained mainly in the cytoplasm. By 24 hpi, it was mainly observed in the nucleus, forming a condensed ring zone at the periphery of the nucleus; small fluorescent dots were spread evenly throughout the central region of the nucleus; significant amounts still could be seen in the cytoplasm. By 36 hpi, the condensed fluorescence ring zone enlarged toward the center of the nucleus. By 48 hpi, the condensed fluorescence ring zone disappeared and fluorescence spread almost evenly throughout the whole nucleus. By 72 hpi, red fluorescence was viewed only in the nucleus, leaving some regions empty of fluorescence (Fig. 3).

FIG 3.

Subcellular localization of AC132 in Sf9 cells infected with AcMNPV. Sf9 cells infected with AcMNPV were sampled at 12, 18, 24, 36, 48, and 72 hpi, respectively. AC132 was blotted with AC132-specific polyclonal antibodies, which were subsequently blotted with rhodamine-conjugated goat anti-mouse IgG to label AC132 (red). The nuclei were stained with Hoechst 33258 (blue). The cells were subjected to confocal microscopy.

Generation of AcMNPV bacmid mutants with ac132 deleted or translocated.

In order to determine if ac132 is essential for replication of the virus, an AcMNPV mutant with orf132 knocked out was constructed based on bMON14272, by using the λ Red homologous recombination system (36). In the recombinant bacmid, named vAc^ac132ko, the first 390 nt at the 5′ end of orf132 (nt 111873 to 112530) was replaced with the cat gene (Fig. 4A). The deletion was confirmed by PCR screening (data not shown). To facilitate detection of viruses in cells, a copy of the polh with the native promoter and the egfp linked with a copy of the AcMNPV p10 promoter was inserted into vAc^ac132ko or bMON14272 through Tn7-mediated transposition at the polh locus, making vAc^{ac132ko-PH-gfp} and vAc^PH-gfp1, respectively (Fig. 4A and B). vAc^PH-gfp1 was used as the wild-type virus in experiments. An ac132 knockout repair bacmid named vAc^{ac132ko-rep-PH-gfp} was constructed, in which a copy of the AcMNPV ac132 with the native promoter, together with a copy of polh with the native promoter and an egfp gene with a p10 promoter, was inserted into the polh locus of vAc^ac132ko (Fig. 5B).

FIG 5.

Analysis of viral reproduction in Sf9 cells. (A) Fluorescence microscopy and phase-contrast microscopy of Sf9 cells transfected with vAc^PH-gfp1, vAc^{ac132ko-PH-gfp}, or vAc^{ac132ko-rep-PH-gfp} at designated time points posttransfection. (B) Fluorescence microscopy of Sf9 cells infected with vAc^PH-gfp1, vAc^{ac132ko-PH-gfp}, or vAc^{ac132ko-rep-PH-gfp} at 24 and 120 hpi. (C) BV growth curves of ac132 knockout, repair, and wt viruses in Sf9 cells. Sf9 cells were infected with each virus at an MOI of 0.1 or inoculated with 1 ml of supernatant from the transfection with vAc^{ac132ko-PH-gfp}. The supernatants were harvested at the designated time points, and virus titers were determined by TCID₅₀ endpoint dilution assays. Each data point represents the average titer of three independent infections. Error bars indicate standard deviations. (D) BV growth curves of ac132 knockout, repair, and wt bacmid-transfected Sf9 cells independent of viral infectivity. DNA was purified from the supernatants collected at the designated time points from the transfections, and the titers were determined by qPCR. Error bars represent standard deviations.

Deletion of ac132 was further verified by Western blotting, using the AC132-specific antiserum. A peptide of about 28 kDa was detected in extracts of the cells transfected by vAc^{ac132ko-rep-PH-gfp} or vAc^PH-gfp1, but it was not present in extracts of the cells transfected by vAc^{ac132ko-PH-gfp} (Fig. 4C).

Ac132 deletion resulted in inefficient BV production.

