Autographa californica Nucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

Abstract

Our previous study showed that the Autographa californica Nucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an α-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A coimmunoprecipitation assay demonstrated the dimerization of Ac76 by high-affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BVs and ODVs. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15 to 48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N terminus exposed to the cytosol and a C terminus hidden in the ER lumen.

INTRODUCTION

The family Baculoviridae encompasses a diverse group of insect-specific viruses that are characterized by rod-shaped, enveloped nucleocapsids with circular double-stranded DNA genomes (1, 2). Autographa californica Nucleopolyhedrovirus (AcMNPV), the archetype species of the Baculoviridae, Alphabaculovirus genus, undergoes a biphasic life cycle with the production of two virion phenotypes: the budded virion (BV) and the occlusion-derived virion (ODV) (3). Although the BV and ODV have a common nucleocapsid structure and carry the same genetic information, they differ in the source and composition of their envelopes, which parallel their different functional roles in the baculovirus life cycle (4–6). The BV is responsible for spreading infections between susceptible insect tissues and between cells in cell culture, and it acquires its envelope from the plasma membrane that is decorated with viral proteins by a strategy similar to other viruses that bud from the cell surface (7). The ODV can initiate primary infection in the midgut epithelium of infected insects and is required for the horizontal transmission of infection among insect hosts. ODV envelope proteins play biological roles in ODV occlusion and interaction with the midgut (5). The ODV is known to obtain its envelope from virus-induced intranuclear microvesicles (7), but the morphogenesis of these intranuclear microvesicles remains unclear. Although there has been some controversy about the source of the intranuclear microvesicles, considerable evidence supports the hypothesis that the formation of these microvesicles is the result of budding of discrete regions of the inner nuclear membrane (INM) into the nucleoplasm (5, 8).

Studies on the morphogenesis of intranuclear microvesicles have been focused predominantly on the integration of integral membrane proteins into the membrane of the endoplasmic reticulum (ER) and the sorting and trafficking pathways of these proteins from the ER membrane to the INM (8, 9). Two viral proteins, FP25K and BV/ODV-E26, along with a cellular protein, importin-α-16, participate in this pathway (9, 10). The ODV envelope protein ODV-E66 is an integral membrane protein (11), and its N-terminal region is sufficient to traffic fusion proteins from the ER to intranuclear microvesicles and the ODV envelope during AcMNPV infection (12), as well as target proteins to the INM in the absence of infection (10). This sequence has been termed an INM-sorting motif (INM-SM) that consists of two distinct features, (i) a hydrophobic domain of approximately 18 amino acids and (ii) within 4 to 8 amino acids from the end of the hydrophobic domain, a positively charged amino acid that is positioned in the cytoplasm or nucleoplasm (8, 10). Thus far, nine baculovirus proteins have been predicted to contain INM-SM-like sequences, and eight of these proteins have been shown to localize to the ODV envelope (8). Although the sorting and trafficking of ODV INM-directed integral membrane proteins from the ER to the nuclear membrane have been distinctly clarified (8, 9), the mechanism by which the nuclear membrane buds into nucleoplasm to form the intranuclear microvesicles remains unknown (8).

ac76 is a highly conserved gene that has been identified in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV (CuniNPV) (2). The ac76 gene encodes a small protein of 84 amino acids with a predicted transmembrane (TM) domain. The deletion of ac76 affects both BV and ODV formation. More importantly, an ac76 knockout virus failed to induce intranuclear microvesicle formation in infected cells (13). However, the function or mechanism by which Ac76 affects intranuclear microvesicle formation is unknown. In this study, we present evidence that Ac76 is an integral membrane protein and forms a stable homodimer by high-affinity self-association. Ac76 is a structural component of both the BV and ODV envelopes and is localized to the plasma membrane, the ER, the outer nuclear membrane (ONM) and inner nuclear membrane (INM), intranuclear microvesicles, and the ODV envelope in virus-infected cells. We further demonstrated that Ac76 contains an atypical INM-SM, which is sufficient to target fusion proteins to the intranuclear microvesicles and ODV envelope in virus-infected cells and to the INM in the absence of infection. Selective permeabilization showed that Ac76 is a type II integral membrane protein.

MATERIALS AND METHODS

Cells and viruses.

Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9 (Sf9) insect cells were cultured at 27°C in TNM-FH medium (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 30 μg/ml streptomycin. The wild type virus, vAcWT, was constructed by the insertion of the gene for polyhedrin and the enhanced green fluorescent protein (GFP) gene into the polyhedrin locus of the bacmid bMON14272 (13). Titers of BV were determined by using a 50% tissue culture infective dose (TCID₅₀) endpoint dilution assay in Sf9 cells (14).

Construction of viruses and plasmids.

To generate the hemagglutinin (HA)-tagged ac76 repair bacmid vAc76:HA, the fragment containing the ac76 promoter sequence and open reading frame (ORF) tagged with three copies of the HA coding sequence at the 3′ end was amplified from the AcMNPV bacmid bMON14272 by overlap PCR with primers ac76-US2 (13), 3×HA-R1 (5′-GGCGTAGTCGGGCACGTCGTAGGGGTAATCTATTGAGCTGGTATTTTT-3′), and 3×HA-R2 (5′-TCTAGATCAGGCGTAATCTGGGACGTCGTATGGGTAGGCGTAGTCGGGCACGTCGTATGGGTAGGCGTAGTCGGGCACGTC-3′ [the XbaI site is underlined]). The fragment containing the ac76 native poly(A) signal was also amplified with primers ac76-PA-F (5′-TCTAGATAAGTGTTTGTTAAAATGTCC-3′ [the XbaI site is underlined]) and ac76-DS1 (13). These two fragments were cloned into the pFB1-PH-GFP vector to generate the donor plasmid pFB1-Ac76:HA-PH-GFP. Then, the HA-tagged ac76 gene, as well as the polyhedrin and GFP genes, was inserted into the polyhedrin locus of the ac76 knockout bacmid bAc76KO (vAc^ac76-KO) (13) via site-specific transposition as previously described (13).

The pIB-Ac76:Flag and pIB-Ac76:HA plasmids were constructed to determine whether the self-association of Ac76 occurs in the absence of viral infection. A fragment containing the ac76 ORF fused with a Flag coding sequence at its 3′ end was amplified with primers Ac76:Flag-F (5′-GGTACCATGAATTTATATTTGTTGTTG-3′ [the KpnI site is underlined]) and Ac76:Flag-R (5′-GGATCCTCACTTATCGTCGTCATCCTTGTAATCATCTATTGAGCTGGTATTTTTG-3′ [the BamHI site is underlined]). The PCR product was digested with KpnI and BamHI and cloned into transient-expression vector pIB/V5-His (Invitrogen Life Technologies), which contains the OpMNPV ie2 immediate early promoter, to generate the pIB-Ac76:Flag expression plasmid. Meanwhile, the Ac76:HA fragment was digested from pFB1-Ac76:HA-PH-GFP and subcloned into pIB/V5-His to generate the pIB-Ac76:HA plasmid.

