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Baculoviruses produce two progeny phenotypes during their replication cycles. The occlusion-derived virus (ODV) is responsible for initiating primary infection in the larval midgut, and the budded virus (BV) phenotype is responsible for the secondary infection. The proteomics of several baculovirus ODVs have been revealed, but so far, no extensive analysis of BV-associated proteins has been conducted. In this study, the protein composition of the BV of Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculoviruses, was analyzed by various mass spectrometry (MS) techniques, including liquid chromatography-triple quadrupole linear ion trap (LC-Qtrap), liquid chromatography-quadrupole time of flight (LC-Q-TOF), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). SDS-PAGE and MALDI-TOF analyses showed that the three most abundant proteins of the AcMNPV BV were GP64, VP39, and P6.9. A total of 34 viral proteins associated with the AcMNPV BV were identified by the indicated methods. Thirteen of these proteins, PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, polyhedron envelope protein (PEP), AC132, ODV-E18, and ODV-E56, were identified for the first time to be BV-associated proteins. Western blot analyses showed that ODV-E18 and ODV-E25, which were previously thought to be ODV-specific proteins, were also present in the envelop fraction of BV. In addition, 11 cellular proteins were found to be associated with the AcMNPV BV by both LC-Qtrap and LC-Q-TOF analyses. Interestingly, seven of these proteins were also identified in other enveloped viruses, suggesting that many enveloped viruses may commonly utilize certain conserved cellular pathways.
During the baculovirus infection cycle, two progeny virion phenotypes are produced, the budded virus (BV) and the occlusion-derived virus (ODV). The two phenotypes are genotypically identical, but each has characteristic structural components to accommodate its respective functions (37). ODVs are responsible for initiating primary infections in the midgut epithelial cells of susceptible hosts, while BVs are responsible for spreading the virus among cells and tissues in the host (48). At the early stage of the infection, the nucleocapsids are transported through the nuclear membranes and migrate across the cytosol to the cell membrane, where the virus buds out (BV). BVs acquire an envelope of virus-encoded proteins as they bud out of the cell membrane (50). In the late stages of the infection, the nucleocapsids within the nucleus are enveloped with a lipid bilayer that resembles, but is not identical to, the inner nuclear membrane (8, 37). Therefore, it is generally believed that the BV and ODV share the same components of the nucleocapsid but differ in their envelopes. However, detailed structures of the BV and ODV have not been elucidated totally.
The availability of genome sequences has facilitated proteomic analyses of baculoviruses, especially by mass spectrometry (MS)-based techniques. Since Braunagel et al. first reported the ODV components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (11), the ODV components of three other viruses, Culex nigripalpus nucleopolyhedrovirus (CuniNPV) (81), Helicoverpa armigera nucleopolyhedrovirus (HearNPV) (21), and Bombyx mori nucleopolyhedrovirus (BmNPV) (62), have also been reported. These data shed light on ODV structure and assembly, but in comparison, little is known about the structural composition of the BV.
In this paper, we described the first comprehensive proteomic analysis of baculovirus BV-associated proteins. By using various complementary mass spectrometry techniques, a total of 34 viral proteins and 11 host proteins were identified in the AcMNPV BV. This information will help us to understand the BV structure as well as shed light on baculovirus infection pathways.
Spodoptera frugiperda (Sf9) cells were maintained at 27°C in Grace's medium (Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (Invitrogen). BVs were purified from the supernatant of AcMNPV (strain C6)-infected Sf9 cells as described previously by Braunagel and Summers (12), with modifications. Briefly, Sf9 cells were infected with AcMNPV at a multiplicity of infection (MOI) of 1. At 72 h postinfection (p.i.), the supernatants were collected, and the cell debris was removed by centrifugation at 2,000 × g for 10 min. The supernatants were then filtered through a 0.45-μm filter (Millipore) and centrifuged at 72,000 × g for 90 min (Beckman SW28 rotor at 20,000 rpm at 4°C) on 5 ml of a 25% (wt/vol) sucrose cushion in 0.1× Tris-EDTA (TE) (pH 7.4). The virion pellet was resuspended in 0.1× TE, layered onto sucrose layers containing 6 ml of 60% sucrose and 6 ml of 25% sucrose in 0.1× TE, and centrifuged at 71,250 × g for 120 min (Beckman SW40 rotor at 20,000 rpm at 4°C). The virus band between the two layers was collected, diluted in 0.1× TE, and centrifuged at 71,250 × g for 60 min (Beckman SW40 rotor at 20,000 rpm at 4°C). The pellet was resuspended in 0.1× TE containing a proteinase inhibitor cocktail (EDTA-free; Roche), a sample of which was immediately negatively stained for electron microscopy (Tecnai G2; FEI), and the rest was stored at −80°C for subsequent SDS-PAGE and MS analyses.
