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DNA Viruses

Identification of protein–protein interactions of the occlusion-derived virus-associated proteins of Helicoverpa armigera nucleopolyhedrovirus

Ke Peng, Minzhi Wu, Fei Deng, Jingjiao Song, Chunsheng Dong, Hualin Wang, Zhihong Hu

  • State Key Laboratory of Virology and Joint Laboratory of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China
  • Correspondence
    Zhihong Hu
    huzh{at}wh.iov.cn

    • Journal of General Virology 2010; 91(3):659–670 · https://doi.org/10.1099/vir.0.017103-0

    View at publisher PubMed

    Abstract

    The purpose of this study was to identify protein–protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II alphabaculovirus in the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct-cross Y2H assays identified 22 interactions comprising 13 binary interactions [HA9–ODV-EC43, ODV-E56–38K, ODV-E56–PIF3, LEF3–helicase, LEF3–alkaline nuclease (AN), GP41–38K, GP41–HA90, 38K–PIF3, 38K–PIF2, VP80–HA100, ODV-E66–PIF3, ODV-E66–PIF2 and PIF3–PIF2] and nine self-associations (IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24). Five of these interactions – LEF3–helicase and LEF3–AN, and the self-associations of IE1, LEF3 and 38K – have been reported previously in Autographa californica multiple nucleopolyhedrovirus. As HA44 and HA100 were two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with a His pull-down assay and the interaction of VP80–HA100 was confirmed by a co-immunoprecipitation assay. A summary of the protein–protein interactions of baculoviruses reported so far, comprising 68 interactions with 45 viral proteins and five host proteins, is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

    • ↵†Present address: Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands.

    • A supplementary table detailing the proteins and primers used for the Y2H assay is available with the online version of this paper.

    INTRODUCTION

    Baculoviruses are members of the family Baculoviridae, double-stranded DNA viruses that specifically infect arthropods of the insect orders Lepidoptera, Hymenoptera and Diptera, and are divided into four genera, Alphabaculovirus, Betabaculovirus, Gammabaculovirus and Deltabaculovirus, as first suggested by Jehle et al. (2006). The alphabaculoviruses are subdivided into groups I and II based on phylogenetic analyses (Herniou et al., 2001). A characteristic feature of members of the Baculoviridae is that they produce two viral phenotypes: occlusion-derived virus (ODV) and budded virus (BV). Packaged in a protein crystal-like structure called an occlusion body (OB), ODVs are responsible for oral infection. From the structural point of view, ODV is quite different from BV. Although BV and ODV appear to share a similar nucleocapsid structure, ODV obtains its envelope from membranes within the nucleus (Braunagel & Summers, 2007), whilst BV obtains its envelope when it buds through the modified plasma membrane (Braunagel & Summers, 1994). Consequently, the composition of the envelope proteins of ODVs is very different from that of BVs (Braunagel et al., 2003; Deng et al., 2007; Perera et al., 2007; Slack & Arif, 2007). In addition to the nucleocapsid and envelope, ODV has a tegument layer sandwiched between them (Slack & Arif, 2007). The structures of ODV and BV are correlated with their functions, and it is therefore important to identify the structural components of ODVs and BVs to elucidate the molecular mechanisms of baculovirus infection.

    Recently, the ODV protein composition of four baculoviruses has been determined by proteomic studies: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (Braunagel et al., 2003), Culex nigripalpus NPV (Perera et al., 2007), Helicoverpa armigera NPV (HearNPV) (Deng et al., 2007) and Bombyx mori NPV (BmNPV) (Liu et al., 2008). These studies showed that ODV contains a number of proteins ranging from 20 to 41 and provided useful information for prediction of ODV assembly and structure. ODV assembly is presumed to be a complex and precisely organized process including viral DNA packaging, nucleocapsid assembly and ODV envelopment, which involves a number of protein–DNA and protein–protein interactions. Therefore, identifying interactions among viral proteins would be beneficial for interpreting the molecular mechanisms of virus assembly and infection.

    In this report, we describe the identification of interactions among ODV proteins by using HearNPV, a group II alphabaculovirus, as a model. To achieve this, the proteins that have been shown to be probable HearNPV ODV components (Deng et al., 2007; Long et al., 2003; Pan et al., 2007) or HearNPV homologues of AcMNPV ODV components (Braunagel et al., 2003; Wu et al., 2008) were cloned into yeast two-hybrid (Y2H) vectors and screened for protein–protein interactions. Two of the identified interactions were verified further by pull-down or co-immunoprecipitation analysis. The results were combined with baculovirus protein–protein interactions reported by others in order to summarize our understanding of the protein interactome of baculovirus.