To determine whether amino acids 15 to 48 of Ac76 (termed SM_15-48) could function as an INM-SM, we generated a series of GFP:SM constructs in which SM_15-48, along with different lengths of flanking sequence, was fused to GFP at its N terminus. First, the GFP ORF lacking a termination codon was amplified from the vAcWT viral sequence with primers GFP-F (5′-AAGCTTCGCCACCATGGTGAGCAAG-3′ [the HindIII site is underlined]; CGCCACC was added as a Kozak sequence) and GFP-R (5′-GGTACCCTTGTACAGCTCGTCCATG-3′ [the KpnI site is underlined]), and the resulting fragment was cloned into pIB/V5-His to generate the pIB-GFP expression plasmid. Then, the SM_15-48 region of Ac76 was amplified with primers SM_15-48-F (5′-TTTTTGGTACCATGCTAGTGTATGACAAAAAG-3′ [the KpnI site is underlined]) and SM_15-48-R (5′-TTTTTGAATTCTTACTCGGTGTTCTTACTTATAATG-3′ [the EcoRI site is underlined]), and the fragment was digested with the corresponding enzymes and cloned into pIB-GFP to generate the pIB-GFP:SM_15-48 (pIB-GS_15-48) expression plasmid. Similarly, SM_1-48 of Ac76 was amplified with primers SM_1-48-F (5′-TTTTTGGTACCATGAATTTATATTTGTTG-3′ [the KpnI site is underlined]) and SM_15-48-R and cloned into pIB-GFP to generate the pIB-GFP:SM_1-48 (pIB-GS_1-48) expression plasmid. The Ac76:Flag fragment was removed from the pIB-Ac76:Flag plasmid with KpnI and BamHI and subcloned into pIB-GFP to generate the pIB-GFP:Ac76:Flag (pIB-G76Flag) plasmid.

To construct the recombinant viruses expressing GFP, GS_15-48, GS_1-48, and G76Flag under the control of the promoter of the gene for polyhedrin, these fragments were amplified and cloned into pFastBac1 to generate donor plasmids pFB1-GFP, pFB1-GS_15-48, pFB1-GS_1-48, and pFB1-G76Flag. The expression cassettes developed were transposed into the polyhedrin locus of bMON14272 to generate recombinant viruses vAcGFP_polh, vAcGS_15-48polh, vAcGS_1-48polh, and vAcG76Flag_polh.

To indicate the localization of the ER in Sf9 cells, a fragment containing the mCherry ORF fused with the signal peptide of AcMNPV chitinase at its N terminus and an ER retention signal (KDEL) at its C terminus (15–17) was amplified from the pCMV-mCherry plasmid (Clontech) with primers mCherry-KDEL-F (5′-GGATCCATGTTGTACAAATTGTTAAACGTTTTGTGGTTGGTCGCCGTTTCTAACGCGATTCCCGGCACGATGGTGAGCAAGGGCG-3′ [the BamHI site is underlined]) and mCherry-KDEL-R (5′-GAATTCTCACAGCTCGTCCTTCTCGCTCTTGTACAGCTCGTCCATGCC-3′ [the EcoRI site is underlined]). The PCR product was cloned into pIB/V5-His to generate the pIB-mCherry-KDEL (pIB-mCh-KDEL) plasmid.

All of the constructs were verified by PCR analysis and DNA sequencing. Bacmid DNAs and transient-expression plasmids were isolated with a Qiagen Large-Construct kit (Qiagen) or an E.Z.N.A. Plasmid Minikit I (Omega), and the concentrations were quantified by determining the optical density.

Analysis of viral propagation.

Sf9 cells (1 × 10⁶/35-mm dish) were transfected with 1 μg of the constructed bacmid (vAc76:HA or vAcWT) by using the Cellfectin II reagent (Invitrogen Life Technologies). At 96 h posttransfection (hpt), the BV-enriched culture supernatants were harvested and the cellular debris was removed by centrifugation at 3,000 × g for 10 min. Titers of BV were determined with a TCID₅₀ endpoint dilution assay in Sf9 cells (14).

Time course analysis of Ac76 expression.

Sf9 cells (1 × 10⁶) were infected with vAc76:HA at a multiplicity of infection (MOI) of 5. At the indicated time points postinfection, the cells were washed twice with phosphate-buffered saline (PBS) and collected by centrifugation at 1,000 × g for 5 min. The pelleted cells were resuspended in PBS with an equal volume of 2× protein sample buffer (0.25 M Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol, 10% 2-mercaptoethanol [2-ME], 0.02% bromophenol blue) and boiled at 100°C for 10 min. The samples were resolved by SDS-15% PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Millipore), and then probed with a mouse monoclonal anti-HA antibody (1:1,000; Abmart) according to the manufacturer's instructions. A goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (1:5,000; Pierce) was used as the secondary antibody. Proteins were visualized with an enhanced chemiluminescence system (ECL; Amersham Biosciences) according to the manufacturer's instructions.

For the detection of dimers and monomers, the cells infected with vAc76:HA were harvested at 72 h postinfection (hpi) and lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS [Thermo Scientific]). The protein concentration was determined with the BCA Protein Assay kit (Pierce) according to the manufacturer's instructions, and the SDS-PAGE and Western blot analyses were performed as described above.

Coimmunoprecipitation.

Sf9 cells (3 × 10⁶) were cotransfected with 2 μg of pIB-Ac76:HA and 2 μg of pIB-Ac76:Flag. To increase the transfection efficiency, a second cotransfection was performed 12 h after the first cotransfection. The cells were further cultivated and harvested at 48 hpt. After being washed once in PBS, the cells were lysed in RIPA buffer (Thermo Scientific) supplemented with 1 mM phenylmethylsulfonyl fluoride (Sigma) and 2 μg/ml complete EDTA-free protease inhibitor cocktail (Roche) and then incubated for 1 h while rotating. The lysate was then centrifuged at 12,000 × g for 15 min at 4°C. The extract was precleared by adding 50 μl of a 50% slurry of protein A/G agarose beads (Abmart), followed by incubation at 4°C for 1 h while rotating. The supernatant was transferred to a fresh 1.5-ml Eppendorf tube and mixed with 20 μl of a 50% slurry of protein A/G agarose beads conjugated to an anti-Flag antibody (Abmart). After incubation at 4°C for 1 h while rotating, the beads were collected by centrifugation at 2,000 × g for 3 min at 4°C, washed four times with 1 ml of RIPA buffer, and boiled in 25 μl of 2× PBS for 5 min. The dissolved immunoprecipitates, as well as the input cell lysates, were analyzed by Western blotting with mouse monoclonal anti-Flag (Abmart) or anti-HA (Abmart) antibodies as described above. Sf9 cells cotransfected with pIB-Ac76:Flag and pIB/V5-His were used as negative controls for the immunoprecipitation reactions.

BV and ODV purification.

BVs and ODVs were purified and fractionated into envelope and nucleocapsid fractions as previously described (18). Western blot analysis was performed as described above, with a mouse monoclonal anti-HA antibody or with one of the following primary antibodies, according to the manufacturer's instructions: monoclonal anti-GP64 AcC6 antibody (1:3,000; eBioscience), polyclonal anti-AcMNPV ODV-E25 antibody (1:2,000) (19), or polyclonal anti-AcMNPV VP39 antibody (1:1,000) (20). A donkey anti-rabbit HRP-conjugated antibody (1:10,000; Amersham Biosciences) or goat anti-mouse HRP-conjugated antibody (1:5,000; Pierce) was used as the secondary antibody.