Polyhedra were purified from infected larvae as previously described (21). ODVs were released by alkaline treatment and purified on continuous sucrose gradients (12). The purified BV and ODV were further fractionated into envelope and nucleocapsid components (38).
Proteins from purified AcMNPV BVs were separated on 12% SDS-PAGE gels and stained with a colloidal blue staining kit (Invitrogen, CA). Protein bands or regions were excised from the gel and subjected to in-gel trypsin digestion as previously described (21), and peptides were analyzed by peptide mass fingerprinting (PMF) techniques using a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) instrument (Voyager DE STR; Applied Biosystems Inc.) or by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) techniques using a triple-quadrupole linear ion trap (Qtrap) 3200 system (Applied Biosystems Inc.) or a Qstar Elite hybrid LC-MS/MS system (Applied Biosystems Inc.).
Upon PMF analysis by MALDI-TOF, the instrument was operated in the positive-ion reflector mode with an accelerating voltage of 20 kV and a delayed extraction of 150 ns, and typically, 200 scans were averaged to generate each spectrum with a 3.0-m flight tube. Mass calibration was achieved by using calibration mixtures provided by Applied Biosystems as an external standard. Data analysis of PMFs was performed by using a Mascot (Matrix Science) search against the National Center for Biotechnology Information nr database.
For electrospray ionization (ESI)-based LC-MS/MS analysis on the Qtrap 3200 instrument (LC-Qtrap), capillary reversed-phase high-performance liquid chromatography (HPLC) separation of protein digests was performed with a 100-μm by 150-mm C18 column packed in-house with C18 beads (YMC ODS-AQ, Waters), and the mobile phase consisted of two components, with component A being 2% acetonitrile with 0.1% acetic acid and component B being 98% acetonitrile with 0.1% acetic acid, operated at a flow rate of 300 nl/min with a gradient starting from 5% component B held for 5 min and then programmed to 60% component B in 40 min and held for another 5 min. MS/MS analysis was operated in a data-dependent mode with optimized nanospray parameters as follows: ion spray (IS) voltage of 2,200 V, declustering potential of 60 V, and temperature of 100°C. The precursor ion's range was chosen from m/z 400 to m/z 1,600, and the product ion's range was chosen from m/z 50 to m/z 1,600. The ion source gas I (GSI), gas II (GSII), curtain gas (CUR), and temperature of GSII were set at 40, 5, 30, and 175°C, respectively.
Upon ESI-based analysis using the Qstar Elite hybrid LC-MS/MS system (LC-quadrupole time of flight [Q-TOF]), capillary reversed-phase HPLC separation of the sample was performed as described above for the Qtrap instrument. MS/MS analysis was operated in a data-dependent mode with the optimized nanospray parameters as follows: IS voltage of 1,800 V, declustering potential of 115 V, and temperature of 100°C. The precursor ion's range was chosen from m/z 400 to m/z 1,600, and the product ion's range was chosen from m/z 50 to m/z 1,600.The temperature of ion source GSI was set at 80°C.
MS/MS data generated by Qtrap and Qstar were analyzed by ProteinPilot software (version 2.0; Applied Biosystems) with several constructed databases, including an AcMNPV open reading frame (ORF) database, a lepidopteran protein database (downloaded from GenBank), and a contaminant database (supplied by Applied Biosystems). By using ProteinPilot software, protein identities with a high confidence equal to or more than 95% (P ≤ 0.05) were considered significant.