    RESULTS

    Construction of clones expressing HearNPV ODV-associated proteins for Y2H screening

    Thirty-nine HearNPV ODV proteins that were determined to be either HearNPV ODV components (Deng et al., 2007; Long et al., 2003; Pan et al., 2007) or HearNPV homologues of AcMNPV ODV components (Braunagel et al., 2003; Wu et al., 2008) were selected for the Y2H screening (see Supplementary Table S1, available in JGV Online). Among these, 21 were encoded by conserved genes found in all baculovirus genomes sequenced so far. The common names of these proteins are also listed in Supplementary Table S1. As the Y2H system used in this study detects protein interactions in the yeast nucleus and thus is not optimal for proteins containing transmembrane (TM) domains, tmhmm software was used to screen the proteins with potential TM domains. Ten of the 39 proteins were predicted to have TM domains (Fig. 1⇓). Most TM-containing proteins were ODV envelope proteins comprising P74, PIF1, PIF2, PIF3, ODV-E18, ODV-E56, ODV-E66 and ODV-E25. For the proteins containing only one predicted TM domain, i.e. ODV-E18, ODV-E25, ODV-E66, VP91, PIF3, PIF2 and PIF1, their truncated open reading frames (ORFs) without the TM domain were amplified by PCR for Y2H screening (Fig. 1⇓). For ODV-E56, in addition to the entire ORF without the TM domain (ODV-E56-T3), two other truncated ORFs, ODV-E56-T1 and ODV-E56-T2, were also constructed (Fig. 1⇓). HA107 (which is unique to HaNPV) was reported to contain multiple TM domains (Pan et al., 2007), and two truncated ORFs, Ha107-T1 and HA107-T2, were constructed (Fig. 1⇓). For P74, which also contains three predicted TM domains, a truncated ORF containing the first 460 aa was constructed (P740T; Fig. 1⇓). Therefore, there were 13 truncated ORFs representing the ten TM domain-containing proteins, plus 29 without a TM domain, making a total of 42 construct pairs. All 42 fragments were cloned into the pGBKT7 and pGADT7 vectors and their fidelity was confirmed by sequence analysis.

    Figure image not available in archive
    Fig. 1.

    Schematic of the truncated TM domain-containing ORFs in this study. The potential TM domains as well as the inside (towards the tegument) and outside (on the ODV membrane surface) orientation of the HearNPV ORFs predicted by tmhmm software are shown. Solid arrows represented the truncated forms of these ORFs constructed in this study.

    Y2H analysis reveals 22 protein–protein interactions

    To systematically identify protein–protein interactions among HearNPV ODV proteins, direct-cross Y2H assays among the 42 construct pairs were performed. Of the 42 proteins tested, 19 were involved in one or more interactions (Table 1⇓). Of these, nine self-associations and 13 reciprocal interactions for a total of 22 interactions were identified. The 13 binary interactions comprised HA9 (homologue of AcMNPV ORF 142=Ac142)–ODV-EC43, ODV-E56–38K, ODV-E56–PIF3, LEF3–helicase, LEF3–alkaline nuclease (AN), GP41–38K, GP41–HA90 (=Ac102), 38K–PIF3, 38K–PIF2, VP80–HA100 (=Ld141/Se52), ODV-E66–PIF3, ODV-E66–PIF2 and PIF3–PIF2, whilst the nine self-associations comprised IE1, HA44 (=Ld55/Se107), LEF3, HA66 (=Ac66/desmoplakin), GP41, CG30, 38K, PIF3 and P24.

    Table 1.

    Summary of the positive results of protein–protein interactions of the HearNPV ODV proteins by a Y2H assay

    The orders of the bait and prey are in accordance with their location in the HearNPV genome, which is shown in the first column. The second column shows the bait, whilst the top line shows the prey. Due to space limitations, some protein names are abbreviated. Reciprocal interactions and self-associations are indicated with the letter R, whilst interactions that could be detected in only one direction are indicated with the letter O. na, Not applicable: combinations in which HA122 served as bait were not considered due to autoactivation activity. Blanks cells indicate negative results.

    All the above interactions are reported for the first time for HearNPV. The two binary interactions of LEF3–helicase (Evans et al., 1999) and LEF3–AN (Mikhailov et al., 2003) and the three self-associations of IE1 (Olson et al., 2002), LEF3 (Evans & Rohrmann, 1997) and 38K (Hefferon, 2003b; Wu et al., 2008) have been reported in AcMNPV. Therefore, 17 interactions, comprising 11 binary interactions and six self-associations, are reported here for the first time.