The effect of the deletion of the ac132 on replication of the virus was first examined by fluorescence microscopy of the Sf9 cells transfected separately with vAc^{ac132ko-PH-gfp}, vAc^{ac132ko-rep-PH-gfp}, and vAc^PH-gfp1. The cells were viewed every hour from 12 to 24 hpt. As shown in Fig. 5A, weak fluorescence was first observed in cells transfected with vAc^{ac132ko-PH-gfp}, vAc^{ac132ko-rep-PH-gfp}, or vAc^PH-gfp1 at 14 hpt. At 24 hpt, more cells with bright fluorescence were present in all transfected cell cultures. No obvious differences in number and intensity of the fluorescent cells were observed between different transfections. By 120 hpt, the majority of the cells in the cultures with vAc^PH-gfp1 or vAc^{ac132ko-rep-PH-gfp} were fluorescent, while there was only a slight increase in the number of fluorescent cells in the culture transfected with vAc^{ac132ko-PH-gfp}. To test if infectious BVs were produced in the transfections, the supernatants collected from each transfection, at 120 hpt, were individually inoculated into fresh cultures of Sf9 cells. By 120 hpi, almost all cells in the dishes inoculated with supernatants of vAc^PH-gfp1 or vAc^{ac132ko-rep-PH-gfp} were fluorescent. In contrast, only 21 and 25 fluorescent cells, respectively, were observed in two dishes inoculated with the supernatant from the transfection with vAc^{ac132ko-PH-gfp} (Fig. 5B). These observations demonstrate that the wt and the ac132 knockout repair bacmid replicated in the transfected cells and produced infectious viruses, whereas there were few infectious BVs released from the cells transfected with the ac132 knockout bacmid. Under a phase-contrast microscope, OBs were observed in most of the cells transfected with vAc^PH-gfp1 or vAc^{ac132ko-rep-PH-gfp} by 96 hpt, but they were found in only a few cells in the culture transfected with vAc^{ac132ko-PH-gfp}, in which the OB-containing cells were isolated, showing no sign of spreading of infection (Fig. 5A).

The effects of ac132 deletion on BV production were further evaluated by virus growth curve analysis. Sf9 cells were infected with infectious supernatants of vAc^{ac132ko-rep-PH-gfp} or vAc^PH-gfp1 at an MOI of 0.1 or inoculated with 1 ml of the supernatant from the transfection with vAc^{ac132ko-PH-gfp}, and the titers of infectious BVs were measured by TCID₅₀ endpoint dilution assays. As shown in Fig. 5C, whereas the titers of infectious BVs increased steadily for both vAc^{ac132ko-rep-PH-gfp} and vAc^PH-gfp1, without a significant difference between these two viral types, the virus titer from the cell culture transfected with vAc^{ac132ko-PH-gfp} cells was too low to be determined at all points.

The titers of both infectious and noninfectious BVs released from transfected cells at selected time points posttransfection were measured by qPCR. As shown in Fig. 5D, there was detectable background of viral genomes present at all time points analyzed due to the presence of genomic DNA from the bacmid transfection. As expected, a steady increase in BV was detected from 24 to 96 hpt for vAc^{ac132ko-rep-PH-gfp} and vAc^PH-gfp1. The BV quantity detected in the culture with vAc^{ac132ko-PH-gfp} had no significant change at the early stage. It started increasing slowly after 48 hpt and increased less than 10-fold by 96 hpt. This result suggested that there were a small number of BVs released from the cells transfected with vAc^{ac132ko-PH-gfp}.

Deletion of ac132 did not have significant effect on virus DNA replication and late gene expression.

To examine the effect that ac132 may have on viral DNA replication, the levels of DNA of vAc^{ac132ko-PH-gfp} and vAc^{ac132ko-rep-PH-gfp} in the cells transfected were measured over a 96-h time course. A gp64-knockout mutant of the AcMNPV wild-type bacmid bMON14272, vAc^gp64ko, was used as a control. Sf9 cells were transfected with the individual bacmids and collected at designated time points, and the total DNA was extracted and analyzed by qPCR. The results are shown in Fig. 6. For all three bacmids, DNA synthesis began increasing at 12 hpt. At 24 hpt, the levels of DNA detected for all three bacmids were similar. After that, the DNA levels measured for the three bacmids rose continuously, but the values differed significantly from each other. The levels of vAc^{ac132ko-rep-PH-gfp} were much higher than those of vAc^{ac132ko-PH-gfp} and vAc^gp64ko, while the levels of vAc^{ac132ko-PH-gfp} were significantly higher than those of vAc^gp64ko at all time points after 48 hpt. Since gp64 is required for infectivity of BV, replication of vAc^gp64ko was limited to the initially transfected cells. The differences in DNA levels between vAc^{ac132ko-PH-gfp} and vAc^gp64ko likely resulted from replication in additional cells infected by the few BVs, which might have been released from the cells transfected by vAc^{ac132ko-PH-gfp}. These results suggested that the deletion of ac132 did not have any effect on viral DNA replication.