Purified BVs and ODVs, as well as their nucleocapsid and envelope fractions, were separated by SDS-PAGE and transferred onto Hybond-N membranes (Amersham) by semidry electrophoresis transfer. Anti-ODV-E18 and anti-ODV-E25 polyclonal antibodies, generated in rabbits, were used as primary antibodies, and alkaline phosphatase-conjugated immunoglobulin G (SABC, China) was used as the secondary antibody. The signal was detected with a 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (BCIP)-nitroblue tetrazolium kit (SABC, China). Polyclonal anti-VP39, anti-GP64, and anti-ODV-E66 antibodies were used as controls for nucleocapsid-, BV envelope-, and ODV envelope-specific proteins, respectively.
It is critical to obtain high-quality purified BVs for MS analysis. Purified BVs were generated from the supernatant of infected Sf9 cells by methods described in Materials and Methods. Electron microscopy of negatively stained BVs confirmed that the BVs generated were morphologically pure and intact (data not shown). Later, when LC-Qtrap and LC-Q-TOF MS/MS data for the BV proteins were analyzed with a contaminant database (supplied by Applied Biosystems), no bovine serum albumin (the most abundant protein in the cell culture supernatant) was identified (data not shown). These results further confirmed the purity of BVs.
Purified BVs were suspended in 0.1× TE buffer, and the BV-associated proteins were separated on 12% SDS-PAGE gels and stained with colloidal blue. As shown in Fig. Fig.1,1, three bands, M1, M2, and M3, were the most intensely stained and abundant AcMNPV BV proteins. These three bands were subjected to in-gel trypsin digestion and MALDI-TOF MS analysis, which revealed that M1, M2, and M3 were the envelope protein GP64 (AC128), the capsid protein VP39 (AC89), and the DNA-binding protein P6.9 (AC100), respectively (data not shown).
The total BV proteins on the gel were divided into 14 regions (L1 to L14) (Fig. (Fig.1)1) and subjected to in-gel trypsin digestion and LC-Qtrap MS analysis (data not shown). A total of 22 viral proteins were identified to be associated with the AcMNPV BV by this method (Table (Table1).1). These proteins include protein tyrosine phosphatase (PTP, AC1), P78/83 (AC9), F protein (AC23), V-ubiquitin (AC35), PP31 (AC36), VP1054 (AC54), AC58/59, AC66, IAP-2 (AC71), AC73, VLF-1 (AC77), VP39 (AC89), ODV-E25 (AC94), BV/ODV-C42 (AC101), VP80 (AC104), AC109, AC114, GP64 (AC128), AC132, ME53 (AC139), 49K (AC142), and BV/ODV-EC27 (AC144). Most proteins were detected in discrete bands around their expected molecular masses (Table (Table11 and Fig. Fig.1).1). However, some proteins were identified in more than one gel band, including V-ubiquitin, VP39, VP80, BV/ODV-C42, GP64, and BV/ODV-EC27 (Table (Table11).
The entire gel regions containing virion proteins excluding the three most abundant bands were subjected to in-gel trypsin digestion. Peptides were pooled together and then analyzed by using the LC-Q-TOF MS/MS technique. Thirty-two proteins were identified to be associated with AcMNPV BV by this method (Table (Table1),1), including PTP (AC1), P78/83 (AC9), F-like (AC23), V-ubiquitin (AC35), PP31 (AC36), AC51, VP1054 (AC54), AC58/59, FP25k (AC61), GP37 (AC64), AC66, IAP-2 (AC71), AC74, VLF-1 (AC77), VP39 (AC89), ODV-E25 (AC94), 38K (AC98), BV/ODV-C42 (AC101), VP80 (AC104), AC109, AC114, AC124, chitinase (AC126), GP64 (AC128), P24 (AC129), polyhedron envelope protein (PEP AC131), AC132, ME53 (AC139), 49K (AC142), ODV-E18 (AC143), BV/ODV-EC27 (AC144), and ODV-E56 (AC148).
By using these two LC-MS/MS techniques, we identified a total of 33 proteins (Table (Table1),1), 21 of which were identified by both LC-Qtrap and LC-Q-TOF, including PTP, P78/83, F-like, V-ubiquitin, 39K/PP31, VP1054, AC58/59, AC66, IAP-2, VLF-1, VP39, ODV-E25, BV/ODV-C42, VP80, AC109, AC114, GP64, AC132, ME53, 49K, and BV/ODV-EC27. AC73 was identified only by LC-Qtrap, while 11 proteins, including AC51, FP25K, GP37, AC74, 38K, AC124, chitinase, P24, PEP, ODV-E18, and ODV-E56, were identified only by LC-Q-TOF.