    Another ten interactions were also identified: HA9 (=Ac142)–HA122 (unique to HearNPV), ODV-E18–HA26 (=Ac26), ODV-E56-T1–ODV-E56-T3, ODV-E56T1–PIF2, P74–ODV-E56-T3, ODV-E66–ODV-E56-T3, PIF3–ODV-E56-T3, HA107-T2–ODV-E56-T3, PIF1–ODV-E56-T3 and PIF2-ODV–E56-T3; however, these could be detected in only one direction in the Y2H assay (Table 1⇑). Many of the interactions were detected when ODV-E56-T3 was used as the prey. HA122 (unique to HearNPV) was found to be able to auto-activate the reporter gene when expressed in the bait vector (Table 1⇑). As HA122 gave rise to false interactions, we cannot exclude the possibility that HA9–HA122 could be a reciprocal interaction. The reliability, if any, of these unidirectional interactions needs further confirmation and they were not included in our discussion. Fourteen proteins, P78/83, ODV-EC27, VP1054, FP25, DNA pol, HA68 (AC74), VLF-1, VP91, VP39, P33, ODV-E25, P6.9, C42 and LEF1, did not interact with any proteins in our Y2H assay. Among all the tested combinations, 12 previously reported interactions were not identified in our study: EC27–EC27, EC27–C42, C42–P78/83, FP25–ODV-E66, ODV-E66–ODV-E25, ODV-E66–VP39, VP39–IE1, VP39–VP39, VP39–ODV-E56, VP39–38K, 38K–VP1054 and 38K–VP80.

    His pull-down assay confirms the self-association of HA44

    As HA44 (=Ld55/Se107) and HA100 (=Ld141/Se52) were the two newly identified ODV proteins that are conserved in other group II viruses (Deng et al., 2007), their interactions were characterized further. To verify the self-association of HA44, His pull-down assays were performed. Full-length HA44 with a 6×His tag at its N terminus (His–HA44) was expressed and purified. The purified protein was then incubated with the lysate of HearNPV-infected and uninfected cells. After incubation, His–HA44 and bound protein(s) were purified by affinity chromatography. Samples were separated by SDS-PAGE and probed with anti-HA44 antiserum. A second negative control using the Ni-NTA beads was set up to exclude the possibility that viral HA44 interacts directly with the NTA resin. The predicted molecular mass of HA44 is 42.8 kDa. As shown in Fig. 2⇓ (lane 1), in the infected HearNPV cells, two forms of HA44 with a migration rate of 44 and 45 kDa were captured by the purified His–HA44. In the His–HA44 pull-down products from the healthy cell lysate (Fig. 2⇓, lane 2), only the 48 kDa His–HA44 (the His tag added approx. 4 kDa) was detected. Almost nothing was detected in the Ni-NTA pull-down products from infected or healthy cell lysate (Fig. 2⇓, lanes 3 and 4). Therefore, the His–HA44 pull-down assay confirmed the HA44–HA44 interaction.

    Figure image not available in archive
    Fig. 2.

    Pull-down assay of His–HA44. HzAM1 cells were infected with HearNPV at an m.o.i. of 5 and collected at 36 h p.i. to perform pull-down assays. His–HA44 was incubated with the lysate from infected or uninfected cells and pulled-down proteins were purified with Ni-NTA beads. Ni-NTA beads alone were used as a negative control. The eluted samples were separated by SDS-PAGE, blotted and probed with anti-HA44 antibody. Lanes: M, Molecular mass marker (kDa); I, lysate of infected cells; 1, proteins pulled down from the lysate of infected cells by His–HA44; 2, proteins pulled down from the lysate of uninfected cells by His–HA44; 3, proteins pulled down from the lysate of infected cells by Ni-NTA; 4, proteins pulled down from the lysate of uninfected cells by Ni-NTA; U, lysate of uninfected cells.

    In vivo co-immunoprecipitation assay verifies the VP80–HA100 interaction

    A co-immunoprecipitation assay was performed to verify the VP80–HA100 interaction. Anti-VP80 or anti-HA100 antiserum was added to the lysate of HearNPV-infected cells. After incubation, protein A was added to capture the immune complex. The precipitated proteins were then separated by SDS-PAGE and probed with anti-VP80 or anti-HA100. Parallel experiments with pre-immune antiserum were performed as negative controls. As shown in Fig. 3⇓, co-immunoprecipitation with anti-VP80 not only precipitated VP80 (80 kDa; Fig. 3a⇓, lane 1), but also precipitated HA100 (60 kDa; Fig. 3b⇓, lane 1). Likewise, co-immunoprecipitation with anti-HA100 precipitated both VP80 (Fig. 3c⇓, lane 3) and HA100 (Fig. 3d⇓, lane 3). The pre-immune antisera did not precipitate any specific proteins. The results verified the bidirectional interaction of VP80–HA100. In the experiments, the heavy chain of the rabbit IgG, which was used to capture target protein, was also readily detected in the Western blot due to direct binding by the goat anti-rabbit secondary IgG. A band of about 60 kDa was also detected when the lysate from infected cells was probed with VP80 antiserum (Fig. 3a, c⇓, lane I). The appearance of a band migrating at a similar low molecular mass was also detected for VP80 of Choristoneura fumiferana MNPV (Li et al., 1997) when the sample from infected cells was probed with VP80 antibody. As this band was detected with anti-VP80 antisera, it might be due to the degradation of VP80. As shown in Fig. 3⇓, this smaller protein did not interfere with the results of the co-immunoprecipitation as it was not present in the co-immunoprecipitated samples.