IEM.

Cells were prepared for immunoelectron microscopy (IEM) with LR White resin (Ted Pella, Inc.) as previously described (21), with some modifications. Briefly, infected or transfected cells (1.5 × 10⁶) were pelleted at 800 × g for 10 min and fixed in a solution containing 1% paraformaldehyde, 0.5% glutaraldehyde, and 0.1 M PBS (pH 7.4) for 10 min at 4°C, followed by a second fixation step in a solution containing 2% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M PBS (pH 7.4) for 1 h at 4°C. The fixed pellets were washed three times with 0.1 M PBS (pH 7.4) for 10 min at 4°C and then dehydrated at −20°C with a 30, 50, 70, and 90% graded ethanol series; each ethanol step lasted for 45 min, and the 30% ethanol dehydration step occurred at 4°C. Samples were infiltrated with 40, 70, and 100% LR White-ethanol series at −20°C for 1 h per step, followed by infiltration with 100% LR White at −20°C overnight. The resin was then polymerized in gelatin capsules by UV irradiation (320 nm) at −20°C for 72 h and at room temperature (RT) for 24 h. Ultrathin sections were immunolabeled and stained as previously described (22). Immunolabeling was performed with a mouse monoclonal anti-HA antibody (1:50; Abmart) or mouse monoclonal anti-GFP antibody (1:50; Abmart), followed by incubation with goat anti-mouse IgG conjugated to 10-nm gold particles as the secondary antibody (1:50; Sigma). The samples were visualized with a JEOL JEM-1400 transmission electron microscope at an accelerating voltage of 120 kV.

Fractionation of integral membrane proteins with Triton X-114.

Sf9 cells (3 × 10⁶) were infected with vAc76:HA at an MOI of 5. At 60 hpi, the cells were collected. Detergent fractionations with Triton X-114 were performed as previously described (11). The resulting aqueous and detergent phases were analyzed by SDS-PAGE and Western blot analysis. Immunoblotting was performed as described above, with a mouse monoclonal anti-HA antibody or rabbit polyclonal anti-VP39 antibody.

Immunofluorescence.

Cells were processed for immunofluorescence microscopy as previously described (23), with some modifications. Briefly, Sf9 cells (3 × 10⁵) were seeded into a 35-mm glass-bottom culture dish (MatTek) and infected with vAcGS_15-48polh, vAcGS_1-48polh, vAcG76Flag_polh, or vAcGFP_polh at an MOI of 5 or cotransfected with 2 μg of pIB-mCh-KDEL and 2 μg of pIB-GS_15-48, pIB-GS_1-48, pIB-G76Flag, or pIB-GFP. At 60 hpi or 36 hpt, the supernatants were removed. After one wash with PBS, the cells were fixed with 3.7% paraformaldehyde for 10 min, washed three times in PBS for 5 min each, and permeabilized in 0.25% Triton X-100 in PBS for 10 min at RT. The cells were then blocked for 1 h in blocking buffer (PBS supplemented with 2% bovine serum albumin), incubated with the primary antibody for 1 h, washed three times in PBS for 5 min each, and then incubated with either donkey anti-mouse IgG conjugated to Alexa Fluor 555 (1:500; Invitrogen/Molecular Probes) or donkey anti-rabbit IgG conjugated to Alexa Fluor 647 (1:500; Invitrogen/Molecular Probes) as a secondary antibody for 1 h at RT, followed by three washes in PBS for 5 min each. Finally, the labeled cells were stained with 2 μg/ml Hoechst 33342 (Invitrogen/Molecular Probes) for 3 min at RT, followed by three washes with PBS prior to analysis. All images were collected with a Leica TCS SP5 confocal laser scanning microscope with the same parameter settings in each experiment. Three primary antibodies were used in this study, a mouse monoclonal anti-lamin B antibody ADL67 (1:200) (24), a mouse monoclonal anti-Flag antibody (1:200; Abmart), and a rabbit polyclonal anti-GFP antibody (1:100; Abmart).

Selective permeabilization.

To investigate the topology of Ac76, Sf9 cells (3 × 10⁵) were seeded into a 35-mm glass-bottom culture dish and transfected with 2 μg of pIB-G76Flag. Digitonin permeabilization was performed essentially as previously described (25). The transfected cells were fixed with 3.7% paraformaldehyde for 10 min at RT, washed three times, and incubated either with 20 μg/ml digitonin (semipermeabilization) or with Triton X-100 as described above (full permeabilization). The primary and secondary antibodies were used as described above. After viewing at least 30 fields, representative cells were imaged with a Leica TCS SP5 confocal laser scanning microscope with the same parameter settings.

RESULTS

Construction of the HA-tagged ac76 repair virus.

Although ac76 is required for intranuclear microvesicle formation, as shown by gene knockout (13), the expression and localization of Ac76 have not been determined because of the lack of a specific antibody. In this study, an HA epitope coding sequence was fused to the 3′ end of the ac76 gene in an ac76 repair construct so that Ac76 could be monitored with a commercially available monoclonal antibody to the HA epitope. A fragment containing the AcMNPV ac76 cassette fused with the 3×HA tag at the 3′ end, as well as the genes for GFP and polyhedrin, was inserted into the polyhedrin locus of bAc76KO (13) to create vAc76:HA (Fig. 1A).

FIG 1.

Construction of the HA-tagged Ac76 repair virus and analysis of its fitness. (A) Schematic representation of the polyhedrin (polh) and ac76 insertion loci of bAc76KO, vAc76:HA, and vAcWT. The genes are indicated on the linear representation of the AcMNPV genome. Arrows denote the protein-coding regions and orientations of transcription of the genes. vAc76:HA contains the ac76 ORF tagged with an in-frame 3×HA epitope sequence (gray triangle) at its 3′ end under the control of its native promoter. (B) Virus production of vAc76:HA and vAcWT. Sf9 cells were transfected with each bacmid. At 96 hpt, the supernatants containing the BVs were harvested and the titers were determined with TCID₅₀ assays. Each value represents the average of three independent transfections. Error bars indicate the standard deviations of the means.

To determine the effect of HA-tagged ac76 on viral propagation, Sf9 cells were transfected with vAc76:HA or vAcWT, and the titers of BV at 96 hpt were determined with a TCID₅₀ endpoint dilution assay. As shown in Fig. 1B, the titers of vAc76:HA and vAcWT were almost equivalent. In addition, electron microscopic analysis showed that cells transfected with vAc76:HA showed characteristics similar to those transfected with vAcWT (data not shown). These results indicated that vAc76:HA was able to rescue the defective phenotype of vAc76KO, as previously described for vAc76 repair (13). The HA-tagged ac76 did not appear to have any effect on viral replication relative to the vAcWT virus; thus, we used HA-tagged ac76 for subsequent analyses.

Ac76 is an integral membrane protein and forms a stable dimer.