GP64 and VP39, which were identified by MALDI-TOF MS, were also confirmed by LC-MS/MS. However, one of the major BV components, P6.9, was detected only by MALDI-TOF but not by LC-MS/MS. P6.9 is composed of 55 amino acids, 22 of which are arginine residues. Theoretically, after complete trypsin digestion, P6.9 could generate one peptide with a length of 11 amino acids and nine short peptides with lengths of 2 to 5 amino acids. These short peptides are not suitable to serve as precursors ions to be detected by LC-MS/MS in our experimental setting.
In summary, a total of 34 viral proteins were found to be associated with the AcMNPV BV. Among the 34 BV-associated viral proteins identified in this study, 21 were previously reported to be BV components in the literature, including PTP (56), P78/83 (111), F-like (11), V-ubiquitin (32), AC51 (114, 125), VP1054 (76), FP25K (7), GP37 (58), VLF-1 (106), VP39 (103), ODV-E25 (89), P6.9 (119), 38K (122), BV/ODV-C42 (9), VP80 (65), AC109 (25), GP64 (118), P24 (121), ME53 (20), 49K (70, 107), and BV/ODV-EC27 (107). Consequently, 13 proteins, including PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, PEP, AC132, ODV-E18, and ODV-E56, were identified as BV-associated proteins for the first time (Table (Table33).
Among the identified BV-associated protein, ODV-E18, ODV-E25, and ODV-E56 were previously considered to be specific for the ODV. In fact, ODV-E25 was reported to also be present in the BV. However, it was thought that its detection in the BV was due to contamination (89). In order to ascertain the locations of the proteins, Western blot analyses using anti-ODV-E18 and anti-ODV-E25 antibodies were performed. The results showed that ODV-E18 and ODV-E25 were detected in the envelope fraction of both BV and ODV (Fig. (Fig.2).2). Unfortunately, we were unable to generate a specific antibody against ODV-E56, and its localization needs to be confirmed later.
The host protein components of purified AcMNPV BV particles were determined based on LC-Qtrap and LC-Q-TOF MS analyses and a data search of an in-house-constructed lepidopteran protein database. The proteins that were identified with high confidence (over 99%) by both LC-MS/MS techniques are summarized in Table Table2.2. Eleven such proteins were identified and could be divided into four categories by their functions: (i) cytoskeleton proteins, including actin and actin-depolymerizing factor 1 (ADF-1); (ii) signal transduction proteins such as 14-3-3 zeta, the casein kinase II alpha subunit (CKIIα), and the Ras-like GTPase Rho1; (iii) vesicular trafficking proteins such as ADP-ribosylation factor (Arf), the Ras-related proteins Rab5 and Rab11, ubiquitin, and annexin B; and (iv) RNA-binding proteins, including the Sr protein.
Of the 11 host proteins, actin (53) and ubiquitin (32) were reported to be associated with baculoviruses, and a total of seven proteins have been reported for enveloped viruses other than baculoviruses (Table (Table2).2). In addition, although ADF-1 was not reported to be virion associated, cofilin, another member of the ADF/cofilin family with similar functions, was reported previously to associate with human immunodeficiency virus (HIV) (16), Epstein-Barr virus (EBV) (43), human cytomegalovirus (HCMV) (110), murine cytomegalovirus (MCMV) (46), influenza virus (96), and herpes simplex virus type 1 (HSV-1) (64). Similarly, although the Sr protein had not been reported to be virion associated, another RNA-binding protein, poly(A)-binding protein (PABP), was identified in Moloney murine leukemia virus (MMLV) (94).
The BV phenotype is responsible for spreading infection within susceptible larval tissues, and therefore, the identification and characterization of its components are critical to an understanding of interactions with permissive cells and infection mechanisms. In this study, we identified 34 viral proteins and 11 host proteins associated with the AcMNPV BV using mass spectrometry techniques. This represents the first MS proteomic study of a baculovirus BV.