FIG 6.

Quantitative PCR analysis of viral DNA replication in Sf9 cells infected by gp64 knockout, ac132 knockout, or ac132 knockout-repaired AcMNPV recombinants. Total DNA was purified from the cells transfected with vAc^gp64ko-gfp, vAc^{ac132ko-PH-gfp}, or vAc^{ac132ko-rep-PH-gfp} at 0, 12, 24, 48, 72, and 96 hpt and digested with DpnI to eliminate input bacmid DNA, and the gp41 sequence was analyzed by real-time PCR. The values displayed represent the averages from infections performed in triplicate with error bars indicating standard deviations.

Western blot assays were performed with antisera separately against AcMNPV VP39 and E25 to determine if viral gene expression for at least vp39 and e25 was affected by ac132 deletion. VP39 is the major capsid protein. E25 is an envelope protein present in both BV and ODV. Both the vp39 and e25 genes are transcribed by viral RNA polymerase (26, 39, 40). Sf9 cells transfected with vAc^{ac132ko-PH-gfp} were harvested at 18, 24, 36, and 48 hpt, and the cell lysates were analyzed. As shown in Fig. 7, VP39 and E25 were detected first at 24 hpt, respectively, in both the cells transfected with vAc^{ac132ko-PH-gfp} and the cells transfected with vAc^gp64ko-gfp, which served as a control. No significant differences were noted for either VP39 or E25 between vAc^{ac132ko-PH-gfp} and vAc^gp64ko-gfp.

FIG 7.

Western blot analysis of VP39 and E25 synthesis in Sf9 cells infected with vAc^{ac132ko-PH-gfp}or vAc^gp64ko-gfp. Sf9 cells were infected at an MOI of 5 and harvested at the designated times postinfection, and cell lysates were prepared for Western blot analysis. Each protein was analyzed with polyclonal antibodies against the protein. The cellular β-actin was probed by a corresponding antibody as a loading control.

Effects of ac132 knockout on virus morphogenesis.

To further determine the effect that ac132 knockout has on virus morphogenesis, thin sections of the cells transfected with vAc^ac132ko-PH or vAc^{ac132ko-rep-PH} were prepared at 48, 72, 80, and 96 hpt and examined by electron microscopy. For vAc^{ac132ko-rep-PH}, VS inundated with rod-shaped nucleocapsids was observed in transfected cells at 48 hpt (Fig. 8Aa). For vAc^ac132ko-PH, VS was not observed until 72 hpt (Fig. 8Ba). Shown in Fig. 8Bb is a section of a vAc^ac132ko-PH-transfected cell at 96 hpt, in which several swirling hair-like structures appeared on both sides of the VS. Only a few nucleocapsids were observed to be associated with the structures. In the cells transfected with vAc^{ac132ko-rep-PH}, enveloped or unenveloped nucleocapsids in cytoplasm and BVs associated with cell membrane were frequently observed (Fig. 8Ab). A few nucleocapsids released from the nucleus into some vesicles (Fig. Bc) were also observed in the cells transfected with vAc^ac132ko-PH. Only a single incomplete BV-like particle associated with cytoplasmic membrane was found in a section of a vAc^ac132ko-PH-transfected cell at 96 hpt (Fig. 8Be). In the cells transfected with vAc^{ac132ko-rep-PH}, enveloped nucleocapsids and OBs filled with virus bundles were commonly found in the nuclei by 72 hpt, and most virions contained multiple nucleocapsids (Fig. 8Ac and -d). In the cells transfected with vAc^ac132ko-PH, only a small part of the nucleocapsids in the nucleus were enveloped through all time points. A few tiny OBs were viewed first at 80 hpt (not shown), complete OBs were not observed until 96 hpt (Fig. 8Bg), and the virions mostly contained a single nucleocapsid (Fig. 8Bc, -f, and -g), although groups of nucleocapsids aligning on the membrane-like structure (Fig. 8Bd) were also observed. There were obviously fewer virions contained in the OBs of vAc^ac132ko-PH than in the OBs of vAc^{ac132ko-rep-PH} (Fig. 8Ad and Bg). These observations showed that ac132 may not be essential for virus replication, but the deletion of ac132 resulted in a delay in the formation of VS and OB and dramatically reduced production of the BV, multiply enveloped ODV, and mature OB.

FIG 8.