To determine whether the HA tag fused to the C terminus of Ac76 was detectable and to analyze the temporal expression of Ac76 in virus-infected cells, vAc76:HA-infected cells were collected at designated time points, resolved by SDS-PAGE with 2-ME present, and analyzed by Western blotting with a mouse monoclonal anti-HA antibody. An immunoreactive band of ∼20 kDa was first detected at low levels at 18 hpi, peaked at 48 hpi, and persisted until 72 hpi (Fig. 2A), demonstrating that ac76 is a late gene, which is consistent with the results of the transcription analyses (13, 26). The apparent molecular mass of Ac76:HA was nearly double its predicted molecular mass, indicating that Ac76 might exist as a dimer. When the protein content was gradually increased, in addition to the high-abundance dimeric band of ∼20 kDa, a low-abundance band of ∼10 kDa, which is in accordance with the predicted molecular mass of Ac76:HA, was also detected and gradually increased with the increase in total protein (Fig. 2B). The above observation indicated that Ac76 might exist as a stable dimer that is resistant to SDS and thermal denaturation (100°C), with a trace amount of monomer present in virus-infected cells.

FIG 2.

Expression and self-association of Ac76. (A) Time course analysis of Ac76 expression. Sf9 cells were mock infected or infected with vAc76:HA at an MOI of 5. At the indicated time points, the cells were collected, resolved by SDS-15% PAGE, and subjected to Western blot analysis with a mouse monoclonal anti-HA antibody. Mi, mock-infected Sf9 cells. (B) Detection of Ac76 dimers and monomers. Sf9 cells were infected with vAc76:HA at an MOI of 5, and the cells were lysed at 72 hpi. The protein concentration of the extract was determined with the BCA Protein Assay kit and subjected to Western blot analysis with a mouse monoclonal anti-HA antibody. Sample concentrations are shown above the lanes. (C) Coimmunoprecipitation assay used to confirm the self-association of Ac76. Sf9 cells were cotransfected twice with pIB-Ac76:Flag and pIB-Ac76:HA. At 48 hpt, the cells were collected and lysed for immunoprecipitation with an anti-Flag antibody. The immunoblot was probed with an anti-Flag antibody to show the expression and immunoprecipitation of Ac76:Flag and with an anti-HA antibody to detect the expression and coimmunoprecipitation of Ac76:HA. Input, input cell lysates; IP, immunoprecipitation with anti-Flag antibody. The antibodies used for Western blot analysis are indicated on the left. (D) Distribution of Ac76 following phase separation. Sf9 cells were infected with vAc76:HA at an MOI of 5. At 72 hpi, the cells were collected, extracted with 1% Triton X-114, and analyzed by Western blotting with an anti-HA or anti-VP39 antibody. Lanes: C, cell lysates; D, detergent phase; A, aqueous phase. (E) Schematic of the heptad repeat model in the Ac76 TM helix and a putative model of Ac76 dimer formation. (a) L²⁶ and L³³ localized to position a and L²⁹, I³⁶, and I⁴³ localized to position d likely form a leucine/isoleucine zipper. (b) The putative helix-helix interaction of Ac76 with itself. The two monomers may be arranged to allow intermolecular contact at positions a and d, such that the leucine and isoleucine residues of one monomer align with the leucine and isoleucine residues of adjacent monomer. The molecular masses (in kilodaltons) of protein standards are indicated on the left of panels A and B and on the right of panels C and D.

Bioinformatics analysis shows that Ac76 contains a highly hydrophobic TM domain that is predicted to form an α-helical TM domain, according to PSIpred (http://bioinf.cs.ucl.ac.uk/psipred/). α-Helical TM proteins are able to form highly ordered oligomers via the TM region interactions in the lipid bilayer (27). Self-association of Ac76 was confirmed by coimmunoprecipitation. Herein, Sf9 cells were simultaneously transfected with plasmids pIB-Ac76:HA and pIB-Ac76:Flag. Ac76:HA was specifically copurified with Ac76:Flag when anti-Flag antibody affinity resin was used to precipitate the protein complexes (Fig. 2C). Notably, the immunoreactive band of ∼18 kDa rather than ∼10 kDa was detected with both the anti-Flag and anti-HA antibodies. This result indicated that Ac76:Flag and Ac76:HA could still form a stable heterodimer in the absence of viral infection.

A basic characteristic of all TM helices is the ability to engage with the translocon complex, thereby ensuring their proper integration into the membrane (28). To determine whether Ac76, which contains a TM helix, is an integral membrane protein, vAc76:HA-infected cells were extracted with Triton X-114 and analyzed by Western blotting with a mouse monoclonal anti-HA antibody. As expected, Ac76 was detected only in the detergent phase and was scarcely detected in the aqueous phase (Fig. 2D). In comparison, VP39, a major structural protein of the virus capsid that lacks TM domains, was found exclusively in the aqueous phase (Fig. 2D). These results demonstrated that Ac76 is an integral membrane protein.

Taken together, the results described above revealed that Ac76 is an integral membrane protein and forms a stable dimer by high-affinity self-association in virus-infected cells.

Ac76 is localized to both BVs and ODVs.

Because Ac76 affects both BV and ODV morphogenesis (13), it is necessary to determine whether Ac76 is associated with virions. Sf9 cells were infected with vAc76:HA, and the virions were purified and fractionated into envelope and nucleocapsid fractions. Ac76 was detected in the envelope fractions of both BVs and ODVs. As a control, the nucleocapsid protein VP39, the BV envelope-specific protein GP64, and the BV/ODV envelope-associated protein ODV-E25 were analyzed in the same samples, and all of these three proteins were detected only in the expected fractions (Fig. 3). These results indicated that Ac76 was associated with BVs and ODVs and localized to the envelope fractions of both BVs and ODVs.

FIG 3.

Western blot analysis of Ac76 from purified and fractionated virions. BVs and ODVs were purified from vAc76:HA-infected cell supernatants and pellets, respectively, and fractionated into envelope and nucleocapsid fractions. Western blot analysis was performed with an anti-HA antibody to detect HA-tagged Ac76, an anti-ODV-E25 antibody to detect the BV/ODV envelope-associated protein ODV-E25, an anti-VP39 antibody to detect the major capsid protein VP39, and an anti-GP64 antibody to detect the BV envelope-specific protein GP64. NC, nucleocapsid fraction; E, envelope fraction.

Ac76 is localized to the plasma membrane, ER, nuclear membrane, intranuclear microvesicles, and the ODV envelope.

To further determine the exact localization of Ac76 in virus-infected cells, the vAc76:HA-infected cells were harvested and processed for IEM. As expected, considerable numbers of gold particles were detected in both the ONM and the INM (Fig. 4A). In the cytoplasm, some gold particles were associated with intracellular cytoplasmic membranes (composed predominantly of the ER) condensed around the periphery of the nuclear membrane, and limited numbers of gold particles were observed associated with the plasma membrane (Fig. 4B). Within the cell nuclei, numerous gold particles were localized to the virus-induced intranuclear microvesicles in the ring zone (Fig. 4C). In addition, a few of the gold particles were localized to the mature ODV envelope (Fig. 4D). As a control, there were nearly no gold particles present in vAcWT-infected cells (data not shown). These results indicated that Ac76 was localized to the plasma membrane, the ER, the ONM, the INM, the intranuclear microvesicles, and the ODV envelope in virus-infected cells. Therefore, Ac76 is a BV/ODV integral envelope protein that has not been previously identified as such.

FIG 4.