Although many proteins were reported previously to be BV-associated proteins based on Western blot analyses or other techniques, our proteomic data significantly expanded this protein list (see Table Table33 for a summary of BV-associated viral proteins). A literature search revealed another 11 BV proteins in addition to the 34 identified in this study, making the total number of BV-associated proteins 45 (Table (Table3).3). Among the 11 reported proteins, 6 were reported to exist in the AcMNPV BV but were not identified by our current study, including BV/ODV-E26 (AC16) (4), PCNA (AC49) (3), V-CATH (AC127) (52), EXON0 (AC141) (24), AC145 (54), and AC150 (54). Another five proteins, including VP91 (ORF86 of Orgyia pseudotsugata multiple nucleopolyhedrovirus [OpMNPV], a homologue of AC83) (90), BRO (ORF25 of Spodoptera litura nucleopolyhedrovirus [SpltNPV], a homologue of AC2) (29), HA33 (ORF33 of HearNPV, a homologue of AC38) (113), BM42 (ORF42 of BmNPV, a homologue of AC53) (1), and BM68 (ORF68 of BmNPV, a homologue of AC82) (41), were found to be associated with BVs of baculoviruses other than AcMNPV. Since their homologues exist in AcMNPV, these proteins are also likely to be associated with the AcMNPV BV. IE-1 (OP145) is an exception. Although it was associated with the OpMNPV BV (102), its homologue, AC147, was not found to be associated with the AcMNPV BV (11). However, IE-1 has been identified as a potential ODV-associated protein in both AcMNPV (11) and HearNPV (21). Therefore, whether IE-1 is a BV-associated protein awaits further analysis.
Although MS is a sensitive protein identification technique, the failure of the identification of some proteins is not unexpected. Similar results have been reported when multiple techniques, including MS, were used to identify the protein composition of the AcMNPV ODV (11). Many factors may contribute to the failure of the identification of all proteins by MS analyses, including a low abundance of the protein in the BV, proteins not amenable to LC-MS/MS techniques, and protein degradation or loss during purification.
The 45 BV-associated proteins were classified into four groups (Table (Table3):3): envelope-associated proteins, proteins essential for nucleocapsid production, other proteins associated with both the BV and ODV, and other BV-specific proteins.
Seven proteins were reported to be envelope associated, including GP64, F-like, V-ubiquitin, GP37, ODV-E25, ODV-E18, and BV/ODV-E26. GP64 is the major envelope protein of BV responsible for membrane fusion (6, 73) and important for efficient BV budding (77), and our study confirmed that it is one of the most abundant proteins in the BV. The F-like protein is localized in the envelopes of both the BV and ODV (11, 79) and is not essential for viral replication (68) but is important for BV infectivity (115). V-ubiquitin is attached to the inner face of the BV membrane by a phospholipid anchor (32) and was identified in several bands of the AcMNPV BV by LC-Qtrap MS/MS (Table (Table1).1). GP37 is a chitin-binding protein, and in SpltNPV, it was found in the envelopes of both the BV and ODV (58). ODV-E25 and ODV-E18 were identified as BV envelope-associated proteins in this study (Fig. (Fig.2).2). Since our BV samples were collected at 72 h p.i., possible contamination with ODVs due to cell lysis was examined. As shown in Fig. Fig.2,2, no ODV-specific bands were detected in BV samples by anti-ODV-E66 antibody. In fact, ODV-E18 and ODV-E25 were detected in BV samples collected at 36 and 48 h p.i. by Western blotting (data not shown). The identification of ODV-E18 as a BV-associated protein corroborates a recent finding that ODV-E18 is essential for BV production (71). The role of ODV-E25 in BV infection needs to be further investigated. BV/ODV-E26 was previously reported to be associated with the envelopes of both the BV and ODV (4) and was reported to have two forms, one of which is palmitoylated and membrane associated (13). We were unable to identify BV/ODV-E26 by either LC-Qtrap or LC-Q-TOF, indicating that the quantities of the protein in the AcMNPV BV are very low. Among the seven BV envelope proteins, the F protein, GP37, ODV-E25, ODV-E18, and BV/ODV-E26 were also reported previously to be ODV associated (11), suggesting that the BV and ODV share not only nucleocapsid proteins but also some envelope proteins.