Transmission electron microscopy of Sf9 cells infected with vAc^{ac132ko-rep-PH} (A) or vAc^ac132ko-PH (B) at 48 (Aa), 72 (Ab, Ad, and Ba), 80 (Ac), or 96 (Bb to -g) hpi. CM, cytoplasmic membrane; NM, nuclear membrane; NC, nucleocapsid; ENC, enveloped nucleocapsid; SHL, swirling hair-like structure; VS, virogenic stroma. Bar, 500 nm.

AC132 interacts with viral envelope protein E18 and DNA-binding protein P6.9.

To explore the mechanism by which ac132 influences assembly and envelopment of nucleocapsids, experiments were performed to detect interaction between AC132 and other viral proteins. The candidate proteins selected for examination included the major capsid protein VP39 (39), the viral genome-binding protein P6.9 (41, 42), BV and ODV envelope proteins E25 (40) and E18 (43), VS-associated proteins IE1 (44) and PP31 (45), and BV/ODV-C42, involved in virus-induced nuclear actin polymerization (46). The extracts of Sf9 cells infected by AcMNPV were immunoprecipitated with AC132-specific antibodies. E18-specific antibodies detected a band of 18 kDa on Western blots of the immunoprecipitates (Fig. 9). P6.9-specific antibodies also detected a weak band, while polyclonal antibodies specific to other proteins did not detect any bands of the corresponding proteins (data not shown). In the columns of cytolysates and supernatant, multiple bands were detected by the P6.9-specific antibodies. The bands with variable sizes represented different phosphorylated and dephosphorylated forms of P6.9 (42). The band detected in the eluate was likely the dephosphorylated form.

FIG 9.

Coimmunoprecipitation of AC132 and ODV-E18 (A) or P6.9 (B). The extracts of Sf9 cells infected by AcMNPV were immunoprecipitated with anti-AC132 polyclonal antibodies (Ab) (1:600). The immunoprecipitates were analyzed by SDS-PAGE and Western blot assays. AC132, E18 (A), and P6.9 (B) were detected with corresponding specific polyclonal antibodies. CL, cytolysates; SP, supernatant; W, washout; E, eluate.

DISCUSSION

In this study, AcMNPV AC132 was determined to be an ODV and BV nucleocapsid-associated protein. In infected cells, AC132 was first detected 12 hpi, and the level peaked at 24 hpi and remained in abundance through the late phase. This is consistent with the accumulation pattern of the ac132 transcript in infected T. ni cells (26). The presence of AC132 in both BV and ODV and its abundance in infected cells imply the importance of the protein in virus replication.

To further elucidate the role of ac132 in the replication cycle of the virus, AcMNPV recombinants with ac132 deleted were constructed and analyzed in comparison with the wild type and ac132 knockout repair mutants. We found that there were few infectious BVs produced in the Sf9 cells transfected with an ac132 knockout mutant (Fig. 5). Detection of total numbers of BVs present in the medium of transfection revealed that almost no BV was released up to 48 hpt. The titer of total BV released from the cells transfected by the ac132 knockout mutant increased less than 10-fold by 96 hpt. The BVs produced by the ac132 knockout mutant were defective in infectivity, since only a few single infected cells were viewed in the culture inoculated with supernatant from the transfection with vAc^{ac132ko-PH-gfp}. To explore the mechanism behind the phenotypic variations resulting from the deletion of ac132, the effects on viral DNA replication and late gene expression were evaluated by qPCR and by Western blot assays. A deletion of ac132 did not affect viral DNA replication (Fig. 6) and expression of two late genes, vp39 and e25 (Fig. 7). The expression of very late genes probably also was not affected by the ac132 deletion since EGFP that was expressed under the control of the promoter of the very late gene p10 appeared at the same time in the cells transfected by vAc^{ac132ko-PH-gfp} as in the cells transfected by the wt or ac132 knockout repair bacmids (Fig. 5A). Electron microscopy revealed nucleocapsids, virions, and virion-containing OBs in the nucleus and a few nucleocapsids budded out of the nucleus, in the cells transfected by the ac132 knockout bacmid vAc^ac132ko-PH, but the formation of OBs was delayed, and the numbers of enveloped nucleocapsids and virions containing multiple nucleocapsids were reduced in comparison within the cells transfected by the ac132 knockout repair bacmid. Similar phenotypic changes were also observed in earlier studies on ac92. Deletion of ac92 resulted in 99.99% reduction of BV production, and infectious BV was almost undetectable (47, 48). An ac92 deletion mutant produced OBs containing singly embedded nucleocapsids or even lacking nucleocapsids, in addition to normal ones (47, 48). In the earlier studies, deletion of ac92 did not affect viral DNA replication or late gene expression either. AC92 protein was shown to be associated with the envelopes of both BV and ODV. Since the deletion of ac132 or ac92 induced similar phenotypic changes, they may function cooperatively in virion assembly.