Immunoelectron microscopy showing the distribution of Ac76:HA in vAc76:HA-infected cells. At 72 hpi, the cells were fixed, dehydrated, embedded in LR White resin, and processed for immunogold labeling with a mouse monoclonal anti-HA antibody. (A) Gold particles localized to the ONM (black diamonds) and INM (open diamonds). (B) Labeling of the plasma membrane (open triangles) and intracellular cytoplasmic membranes (black arrows). (C) Gold particles localized to the different sizes of microvesicles. (D) Labeling of the ODV envelope (black triangles). c, cytoplasm; n, nucleus; PM, plasma membrane. Scale bars, 500 nm.

Ac76 contains an atypical INM-SM.

A comparison of the predicted amino acid sequences showed that Ac76 contained a sequence similar to the INM-SM of ODV-E66 (13). However, the putative INM-SM-like sequence of Ac76 had some differences compared with other INM-SM-like sequences. To date, all of the predicted INM-SM-like sequences in baculovirus are located at the N terminus of the proteins, and the positively charged amino acids are close to the C-terminal end of the hydrophobic sequence (8). In contrast, in Ac76, the hydrophobic TM domain, which is composed of the 20 amino acids from residues 24 to 43, is located in the middle of the full-length protein sequence of 84 amino acids. Furthermore, the two positively charged amino acids within 4 to 8 amino acids of the end of the hydrophobic domain are located in the N-terminal flanking region. To determine whether amino acids 15 to 48 from Ac76 (SM_15-48) could function as an INM-SM, this sequence was fused in frame to the C terminus of GFP, and its transport and localization during transient expression following transfection or viral infection were traced by GFP autofluorescence. A transient-expression plasmid, pIB-GFP:SM_15-48 (pIB-GS_15-48), was generated, that expressed the fusion protein under the control of the OpMNPV ie2 gene promoter. In addition, a recombinant virus, vAcGS_15-48polh, was generated in which the GFP:SM fusion protein was highly expressed under the control of the polyhedrin promoter. To further investigate whether the N-terminal 14 amino acids have any effect on the localization of the putative INM-SM and to compare the function of INM-SM with full-length Ac76, two other fusion proteins, GFP:SM_1-48 (GS_1-48) and GFP:Ac76:Flag (G76Flag), were also expressed by transient expression or recombinant viruses. GFP alone was expressed in the same systems as a control. A schematic diagram of the SM fusion constructs is shown in Fig. 5A.

FIG 5.

Construction and expression of SM fusion proteins. (A) Schematic diagram of SM fusion expression cassettes. The predicted TM amino acid sequences are indicated by horizontal lines at the top of each sequence; stars indicate the positively charged amino acids flanking the TM sequence. The amino acid region between residues 15 and 48 of Ac76 was termed SM_15-48, and the amino acid region between residues 1 and 48 was termed SM_1-48. SM_15-48 and SM_1-48 were fused with GFP at their N termini; Ac76 was fused with GFP at its N terminus and a Flag tag at its C terminus. These SM fusion expression cassettes were cloned into the pIB/V5-His plasmid for transient expression or transposed into the polh locus of bMON14272 to generate recombinant viruses. (B) Transient expression of SM fusion proteins. (C) Expression of SM fusion proteins in virus-infected cells. Mock, mock-infected Sf9 cells.

Cells transfected with the selected expression plasmids were collected and analyzed by Western blotting with a mouse monoclonal anti-GFP antibody. Immunoreactive bands of ∼27, ∼31, ∼33, and ∼37 kDa were detected, and these bands were consistent with the predicted molecular masses of the monomer forms of GFP, GS_15-48, GS_1-48, and G76Flag, respectively (Fig. 5B). Correct expression of these fusion proteins in the recombinant viruses was also verified by Western blotting (Fig. 5C).

The localization of the SM fusion proteins in the absence of viral infection was determined by transient expression. Cells were transfected with the selected plasmids and examined for GFP-specific fluorescence at 36 hpt. The fluorescence of G76Flag colocalized with the ER marker mCh-KDEL in the cytoplasm and formed a smooth ring that was closely juxtaposed with the inner nuclear marker, lamin B, which underlies the nucleoplasmic face of the INM (Fig. 6A). The distribution patterns of both GS_1-48 (Fig. 6B) and GS_15-48 (Fig. 6C) were similar to that of G76Flag. The fluorescence of these three fusion proteins was slightly distributed along the plasma membrane in less than 5% of more than 200 cells observed (data not shown). As expected, GFP fluorescence for the GFP-only plasmid was uniformly distributed throughout the cytoplasm and nucleus (Fig. 6D). The localization of the SM fusion proteins in the ER, ONM, and INM was further confirmed by IEM with a mouse monoclonal anti-GFP antibody (Fig. 7).

FIG 6.

Subcellular localization of SM fusion proteins in cells during transient expression. Sf9 cells were cotransfected with mCh-KDEL and SM fusion constructs of G76Flag (A),S_1–48 (B), GS_15–48 (C), or GFP (D). At 36 hpt, the cells were fixed, incubated with anti-lamin B antibody (ADL67), and visualized with a donkey anti-mouse IgG conjugated to Alexa Fluor 647 (purple) as the secondary antibody. SM fusion proteins and mCh-KDEL were detected by visualizing the autofluorescence of GFP (GFP Auto, green) and mCherry (red), respectively. Hoechst 33342 was used to identify the nucleus and DNA-rich regions (blue).

FIG 7.

Immunogold labeling of SM fusion proteins in cells during transient expression. Sf9 cells were transfected with the SM fusion constructs of G76Flag (A), GS_1-48 (B), GS_15-48 (C), or GFP (D). At 36 hpt, the cells were prepared for immunogold labeling with a mouse monoclonal anti-GFP antibody as the primary antibody and goat anti-mouse IgG conjugated to 10-nm gold particles as the secondary antibody. c, cytoplasm; n, nucleus. Scale bars, 500 nm.

To further determine whether the putative INM-SM of Ac76 can target fusion proteins to the virus-induced intranuclear microvesicles and ODV envelopes during viral infection, the cells infected with the selected recombinant viruses were examined for GFP-specific fluorescence at 60 hpi. The fluorescence of G76Flag was concentrated as discrete foci within the nucleus, which should be the ring zone, the site of several morphogenic processes that are closely associated with ODV development, with a slight distribution along the nuclear membrane rim in close juxtaposition with the inner nuclear marker, lamin B. In addition, some G76Flag accumulated along the outer periphery of the nuclear membrane, which corresponds to the ER (Fig. 8A). This localization pattern was similar to that of Ac76-GFP, which was expressed under the control of the ac76 promoter in the ac76 knockout virus (13). In these two different experiments, the GFP fusion was at the N versus the C terminus, yet the results were similar. The distribution pattern of GS_1-48 was similar to that of G76Flag except that GS_1-48 showed a stronger smooth ring at the nuclear membrane and less discrete foci in the intranuclear ring zone (Fig. 8B). For GS_15-48, fluorescence was found to form a very strong nuclear rim. The accumulation of fluorescence along the outer periphery of the nuclear membrane or within the nucleus as discrete foci was also observed for GS_15-48, but the fluorescence level within the nucleus was much lower than that of G76Flag (Fig. 8C). Similar to the transfected cells, the fluorescence of these three fusion proteins was slightly distributed along the plasma membrane in less than 5% of more than 200 cells observed (data not shown). As expected, GFP fluorescence for the GFP-only virus was uniformly present throughout the cytoplasm and nucleus (Fig. 8D). Thus, the distribution patterns of the fusion proteins were due to the fused sequences selected from Ac76.