So far, 11 proteins have been reported to be nucleocapsid components that are essential for nucleocapsid formation. These proteins are P6.9 (105, 119), VP39 (80, 103), VP1054 (76), VLF-1 (106), 38K (123), AC109 (25, 60), 49K (107), BV/ODV-EC27 (107), P78/83 (116), BV/ODV-C42 (107), and VP80 (100). All the proteins have been identified in both the BV and ODV (Table (Table3).3). P6.9, a small basic DNA-binding protein conserved in all baculoviruses, is considered to be a DNA core component inside the capsid (105, 119). VP39 is the major capsid protein, and it is conserved in all baculoviruses (80, 103). Our study has shown that P6.9 and VP39 are the most abundant proteins along with GP64, which is consistent with their respective functions. VP1054 (76), VLF-1 (106), 38K (123), AC109 (60), 49K (70, 107), and BV/ODV-EC27 (10, 107) are conserved baculoviral proteins found, directly or indirectly, to play important roles in nucleocapsid assembly. P78/83 is a phosphoprotein associated with the end structure of nucleocapsid (82, 88, 111). It nucleates actin polymerization, which may be involved in the translocation of nucleocapsid to the nucleus (28, 116). BV/ODV-C42 is a capsid-associated protein that contains a conserved nuclear localization signal and interacts directly with P78/83 to mediate its entry into the nucleus (117). VP80 was reported to be a nucleocapsid protein (65, 74) essential for BV production and nucleocapsid maturation (100). All 11 BV proteins essential for nucleocapsid morphogenesis were confirmed to be BV components by this study.
There are other 17 proteins associated with both the BV and ODV, including PTP, AC51, AC58/59, FP25K, AC66, AC74, AC114, P24, AC132, ME53, ODV-E56, BRO, PCNA, VP91, EXON0, AC145, and AC150 (Table (Table3).3). The roles of these proteins in the BV/ODV remain to be elucidated.
Ten proteins have been reported to be associated with the BV but not with the ODV, including PP31, IAP-2, AC73, AC124, chitinase, PEP, AC38, AC53, AC82, and V-CATH. Two proteins, chitinase and V-CATH, are required for the liquefaction of host insects (18, 36, 52). It was suggested previously that the association of V-CATH with the nucleocapsid and envelope of the BV contributes to the degradation of actin as well as the liquefaction of infected insects (52). PEP is considered to be the component of the polyhedron envelope (31, 108). It is abundantly produced during the late phase of infection; therefore, it may be nonspecifically trapped in the BV. Recently, it was shown that AC53 is involved in nucleocapsid assembly and is essential for virus production (61). The roles of the rest of the BV-associated proteins need to be investigated further.
Among the 45 BV-associated proteins (Table (Table3),3), 12 are conserved in all sequenced baculoviruses, including ODV-E18, ODV-E56, VP39, P6.9, VP1054, VLF-1, 38K, AC109, 49K, BV/ODV-EC27, AC66, and VP91 (35, 42, 85, 109). It is interesting that all these conserved proteins exist in both the BV and ODV, and it is likely that these are the core proteins for BV and ODV synthesis.
During the LC-Qtrap analysis, several proteins were identified in more than one gel band not always corresponding to their expected molecular masses. These proteins include V-ubiquitin, VP39, VP80, BV/ODV-C42, GP64, and BV/ODV-EC27 (Table (Table1).1). Similar phenomena were observed by proteomic studies of ODVs of CuniNPV (81) and HearNPV (21). Various factors could be responsible for this, such as a posttranslational modification of proteins, an incomplete denaturation of virions, an incomplete breakdown of protein complexes, or protein degradation during the experimental procedures. The presence of ubiquitin in two higher-molecular-mass fractions suggests the potential presence of ubiquitin-conjugated proteins in those regions. The appearance of VP39, GP64, and VP80 in multiple bands likely reflects protein degradation during the experimental procedures. Our result for BV/ODV-C42 is consistent with a previous report showing that the protein was detected in infected cells as two bands with molecular masses of 41.5 and 30 kDa by Western blotting (9). Similarly, BV/ODV-EC27 has been recognized as bands of 27 and 35 kDa in Western blots of the ODV (10). Further investigations are needed to address the significance of the multiple forms of these proteins.