    Figure image not available in archive
    Fig. 3.

    Co-immunoprecipitation assays for VP80 and HA100. HzAM1 cells were infected with HearNPV at an m.o.i. of 5. At 36 h p.i., the lysates of infected and uninfected cells were immunoprecipitated (IP) with the indicated antibodies and corresponding pre-immune serum. Samples were separated by SDS-PAGE, blotted and probed with the indicated antibodies. Lanes: M, Molecular mass marker (kDa); I, lysate of infected cells; 1, proteins of infected cells precipitated by anti-VP80 antibody; 2, proteins of infected cells precipitated by VP80 pre-immune serum; 3, proteins of infected cells precipitated by anti-HA100 antibody; 4, proteins of infected cells precipitated by HA100 pre-immune serum; U, lysate of uninfected cells. The positions of VP80, HA100 and the heavy chain of co-eluting rabbit IgG (IgG-H) recognized by the goat anti-rabbit secondary IgG are indicated. The antibodies used for Western blotting are indicated below each blot.

    DISCUSSION

    This study aimed to identify the protein–protein interactions of potential ODV components of HearNPV. Using direct-cross Y2H assays covering 1764 (42×42) combinations, 22 interactions comprising 13 binary interactions and nine self-associations were identified. To verify the Y2H data further, two interactions involving newly identified ODV components of HearNPV, the self-association of HA44 and the HA100–VP80 interaction, were verified using pull-down and co-immunoprecipitation assays, respectively. The remaining interactions need to be confirmed further with other methods.

    To date, a number of protein interactions have been reported for baculoviruses, mainly in AcMNPV, which is generally used as a model baculovirus. In a recent review, Braunagel & Summers (2007) reported direct-cross Y2H assays of 14 ODV-associated proteins: polyhedrin (POLY), P39, P78/83, BV/ODC-C42 (C42), FP25, BV/ODV-EC27 (EC27), IE1, ODV-E66, ODV-E25, ODV-E56, BV/ODV-E26 (E26), GP41, PIF3 and Ac91. Eight interactions comprising VP39–POLY, VP39–ODV-E56, IE1–VP39 and IE1–E26 and self-associations of POLY, VP39, EC27 and IE1 were reported. In the same review, they also presented the results of a Y2H library screening. As most of the interactions identified by library screening were observed in only one direction, they were not included in our discussion. Many other interactions have been reported when individual or a few proteins have been studied by Y2H assays, co-immunoprecipitation or other methods. With our new protein–protein interaction data, we tried to update the protein interactome of baculovirus. The protein–protein interactions of baculoviruses reported so far including our results are summarized in Fig. 4⇓. A total of 68 protein–protein interactions involving 45 viral proteins and five host proteins have been identified. The proteins involved in interactions are classified into three groups: DNA replication, transcription and structure-associated, and are described further below.

    Figure image not available in archive
    Fig. 4.

    Summary of interactions of baculovirus proteins. The figure shows a schematic of the currently known protein–protein interactions of baculoviruses. The interactions were classified into three groups: DNA replication, transcription and structural. The classification is not absolute in that some replication/transcription proteins were listed in the structural group because they have interactions with structural proteins. Baculoviral proteins included in the current Y2H study are shown as shaded circles, and those previously published but not included in our study are shown as open circles. An asterisk indicates proteins conserved in baculoviruses. Host proteins are shown as open squares. The interactions identified in this study are indicated as dashed lines, and interactions reported previously in the literature are indicated as solid lines.