FIG 8.

Subcellular localization of SM fusion proteins in virus-infected cells. Sf9 cells were infected with vAcG76Flag_polh (A), vAcGS_1-48polh (B), vAcGS_15-48polh (C), or vAcGFP_polh (D) at an MOI of 5. At 60 hpi, the cells were permeabilized, incubated with a mouse monoclonal anti-lamin B antibody (ADL67), and visualized with donkey anti-mouse IgG conjugated to Alexa Fluor 555 (red) as the secondary antibody. SM fusion proteins were detected by GFP autofluorescence (GFP-Auto, green). Hoechst 33342 was used to identify the nucleus and DNA-rich regions (blue).

To further determine whether the SM fusion proteins were localized to the nuclear membrane, intranuclear microvesicles, and the ODV envelope, cells infected with the selected recombinant viruses were harvested at 60 hpi and processed for IEM. As expected, in cells expressing G76Flag, most of the gold particle-labeled virus-induced microvesicles. In addition, a few gold particles labeled the nuclear membrane and intracellular cytoplasmic membranes (composed predominantly of the ER) (Fig. 9A), which was consistent with the result of confocal microscopy (Fig. 8A). The labeling pattern of GS_1-48 was not significantly different from the labeling pattern of G76Flag, except for less label with intranuclear microvesicles and more association with the ER and nuclear membranes (Fig. 9B). For GS_15-48, significant numbers of gold particles were localized to the ONM, INM, and the intracellular cytoplasmic membranes. In addition, an abundance of gold particles was also found associated with intranuclear microvesicles and the ODV envelope (Fig. 9C). The localization of the fusion proteins to the ER and intranuclear microvesicles was consistent with the discrete fluorescent foci observed along the outer periphery of the nuclear membrane or within the nucleus by confocal microscopy (Fig. 8). As expected, when not linked to the sequences selected from Ac76, gold particle labeling revealed that GFP was uniformly distributed throughout the cytoplasm and nucleus (data not shown).

FIG 9.

Immunogold labeling of SM fusion proteins in virus-infected cells. Sf9 cells were infected with vAcG76Flag_polh (A), vAcGS_1-48polh (B), or vAcGS_15-48polh (C) at an MOI of 5. At 60 hpi, the cells were prepared for immunogold labeling as described in the legend to Fig. 7. The black arrowheads indicate the labeling of the ODV envelope. c, cytoplasm; n, nucleus; NM, nuclear membrane; m, microvesicles. Scale bars, 500 nm.

Taken together, these results demonstrated that SM_15-48, as well as SM_1-48, was sufficient to target the fusion proteins to the ER and the nuclear membranes in the absence of viral infection and to the nuclear membranes, virus-induced microvesicles, and ODV envelope in virus-infected cells. The localization patterns of GS_15-48 and GS_1-48 were similar to those of G76Flag and Ac76:HA; therefore, SM_15-48 is an atypical INM-SM.

Ac76 is a type II integral membrane protein.

There is a strong correlation between topology and function (28). In most cases, the topology of a TM protein is determined during the initial insertion of the polypeptide chain into the membrane (28). The positively charged amino acid residues in the 15 residues flanking the TM domain greatly affect the orientation (29, 30). Ac76 contains two positively charged lysines in the 15 residues flanking both sides of the TM helix. To determine the topology of Ac76, cells transfected with the plasmid pIB-G76Flag were selectively permeabilized with Triton X-100 (full permeabilization) or digitonin (semipermeabilization) and processed for immunofluorescence analyses with antibodies against lamin B, GFP, or Flag. As expected, the lamin B antibody was not bound when the cells were treated with digitonin, which selectively permeabilizes the plasma membrane and leaves the ER and nuclear envelope intact (Fig. 10A). In contrast, lamin B was clearly visible when the cells were fully permeabilized with Triton X-100, which resulted in perforation of the plasma membrane, ER, and nuclear envelope (Fig. 10A). Antibodies to GFP showed the same distribution pattern as autofluorescence in either fully permeabilized or semipermeabilized cells (Fig. 10A), indicating that the GFP tag (which is on the N terminus of Ac76) was exposed to the cytoplasmic face of the ER. However, the Flag tag (which is on the C terminus of Ac76) was detectable only in fully permeabilized cells and exhibited a distribution pattern similar to that of both the anti-GFP antibody and autofluorescence (Fig. 10B), indicating that the Flag tag was hidden in the lumen of the ER. Because the GFP-tagged N terminus was exposed to the cytoplasmic face of the ER and the Flag-tagged C terminus was hidden in the lumen of the ER, Ac76 is a type II integral membrane protein (31). A schematic of the orientation of Ac76 in the ER, ONM, and INM is shown in Fig. 10C.

FIG 10.

Topology of Ac76. Sf9 cells were transfected with pIB-G76Flag. At 36 hpt, the cells were incubated either with 20 μg/ml digitonin for semipermeabilization or with 0.25% Triton X-100 for full permeabilization. The cells were then probed with a mouse monoclonal anti-lamin B antibody (ADL67) and a rabbit polyclonal anti-GFP antibody (A) or a mouse monoclonal anti-Flag antibody and a rabbit polyclonal anti-GFP antibody (B) as the primary antibodies. Donkey anti-mouse IgG conjugated to Alexa Fluor 555 (red) and donkey anti-rabbit IgG conjugated to Alexa Fluor 647 (purple) were used as the secondary antibodies. G76Flag was detected by GFP autofluorescence. Hoechst 33342 was used to identify the nucleus and DNA-rich regions (blue). (C) Schematic drawing of the orientation of Ac76 in the ER, ONM, and INM. NPC, nuclear pore complex; N, N terminus; C, C terminus.

DISCUSSION

Our previous studies have shown that ac76 is involved in intranuclear microvesicle formation (13), but the exact role of Ac76, particularly whether the defect in intranuclear microvesicle formation in the ac76-null virus results from the lack of a structural protein involved directly in this process or of pleiotropic effects, remains unknown. The characterization of the expression, distribution, and topology of Ac76 may help to shed light on the mechanism by which Ac76 is involved in intranuclear microvesicle morphogenesis. In the present study, we demonstrated that Ac76 is an integral membrane protein, associated with both the BV and ODV envelopes, and localized to the plasma membrane, ER, ONM, INM, intranuclear microvesicles, and ODV envelope. The 34 amino acid residues at positions 15 to 48 of Ac76 indicate an atypical INM-SM and are sufficient to target fusion proteins to the above-mentioned positions. Our data indicate that the N terminus of Ac76 is exposed to the cytoplasm, and the C terminus is hidden in the lumen of the ER or nuclear envelope and therefore that Ac76 represents a type II integral membrane protein.