In addition to the viral proteins, 11 BV-associated cellular proteins were identified by both LC-Qtrap and LC-Q-TOF. Host cellular proteins can be incorporated into viral particles either randomly or specifically. Early studies have shown that baculoviruses can trap heterologous factors such as chloramphenicol acetyltransferase (CAT) into BV particles nonspecifically (15). It was suggested previously that there was an affinity between CAT and the membrane of the virus particle (15). Likewise, proteins associated with the plasma membrane could also be included into the membrane envelope of the BV nonspecifically. However, the incorporation of certain host proteins has been shown to be important for virus infection. For example, actin, which was detected on budded virions, was shown previously to be important for baculovirus infection and replication both during nucleocapsid transport and after viral gene expression (112). Most of the actin was nonspecifically trapped within the BV and fractionated with the envelope fraction. However, the remaining actin appeared to be tightly and specifically attached to the nucleocapsid (53). Actin and proteins of the ADF/cofilin family are associated with many viruses (Table (Table2),2), indicating that the usurping of host cytoskeletal machinery by viruses is likely to be a common theme in enveloped virus egress (43, 67). It was previously suggested that because of the proximity of the cytoskeletal proteins to virus assembly, transport, and budding sites, they could be inevitably incorporated inside virions, and their presence may play a role in infectivity (78).
Three signal transduction proteins were identified in the AcMNPV BV. Rho proteins are regulated downstream from growth factor receptors and cell adhesion molecules. They affect different cell responses such as transcriptional regulation, cell cycle control, cell migration, and even survival. It was reported previously that Rho proteins are the key transducers that result in changes to the actin cytoskeleton (14, 84). Therefore, Rho1 could play a role in the regulation of the cell skeleton apparatus in AcMNPV infection. The 14-3-3 proteins bind specifically to phosphoserine/phosphothreonine motifs with over a hundred interacting partners identified. These proteins are involved in apoptosis, signal transduction, the stress response, and malignancy (19). CKII is a serine/threonine kinase that digests a number of substrates and is involved in gene transcription, the control of cell growth, tissue morphogenesis, and the regulation of the cell cycle (22). The exact roles of these signal transduction proteins in AcMNPV infection need to be further investigated.
Rab5 and Rab11 of the small GTPase superfamily were identified in the AcMNPV BV. AcMNPV enters the host cell via clathrin-mediated endocytosis (63). It was shown previously that the Rab5 protein plays an important regulatory role in early endocytosis (75, 93) and is important for infection by influenza virus, vesicular stomatitis virus, dengue virus, and West Nile virus (51, 98). Rab11 regulates trafficking through specialized endosomal compartments called recycling endosomes (75, 93), and it was previously reported to be involved in viral protein transport and virion release of Mason-Pfizer monkey virus (95) and hantavirus (87). The identification of these GTPase proteins in the AcMNPV BV provides valuable information for future studies of the entry and egress pathways of baculoviruses.
It is noteworthy that many of the host proteins identified in this study have also been reported to be present in virions of diverse enveloped virus families (e.g., herpesviruses, poxviruses, and retroviruses) (Table (Table2).2). Considering that these studies were performed independently using different cell types and different mass spectrometry methods, this similarity is not likely to be because of contamination or accidental packaging into virions (96). The most likely explanation is that the incorporated host proteins common to all of these enveloped viruses play some role in virus assembling, transport, budding, and infection. Data from the current study agree with data from other studies suggesting that conserved cellular pathways are utilized by many enveloped viruses. Therefore, it is fundamentally important to identify these cellular pathways and their utility in the infection process.
This research was supported by grants from the National Science Foundation of China (30630002 and 30770084), 973, grants (2009CB118903, 2007CB914204, and 2010CB530103) and Programme Strategic Scientific Alliances between China and the Netherlands (grant 2008DFB30220).
We acknowledge the State Key Laboratory of Virology Proteomics/MS Center (Wuhan University) for technical support. We thank Jia Zhu and Jianlan Yu for assistance with the experiments. We thank Basil M. Arif for critical editing of the manuscript.
Published ahead of print on 5 May 2010.