    DNA replication

    Ten protein–protein interactions have been reported to be important for AcMNPV DNA replication, among which are seven essential proteins: DNA polymerase (DNA pol), helicase, LEF3 (single-stranded DNA-binding protein), IE1 (transcriptional factor, binds the origin of replication), LEF1 (primase), LEF2 (primase-associated factor) and LEF11, and three stimulatory proteins: IE-2 (transcriptional factor and cell-cycle-arrest gene), PE-38 (transcriptional factor and possible ubiquitin ligase) and P35 (apoptosis inhibitor) (Lin & Blissard, 2002; Lu & Miller, 1995; Mainz et al., 2002). In addition, DNA-binding protein (DBP) has also been reported to be a replication factor for BmNPV (Mikhailov et al., 1998). The interactions among these replication-associated proteins have been investigated in several studies and the interactions of LEF1–LEF2 (Evans et al., 1997) and LEF3–helicase (Evans et al., 1999) and the self-associations of LEF3 (Evans & Rohrmann, 1997), IE1 (Olson et al., 2001; Rodems & Friesen, 1995), IE2 (Imai et al., 2000) and DBP (Mikhailov et al., 2008) have been reported. In addition, some interactions in which replication-associated proteins were involved were also reported, including LEF3–AN (Mikhailov et al., 2003), IE1–VP39 (Braunagel & Summers, 2007), IE1–E26 (Kang et al., 2005; Nie et al., 2009) and IE0–E26 (Nie et al., 2009). Among the proteins involved in baculovirus DNA replication, DNA pol, helicase, LEF3, IE1 and LEF1 have been reported to be ODV components and were included in our study. Our results confirmed the interactions of LEF3–helicase and LEF3–AN and the self-associations of IE1 and LEF3 in HearNPV (Table 1⇑; Fig. 4⇑).

    Transcription

    Many proteins have been reported to be involved in baculovirus transcription (Passarelli & Guarino, 2007). The late baculovirus genes are transcribed by a viral-encoded RNA polymerase with four components: LEF4, LEF8, LEF9 and P47 (Guarino et al., 1998). Eight interactions among the viral RNA polymerase components were identified including five binary interactions (P47–LEF4, P47–LEF8, P47–LEF9, LEF4–LEF9 and LEF8–LEF9) and three self-associations (P47, LEF8 and LEF9) (Crouch et al., 2007). In addition to the above interactions, self-association of LEF5 (Harwood et al., 1998) and host cell-specific factor 1 (HCF1) (Hefferon, 2003a) have also been reported. In addition, protein kinase 1 (PK1) has been shown to be involved in very late gene expression (Fan et al., 1998) and the interaction of PK1 with the protein kinase-interacting protein (PKIP) has been reported (Fan et al., 1998).

    Structure-associated

    More than 40 proteins have been reported to be associated with baculovirus structures (ODV, BV and OB) and some have been studied in great detail. When the reported interactions are summarized, it appears to be a complicated network (Fig. 4⇑). We tried to categorize these interactions into several groups, as follows.

    (i) EC27, C42 and P78/83.

    EC27, encoded by Ac144, has amino acid similarity to cellular cyclins and had been suggested to be a multifunctional cyclin (Belyavskyi et al., 1998). EC27 can interact directly with either cellular cyclin kinase cdc2 or cdk6, and the protein complex EC27–cdk6 also binds to viral proliferating cell nuclear antigen (PCNA) (Belyavskyi et al., 1998) (data not included in Fig. 4⇑). Self-association of EC27 was identified by Y2H assay (Braunagel & Summers, 2007). C42 of AcMNPV contains the canonical binding motif for pocket proteins and interacts with EC27 and P78/83 (Braunagel et al., 2001). C42 contains a nuclear localization signal at its C terminus and the interaction of C42 and P78/83 is critical for transporting P78/83 to the nuclei (Wang et al., 2008). P78/83 is a phosphorylated structural protein located at the basal region of the nucleocapsid (Russell et al., 1997). It is a Wiskott–Aldrich syndrome protein (WASP)-like protein, which can bind to actin (Lanier & Volkman, 1998) and host cell actin-related protein 2/3 (Arp2/3) complex (Goley et al., 2006).

    (ii) FP25, and trafficking of BV and ODV.

    Normally, in a baculovirus-infected cell, BVs are produced first, and at the late stage of infection the BV production is curtailed in favour of ODV formation (Miller, 1997). To date, the mechanism of switching from BV to ODV production remains unclear, but it has been shown that FP25 plays an important role. FP25 is a highly conserved baculovirus protein containing a coiled-coil region and a putative actin-binding helix (Braunagel et al., 1999). When fp25 is deleted, BV production increases and ODV production decreases (Wu et al., 2005). FP25 interacts with E26 (Beniya et al., 1998), ODV-E66 and GP64 (Braunagel et al., 1999). When it interacts with E26, cellular actin is also a component of the protein complex (Beniya et al., 1998) (data not included in Fig. 4⇑). Most ODV envelope proteins contain an inner nuclear membrane sorting motif (INM-SM), which is important for the protein to be localized into the INM and eventually into the ODV envelope (Braunagel et al., 2009). It has been shown that FP25 is involved in trafficking of ODV-E66 to the nucleus (Rosas-Acosta et al., 2001). Braunagel et al. (2009) showed that FP25 could cross-link to all of the INM-SMs derived from ODV envelope proteins, suggesting that it plays an important role in trafficking the ODV envelope proteins. Interestingly, in our current study, we did not detect any interactions associated with FP25, which is consistent with the recent report by Braunagel & Summers (2007) that no binary interactions were observed among FP25 and ODV envelope proteins when direct-cross Y2H assays were performed (Braunagel & Summers, 2007). So far, very little is known about BV egress. GP64 is the major envelope protein of group I BVs; it forms a trimer (Oomens et al., 1995) and is responsible for viral entry (Li & Blissard, 2009) and BV budding (Oomens & Blissard, 1999). The interaction of FP25 with GP64 was detected by co-immunoprecipitation analysis (Braunagel et al., 1999). EXON0 is a structural protein of the nucleocapsid and is required for efficient production of BV (Dai et al., 2004; Fang et al., 2007). EXON0 forms dimers and interacts with C42 and FP25 (Fang et al., 2008). Furthermore, EXON0 was found to interact with cellular β-tubulin (Fang et al., 2009). It is interesting to note that both EXON0 and GP64, which are involved in BV egress, were found to interact with FP25; the significance of these interactions is currently unknown. Recently, it was shown that Ac66 is required for the egress of nucleocapsid from the nucleus and for general synthesis of pre-occluded virions (Ke et al., 2008). In this study, we found that HA66, the homologue of Ac66, self-associated.