Ac76 contains a predicted α-helical TM domain, which might engage with the translocon complex and integrate into the lipid membrane to form an integral membrane protein (28). As expected, Ac76 was fractionated into the detergent phase of a Triton X-114 extraction, demonstrating that Ac76 is an integral membrane protein. The apparent molecular mass of most of the Ac76:HA detected by Western blotting was nearly double the predicted molecular mass, with a trace amount of protein matching the predicted molecular mass. Although SDS is a very strong denaturing agent that can break most weak interactions and denature some membrane proteins, many small membrane proteins maintain their oligomeric structure in the presence of SDS and boiling (32), e.g., dimeric glycophorin A (33) and pentameric phospholamban (34). The migration of the TM peptides at an apparent molecular weight greater than 2-fold the predicted molecular weights on SDS-PAGE is often considered evidence of high-affinity self-association (35). Thus, Ac76 might exist as a dimer in virus-infected cells by high-affinity self-association that is resistant to SDS, 2-ME, and thermal denaturation (100°C). Self-association of Ac76 was confirmed by coimmunoprecipitation by SDS-PAGE with the reducing agent 2-ME in all samples (Fig. 2C). It is worth noting that Ac76:Flag and Ac76:HA could still form a stable dimer in the absence of viral infection. However, SM fusion proteins were detected at the predicted molecular masses rather than the dimeric sizes by SDS-PAGE. This may be owing to the large size of these chimeric proteins, as all of these proteins are much larger than the small proteins, which are usually less than 200 amino acids in length (36). Thus, their self-association may be unstable and can be broken by SDS. Ac76 does not contain any cysteine residues, and therefore, dimer formation via disulfide bonds is not possible. TM α-helices can associate with one another in the lipid bilayer (27), and this association is important to the folding and oligomerization of many integral membrane proteins (37, 38). Ac76 contains a TM α-helix; thus, its dimerization might be through helix-helix interactions. The forms of TM helix-helix interactions include mainly the leucine zipper, polar residues, and the GXXXG motif (35, 39). According to the heptad repeat model (abcdefg)_n in the TM helix of phospholamban, Leu³⁷, Leu⁴⁴, and Leu⁵¹ occupy the a position and Ile⁴⁰ and Ile⁴⁷ occupy the d position in the motif; these residues create a leucine/isoleucine zipper (34, 40). The leucine- and isoleucine-rich sequence ²⁶LYLLILFLVFIIVSPAII⁴³ in the TM helix of Ac76 shows similar characteristics, as L²⁶ and L³³ are localized to position a and L²⁹, I³⁶, and I⁴³ are localized to position d (Fig. 2E); thus, this sequence likely forms a leucine/isoleucine zipper to mediate the helix-helix interaction of Ac76 with itself (Fig. 2E). In addition, polar residue S³⁹ in the helix might form an interhelical hydrogen bond (41) to strengthen the helix association by cooperating with the leucine/isoleucine zipper (42). Point mutation studies are needed to determine which amino acid residues are involved in the dimerization of Ac76.

Immunogold-labeled Ac76 was localized to the ER, ONM, INM, intranuclear microvesicles, and ODV envelope in infected cells. This localization pattern is very similar to that of another ODV envelope protein, ODV-E66 (12, 21). ODV-E66 is an integral membrane protein (11) and contains an INM-SM that is sufficient to traffic fusion proteins from the ER to the INM or ODV envelope (10, 12). These trafficking mechanisms have been identified by using ODV-E66 as a model (8). Thus far, nine baculovirus proteins containing INM-SM-like sequences have been revealed by sequence comparison, and all of the INM-SM-like sequences are located in the N termini of these proteins, with positively charged amino acids close to the C-terminal end of the hydrophobic sequence (8). A comparison of the predicted amino acid sequence showed that Ac76 contained a sequence pattern similar to the INM-SM of ODV-E66 (13). However, the putative INM-SM-like sequence of Ac76 is located in the middle of the protein and the positively charged lysine residues within 4 to 8 amino acids from the end of the hydrophobic sequence are in the N-terminal flanking region. To investigate whether this sequence functions as an INM-SM and localizes Ac76 to the ER, ONM, INM, intranuclear microvesicles, and ODV envelope, a series of fusion proteins were expressed during viral infection or in the absence of viral infection. Our results showed that SM_15-48 was sufficient to direct fusion proteins to the above-mentioned positions in infected cells. In the absence of infection, the fusion proteins were localized along the ER membrane and accumulated at the INM. These results demonstrated that the 34 amino acids located in the middle of Ac76 function as an atypical INM-SM, which had not been previously discerned.

The INM-SM plays a fundamental role in the trafficking of baculovirus integral membrane proteins from the ER to the ODV envelope (8). A review of the amino acid sequences of the ODV integral envelope proteins with TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) revealed that, in addition to the nine proteins that contain typical INM-SM-like sequences, there are some other proteins that contain a TM helix (helices) associated with positively charged amino acids close to the N- or C-terminal end of the TM helix, including ODV-E18, PIF5, PIF6, Ac108, Ac78, F-protein, and P74 (Fig. 11A). Considering the localization of these proteins (43–49), along with the identification of the INM-SM-like sequence of residues 15 to 48 of Ac76, we predicted that these proteins could also contain atypical INM-SM-like sequences. Thus, the INM-SM of baculovirus should be subdivided into two types, (i) the classical type, in which the TM domain is located at the N terminus of a protein with associated positively charged amino acids close to the C-terminal end of the TM domain, such as ODV-E66, PIF1, PIF2, PIF3, PIF4, Ac83, Ac91, and Ac150, except for ODV-E25, which lacks the positively charged residues, and (ii) the atypical type, in which the TM domain can be located in the middle or nearly at the C terminus of the protein, with associated positively charged amino acids close to the N- and/or C-terminal end of the TM domain, such as Ac76, ODV-E18, PIF5, PIF6, Ac108, Ac78, F-protein, and P74.

FIG 11.

Sequence analysis and orientation of the ODV integral envelope proteins. (A) The TM helices and flanking sequence of some ODV integral envelope proteins. The TM helices are blue. The numbering indicates the start and end sites of the TM helices. The positively charged amino acids flanking the TM helices are red. aa, amino acids. (B) Model of the orientation of the major ODV integral envelope proteins with predicted INM-SMs in the ODV envelope. This drawing is in the context of AcMNPV. The blue curves represent the TM helices of these proteins. The different protein sizes correspond to their molecular masses. Ac76 and ODV-E18 (43) are shown as homodimers in the envelope. N, N terminus; C, C terminus.

A fundamental characteristic of all of the integral membrane proteins is their topology, including the number of times they span the membrane and their overall orientation relative to the membrane (28). Identification of Ac76 topology will provide insight into its function in microvesicle formation. All integral membrane proteins are initially assembled in the ER, where the TM domains are integrated into the membrane, and the final topology is determined (31). Charge differences in the 15 residues flanking both sides of the TM domain have been proposed to determine the orientation (30). The positively charged amino acid residues in the flanking regions greatly affect the orientation (29). Ac76 contains two positively charged lysine residues in the 15 residues flanking both sides of the TM helix, and selective permeabilization showed that Ac76 is a type II integral membrane protein with its N terminus facing the cytosol and its C terminus facing the ER lumen. The positive charge distribution pattern of Ac76 is similar to that of asialoglycoprotein (50), which also has a type II topology. These results are consistent with the statistical and experimental studies that showed that a membrane protein with an N-terminal signal composed in part of positive charges is oriented with its N terminus facing the cytosol (51, 52).