Another notable phenotypic change caused by ac132 deletion was that the formation of the VS was delayed. The VS is known to be the site of viral DNA replication and the subsequent packaging of DNA and assembly of nucleocapsids (9, 10). In Bombyx mori nucleopolyhedrovirus (BmNPV), it has been shown that IE1 and hr sequences cooperatively form an initial scaffold for the VS, and then, helicase, LEF3, and DNA-binding protein join in to form a subnuclear structure capable of recruiting other replication factors (44, 49, 50). The major capsid protein VP39, another structural protein, BV-ODV-E26, and a nonstructural protein, PP31, are also associated with the VS (45, 51). PP31 is a phosphorylated DNA-binding protein. Deletion of pp31 of BmNPV resulted in deficiency in the formation of VS and OBs and production of virions. Deletion of AcMNPV pp31 caused significant reduction in both BV production and transcription of a large number of viral genes (52). The role that AC132 plays in the formation of VS remains to be elucidated.

Besides the VS, baculoviruses generate another subnuclear structure, named the peristromal region (PR), which forms later than the VS and localizes to the periphery of it. The region is the site of intranuclear envelopment of nucleocapsids to produce ODVs (53). The transition from BV production to ODV production is hypothesized to relate to the formation of the peristromal region, which prevents nucleocapsids from reaching the nuclear membrane, so that the nucleocapsids assembled in the later stages are mostly trapped in this region and get enveloped by the intranuclear membranes (54). In this study, swirling hair-like structures were found in a section of a cell transfected by vAc^ac132ko-PH and localized outside the VS. The swirling hair-like structures were first observed in the cells infected by Heliothis armigera nucleopolyhedrovirus and thought to be involved in the intranuclear envelopment of nucleocapsids (55). However only a few nucleocapsids were observed to be associated with the swirling hair-like structures shown in Fig. 8Bb, implying that few nucleocapsids were assembled before PR formation in this case. That could account for the extremely low BV production in the culture transfected by the ac132 knockout mutant and suggested that ac132 is likely involved in assembly of nucleocapsids. The results from immunoprecipitation assays showed that P6.9 coimmunoprecipitated with AC132. P6.9 is the sole viral DNA-binding protein. During nucleocapsid assembly, viral DNA is condensed and packaged with P6.9 to form a DNA-protein core within the capsid (41, 56). At least a small part of P6.9 is present in the VS of infected Sf9 cells (42). In this study, a small part of AC132 was also observed in the center of the nucleus in infected cells as early as 12 hpi. These data imply that AC132 may be involved in early events of nucelocapsid assembly through interaction with P6.9.

Immunoconfocal microscopy revealed that AC132 formed a ring zone in the periphery of the nucleus in infected cells, which is similar to the intranuclear localization of AcMNPV E25, E26, AC76, AC92, and AC109 (34, 47, 48, 57–61). E25, E26, and AC76 are associated with virus-induced microvesicles in the nucleus (57–59). The intranuclear microvesicles are present in the periphery region and are considered to be precursors of the envelopes of ODV virions (62). E25, E26, and AC92 are viral envelope proteins (40, 47, 48, 57). Additional viral envelope proteins E18, ODV-E56, and ODV-E66 are also enriched in intranuclear microvesicles (43, 63, 64). Deletion of E25, E18, AC76, AC92, and AC109 resulted in either no or reduced ODV production (47, 48, 59–61, 65–67). These proteins are considered to be involved in envelopment of ODV. The mechanisms for occurrence of intranuclear microvesicles and envelopment of nucleocapsids have not been understood yet. They must involve interactions between the proteins of the nucleocapsid and the proteins on the intranuclear microvesicles or envelopes. In this study, AC132, as a nucleocapsid-associated protein, was found to interact with E18. This may represent a way in which AC132 functions in envelopment of ODV. Since AC132 is present in the nucleocapsids of both ODV and BV and E18 is an envelope protein of both ODV and BV, it may also affect envelopment of BV through interaction with E18.