    (iii) 38K and its associations.

    38K encoded by Ac98 is a striking protein, and has the most extensive interactions with other proteins (Fig. 4⇑). It is a conserved gene in baculoviruses and is essential for nucleocapsid assembly (Wu et al., 2006). Recently, the 38K protein was found to interact with VP39, VP80, VP1054 and itself, reflecting its important role in nucleocapsid assembly (Wu et al., 2008). Our research confirmed the self-association of 38K and identified another four binary interactions of 38K (38K–GP41, 38K–ODV-E56, 38K–PIF2 and 38K–PIF3) in HearNPV. Domain searches revealed a haloacid dehalogenase-like (HAD) superfamily domain conserved in all 38K homologues of baculoviruses (data not shown). The majority of the enzymes in the HAD superfamily are involved in phosphoryl transfer (Burroughs et al., 2006). If 38K is a functional phosphatase, then the interactions with different structural proteins might be its interactions with multiple substrates. Whether 38K is a functional phosphatase and whether its interactions with structural proteins are essential for viral assembly are interesting questions that remain to be investigated.

    (iv) ODV envelope proteins.

    ODVs acquire their envelope from membranes within the nucleus and are responsible for primary infection. A number of ODV envelope proteins have been identified, including P74, PIF1, PIF2, PIF3, ODV-E18, ODV-E25, ODV-E28, ODV-E56 and ODV-E66 (Braunagel & Summers, 2007; Slack & Arif, 2007). P74, PIF1, PIF2 and PIF3 are essential proteins for per os infectivity (PIF) (Haas-Stapleton et al., 2004; Ohkawa et al., 2005). P74, PIF1, PIF2 and PIF3 are conserved baculovirus genes and are also found in other invertebrate viruses such as salivary gland hypertrophy virus (SGHV) (Garcia-Maruniak et al., 2009) and nudiviruses (Wang & Jehle, 2009). Recently, a new PIF factor, PIF4, encoded by Ac96 was also identified in AcMNPV (Fang et al., 2009). PIF4 is conserved in baculoviruses, and a homologue of PIF4 is found in the distantly related Hz-1 virus (Fang et al., 2009). ODV-E66 is a hyaluronan lyase and may be important for penetration of extracellular barriers during primary infection (Vigdorovich et al., 2007). ODV-E66 is conserved in lepidopteran baculoviruses (Herniou et al., 2003) and is also found in SGHV (Garcia-Maruniak et al., 2009) and nudivirus (Wang & Jehle, 2009). The conservation of these proteins suggests a conserved mechanism of midgut infection by invertebrate viruses. ODV-E66 was found to interact with ODV-E25, FP25, E26 and VP39 (Braunagel et al., 1999), and in the current study, it was found to interact with PIF2 and PIF3. Interactions of PIF2–PIF3 and PIF3–ODV-E56 and self-association of PIF3 were also identified in the current study. As PIF4 is newly identified, it was not included in our study. It has been suggested that the factors involved in PIF may function in concert as a protein complex (Song et al., 2008) and thus it would be very interesting to investigate further the interactions among the ODV envelope proteins including the PIF proteins and their roles in primary infection.

    (v) Other structure-associated interactions.

    There were a few structure-associated interactions that were not included in the above classification. Among these, many were self-interactions, such as those of VP39 (Braunagel & Summers, 2007), POLY (Braunagel & Summers, 2007), P10 (Dong et al., 2005), GP41, HA44, CG30 and P24 (this study). In Spodoptera litura NPV, it has been suggested that there is either homo- or hetero-oligomerization of P24 (Li et al., 2005). Apart from the self-associations, some binary interactions of structural proteins such as VP39–POLY and VP39–ODV-E56 (Braunagel & Summers, 2007), VP39–actin (Lanier & Volkman, 1998), P10–tubulin (Patmanidi et al., 2003), HA9 (Ac142)–ODV-EC43, VP80–HA100 and GP41–HA90 (Ac102) (this study) have also been identified.