The topology of integral membrane proteins is determined by a combination of local sequence features, including hydrophobicity and flanking charges, and global constraints, including the length and structure of the N-terminal domain (31). The overarching determinant of integral membrane protein topology is the positive-inside rule, in which positive charges flanking a TM domain are statistically significantly most often found in the cytoplasm (30, 31, 53). According to these factors, combined with the known characteristics of some ODV envelope proteins, we suggest that the topology of other ODV integral envelope proteins could also be predicted. Previous studies have shown that ODV-E66 is a type I integral membrane protein (10). The positively charged lysine residues of ODV-E66 that are close to the C-terminal end of the TM domain are exposed in the cytoplasm or nucleoplasm, which is in agreement with the positive-inside rule. The ODV envelope proteins containing INM-SM-like sequences similar to that of ODV-E66, such as ODV-E25, PIF1, PIF2, PIF3, PIF4, Ac83, Ac91, and Ac150, should also be type I integral membrane proteins. ODV-E18 and Ac108, small proteins that contain internal TM helices with more positive charges close to the N-terminal end, could be type II integral membrane proteins obeying the positive-inside rule. Ac78, a small protein containing an internal TM helix near the C terminus with a positively charged amino acid close to the C-terminal end, could be a type I integral membrane protein. PIF5 and PIF6, which contain long N-terminal sequences that precede the TM helix with equal positively charged residues flanking both sides, could present a type II topology (31). On the basis of the examination of natural proteins, integral membrane proteins with approximately 50 to 60 residues upstream of the TM domain favor type II topology (31, 54). P74, a large protein that contains two TM helices at the C terminus and a lengthy N terminus, which strongly disfavors its N terminus translocation into the lumen of ER, most likely leaves the bulk of its N terminus in the cytosol. The double TM domain motif should form a “hairpin” or folded TM domain in the membranes (5). F-protein, which contains an internal TM helix near the C terminus with a long cytoplasmic tail domain close to the C-terminal end of the TM helix, has been shown to be a type I integral membrane protein with the bulk of its N terminus hidden in the ER lumen (55, 56). INM-SM-directed proteins are trafficked from the ER to the ONM and INM. With the budding of the INM into the nucleoplasm to form intranuclear microvesicles and the further envelopment of nucleocapsids into microvesicles to form ODV, proteins containing the INM-SM are finally localized to the ODV envelope (8). Although the details of ODV envelopment remain unknown, the orientations of the ODV integral envelope proteins could still be speculated on the basis of their topology in the ER and the known functions of some of these proteins. P74, ODV-E25, PIF1, PIF2, and PIF3 have been reported to be on the ODV surface (57, 58), and all of these proteins are predicted to be exposed to the cytoplasmic face of the ER. Thus, membrane proteins oriented toward the cytoplasmic face of the ER are consequently exposed on the ODV surface. According to this rule, ODV-E66, PIF4, PIF5, PIF6, Ac83, Ac91, and Ac150 should be exposed on the ODV surface, and F-protein and Ac78 should be oriented on the inner side of the ODV envelope. For Ac76, ODV-E18, and Ac108, the N terminus is exposed on the ODV surface, and the C terminus is toward the inner side of the ODV envelope (Fig. 11B). The orientations of these proteins are consistent with their known functions. PIF1 and PIF2, as well as P74, play fundamental roles in ODV binding to insect midgut cells (59); therefore, they should be on the ODV surface. ODV-E66 has been shown to have chondroitinase activity (60) and plays an important role in the oral infection process (61). This protein also ought to be on the ODV surface to digest the chondroitin sulfate barrier of the peritrophic matrix of the host midgut, thus facilitating viral infection of epithelial cells (60). Ac83 is the only component with a chitin-binding domain in the PIF complex on the ODV envelope (46, 62). Deletion of the chitin-binding domain of Ac83 results in the failure of AcMNPV oral infection of Trichoplusia ni larvae. Therefore, Ac83 may be on the ODV surface and may be involved in ODV binding to the peritrophic membrane and/or other chitin-containing insect tissues (62). As the topology of integral membrane proteins is highly complex and easily influenced by a combination of factors, our illustration of the ODV integral envelope proteins is speculative and much work remains to be performed to determine the orientation of these proteins.

Semipermeabilization assays showed that Ac76 is a type II integral membrane protein. In this assay, the high concentrations of cholesterol in the plasma membrane of a cell allow the plasma membrane to be selectively permeabilized by low concentrations of digitonin, leaving the ER/nuclear envelope intact (8, 63). It is noteworthy that the fluorescent signal of lamin B was visible in most of pIB-G76Flag-transfected cells, even with digitonin concentrations as low as 30 μg/ml (data not shown), the concentration at which Braunagel et al. (10) performed semipermeabilization assays with Sf9 cells expressing 33-GFP, a chimeric protein containing the N-terminal 33 amino acids of ODV-E66 fused to GFP. When the concentration was reduced to 20 μg/ml, digitonin selectively permeabilized the plasma membrane and lamin B was undetectable. This result suggested that in cells expressing G76Flag, the concentration of cholesterol in the ER/nuclear envelope might be higher than that in normal cells. The lipid raft is a cholesterol-rich microdomain on the cell surface (64, 65) and participates in cell surface receptor-mediated signal transduction, protein sorting, and membrane transport (66, 67). One specialized type of lipid raft is known as caveolae (67). A large meshwork of caveolin oligomers act synergistically with cholesterol to distort the membrane and drive the invagination of a subset of lipid rafts to form caveolae (68). Lipid rafts are implicated in the entry and egress of many virus species (69, 70). Influenza A virus ion channel protein M2, which contains a cholesterol recognition and/or interaction amino acid consensus CRAC motif [-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-, where X denotes any amino acid] (71) in its highly conserved amphipathic helix, is localized to the neck of budding virions, and mutation of the M2 amphipathic helix results in the failure of the virus to undergo membrane scission and virion release (72). A recent study showed that cell surface cholesterol plays important roles in the internalization of viral particles bearing GP64 into mammalian cells through direct interaction with GP64 (73). Lipid rafts exist not only on the plasma membrane but also on the ER, ONM, and other intracellular membranes (74–79). There are still no reports about the distribution of lipid rafts in the INM. Baculovirus infection has been shown to induce remodeling of the lipid composition of the nuclear envelope by increasing its cholesterol concentration, and the ODV envelope is densely packed with cholesterol (8). This suggests that some cholesterol-binding viral proteins are transported from the ER to the ONM and INM, which leads to an enrichment of cholesterol in the INM, thereby forming lipid raft platforms that might be the source of intranuclear microvesicles. We found that Ac76 contains the potential CRAC motif LVYDKK located in the pre-TM domain. Ac76 is a type II integral membrane protein and exists as a stable dimer with the predicted CRAC motif facing the nucleoplasmic leaflet of the INM. We propose a model in which Ac76 could be involved in remodeling of the INM by enriching its cholesterol concentration in the nucleoplasmic leaflet, which could, in turn, result in bending of the INM toward the nucleoplasm to form the intranuclear microvesicles. Further studies of Ac76 are required to test this model.