    In addition to the above interactions, the self-association of P26 (=Ac136) (Goenka & Weaver, 2008), which does not fit into any of the three categories of replication, transcription or structure-associated interactions, is also presented in Fig. 4⇑.

    In addition to the interactions mentioned above, certain interactions that were not directly confirmed, such as that of the actin rearrangement-inducing factor (ARIF-1) with F-actin (Dreschers et al., 2001), IE2 with the promyelocytic leukaemia protein (PML) (Murges et al., 2001), INM-directed proteins with importin-α-16 (Braunagel et al., 2009) and fibroblast growth factor (FGF) with its receptor breathless (Btl) (Katsuma et al., 2006), were not included in Fig. 4⇑.

    Fig. 4⇑ shows that many baculoviral proteins interact with more than one protein: 38K interacts with eight proteins including itself; ODV-E66 with six, VP39 with seven including itself; PIF3 with five including itself; FP25 and E26 with four; P47, LEF9, EC27 and EXON0 with four including self-association; C42, ODV-E56 and P78/83 with three; LEF3, LEF8, IE1 and GP41 with three including self-association; LEF4 and VP80 with two; and GP64, P10 and POLY with two including self-association. The data indicate that the interactions of baculovirus ODV proteins are complex. Therefore, further confirmation of these interactions and elucidation of the corresponding functions will significantly improve our understanding of the mechanisms of baculovirus infection.

    It is important to point out that Fig. 4⇑ is a summary of results from different baculoviruses and thus the details may vary among different viruses. For example, E26 (group I unique), HA44 (conserved in group II Alphabaculovirus and Betabaculovirus) and HA100 (group II unique) exist in only certain group(s) of baculovirus and therefore their protein interactions would be limited to the corresponding group(s). Also, certain interactions may exist only in certain viruses and not in others.

    Fig. 4⇑ also shows that many interactions reported previously were not identified in our Y2H study. Among all of the tested combinations, 12 previously reported interactions were not identified in this study. These were EC27–EC27, EC27–C42, C42–P78/83, FP25–ODV-E66, ODV-E66–ODV-E25, ODV-E66–VP39, VP39–IE1, VP39–VP39, VP39–ODV-E56, VP39–38K, 38K–VP1054 and 38K–VP80. It is generally understood that the Y2H system cannot identify all native protein interactions and that interactions identified by other methods might not be reproduced in Y2H assays (Fields, 2005). The Y2H system may have different post-translational modifications on proteins compared with the natural environment where virus proteins are produced. Consequently, for those interactions that are dependent on the modifications, the Y2H screen may not be able to identify them. Some interactions have been identified by others using a Y2H system, but not identified in our study. This may due to the detailed differences in the Y2H system used. For example, the reported 38K–VP80 interaction was detected under the less stringent selection condition but not detected in the most stringent selection condition (Wu et al., 2008), whilst the most stringent selection condition (lacking tryptophan, leucine, histidine and adenine) was used for all of our combinations. In fact, employing multiple techniques to identify protein interactions is important and indispensable to obtain a complete protein interactome.

    In summary, we identified 22 interactions among potential ODV proteins of HearNPV by Y2H assay. With these data, we summarized the protein–protein interactions in baculoviruses reported to date, which include 68 interactions with 45 viral proteins and five host proteins. This research will shed light on the mechanisms of ODV assembly and oral infection. However, the interactome of baculoviruses is still far from complete, as viral proteins other than ODV structural proteins also need to be studied systematically. In addition, knowledge of virus–host protein interactions is critical for an understanding of viral infections, yet very few interactions with host proteins have been identified to date. Currently, we are constructing a Y2H library of host genes to analyse further the virus–host interactions of baculoviruses.

    METHODS

    Virus, cells and antibodies.

    An in vivo-cloned strain (G4) of HearNPV (Chen et al., 2001) was used as the wild-type virus. The HzAM1 cell line derived from Heliothis zea (McIntosh & Ignoffo, 1983) was used in the pull-down and co-immunoprecipitation assays. The anti-HA44, anti-HA100 and anti-VP80 antibodies used in this study have been described previously (Deng et al., 2007).

    Y2H assays.

    The 39 proteins used in the Y2H study are listed in Supplementary Table S1. tmhmm software () was used to screen the potential TM domains as well as the inside and outside orientation of the ORFs. The entire ORFs of the 29 proteins that did not contain a predicted TM domain were amplified from the HearNPV G4 genome (Chen et al., 2001) using the primers listed in Supplementary Table S1. For the ten proteins that contained a potential TM domain, primers were designed to generate the truncated ORFs without a TM domain (Supplementary Table S1 and Fig. 1⇑). PCR products were purified and digested with corresponding restriction enzymes and ligated into pGBKT7 and pGADT7 vectors (Matchmaker Two-Hybrid System 3; Clontech). Clones were verified by enzyme digestion and sequencing. The Y2H assays were performed according to the protocol of the manufacturer. In principle, the yeast strains of bait were constructed by transformation of bait clones (pGBKT7 clones) into yeast strain AH109 (Clontech) according to a method described previously (Gietz & Woods, 2002) and selected on Trp− synthetic dextrose (SD) plates. For Y2H screening, each yeast strain of bait was transformed individually with each prey clone (pGADT7 clone) and selected on Trp−, Leu−, Ade−, His− SD plates. Each pair of positive combinations was repeated twice in the Y2H assay and negative combinations were repeated once.

    Confirmation of HA44 self-association by His pull-down assay.

    To confirm HA44 self-association, the ha44 gene was PCR amplified from the HearNPV G4 genome using primers HA44-PD-F (5′-GCGGAATTCATGAGCAATCCCAGCAAACAATC-3′) and HA44-PD-R (5′-GCGGAATTCTCAATAGCGCAAACGAGTTTCG-3′) (BamHI digestion sites underlined). The PCR product was purified and cloned into pET28a vector (Novagen) and the correct clone was transformed into Escherichia coli BL21 strain to express His-tagged HA44 (His–HA44). The soluble His–HA44 was purified by affinity chromatography using Ni-NTA resin (Qiagen). To perform the pull-down assay, 3×106 HzAM1 cells were infected with HearNPV G4 at an m.o.i. of 5. At 36 h post-infection (p.i.), the infected and control uninfected cells were washed three times with PBS and incubated in 1 ml PD buffer [1× PBS (pH 7.2), 10 % glycerol, 1 % Triton X-100, Protease Inhibitor Cocktail (Sigma)] on ice for 30 min. The swollen cells were then collected and sonicated in an ice-cold water bath and centrifuged at 20 100 g for 15 min at 4 °C. The supernatant was mixed with 50 μg purified His–HA44 and incubated at 4 °C with gentle agitation for 3 h. Ni-NTA resin (20 μl) was added to the mixture and incubated at 4 °C for another 1 h. After washing three times with washing buffer [20 mM Tris/HCl (pH 7.9), 300 mM NaCl, 30 mM imidazole, 10 % glycerol], the protein was eluted from the resin with elution buffer [20 mM Tris/HCl (pH 7.9), 300 mM NaCl, 200 mM imidazole, 10 % glycerol]. The experiment was performed simultaneously in the absence of His–HA44 as a negative control. Samples were separated by SDS-PAGE, followed by Western blotting with anti-HA44 antiserum (Deng et al., 2007).

    Confirmation of the HA100–VP80 interaction by a co-immunoprecipitation assay.

    For each experiment, 3×106 HzAM1 cells were infected with HearNPV G4 at an m.o.i. of 5. At 36 h p.i., cells were washed three times with PBS and incubated in 1 ml TEP buffer [1× PBS (pH 7.2), 2 mM EDTA, 10 % glycerol, 1 % Triton X-100, Protease Inhibitor Cocktail (Sigma)] in an ice-cold water bath for 30 min. The swollen cells were collected and sonicated in an ice-cold water bath. Samples were then centrifuged at 20 100 g for 20 min at 4 °C and the supernatant was mixed with the indicated antiserum for 3 h at 4 °C with gentle agitation. Protein A–agarose beads (20 μl; Calbiochem) were added to the mixture and incubated for another 1 h at 4 °C with agitation. The beads were then collected by centrifugation and washed three times with TEP buffer. The bound proteins were eluted from the agarose beads with E buffer [10 mM glycine/HCl (pH 3.0), 0.01 % sodium azide] and immediately neutralized with an equal amount of N buffer (500 mM Tris base, 0.01 % sodium azide). Samples were boiled in SDS-PAGE sample buffer and subjected to SDS-PAGE followed by Western blotting with anti-VP80 or anti-HA100 antiserum (Deng et al., 2007) as the primary antibody and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) as the secondary antibody. Parallel experiments with pre-immune antiserum were performed simultaneously as negative controls.

    Acknowledgments

    The work was supported by a key project from NSFC grants (30630002), a 973 project (2009CB118903) and a PSA project from MOST and KNAW (2008AA000238). We would like to thank Dr George F. Rohrmann for critically reviewing the manuscript and Dr David A. Theilmann for sharing information about baculovirus protein–protein interactions.

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