Abstract
The envelope fusion proteins of baculoviruses, glycoprotein GP64 from group I nucleopolyhedrovirus (NPV) or the F protein from group II NPV and granulovirus, are essential for baculovirus morphogenesis and infectivity. The F protein is considered the ancestral baculovirus envelope fusion protein, while GP64 is a more recent evolutionary introduction into baculoviruses and exhibits higher fusogenic activity than the F protein. Each of the fusion proteins is required by the respective virus to spread infection within larval tissues. A recombinant Helicoverpa armigera NPV (HearNPV) expressing GP64 from Autographa californica multiple nucleopolyhedrovirus, vHaBac-gp64-egfp, was constructed, which still retained the native F protein, and its infectivity was assayed in vivo and in vitro. Analyses by one-step growth curve to determine viral titre and by quantitative PCR to determine viral DNA copy number showed that vHaBac-gp64-egfp was more infectious in vitro than the control, vHaBac-egfp. The polyhedrin gene (polh) was reintroduced into the recombinant viruses and bioassays showed that vHaBac-gp64-polh accelerated the mortality of infected larvae compared with the vHaBac-egfp-polh control, and the LC50 (median lethal concentration) of vHaBac-gp64-polh was reduced to approximately 20 % of that of vHaBac-egfp-polh. Therefore, incorporation of GP64 into HearNPV budded virions improved virus infectivity both in vivo and in vitro. The construction of this bivalent virus with a more efficient fusion protein could improve the use of baculoviruses in different areas such as gene therapy and biocontrol.
Introduction
Baculoviruses are a large family characterized by an infection cycle that produces two progeny phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). Distinct in structure and function (Blissard, 1996), BV is highly infectious in cell culture and is responsible for cell-to-cell transmission in infected animals, while ODV initiates infection in the midgut epithelial cells via direct membrane fusion (Summers, 1971). BV contains abundant envelope fusion proteins that are necessary for virus entry into host cells via the endocytic pathway (Volkman & Goldsmith, 1985).
The family Baculoviridae is divided into four genera: Alphabaculovirus, Betabaculovirus, Gammabaculovirus and Deltabaculovirus. The genus Alphabaculovirus (formerly known as nucleopolyhedroviruses, NPVs) contains two distinct groups (I and II) based on their host and major fusion protein (Hayakawa et al., 2000; Herniou et al., 2001). Glycoprotein GP64 was identified from the members of group I NPVs (Blissard & Rohrmann, 1989; Whitford et al., 1989) and was determined to be essential for virus entry into cells and for an efficient budding process (Hefferon et al., 1999; Oomens & Blissard, 1999). GP64 is also responsible for inducing low-pH-dependent fusogenic activity, the event believed to be essential for virus entry into host cells via the endocytic pathway (Blissard & Wenz, 1992). The F protein was found in the members of group II NPVs and plays similar roles to GP64, such as cellular receptor recognition and association, membrane fusion and efficient budding (IJkel et al., 2000; Long et al., 2006; Pearson et al., 2000). The F protein could rescue gp64-null Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and thus is believed to be a functional analogue of GP64 (Long et al., 2006; Pearson et al., 2000). It was reported that GP64 failed to rescue f-null Spodoptera exigua MNPV (SeMNPV) virus (Westenberg & Vlak, 2008). However, it was recently shown that GP64 could partially rescue the function of the Helicoverpa armigera NPV (HearNPV) F protein (Wang et al., 2010).
An F-like protein is hypothesized to be the ancestor of the fusion protein (Pearson et al., 2000), and it is postulated that GP64 was captured by an ancestral group II baculovirus, which evolved into group I NPVs (Pearson & Rohrmann, 2002). As group I NPVs still carry an F protein homologue (F-like protein) (Lung et al., 2003; Pearson et al., 2001), it is logical to suggest that a certain function is furnished by the F-like protein to group I NPVs. GP64 has a highly effective fusogenic activity (Blissard & Wenz, 1992; Wang et al., 2010) and AcMNPV could be transduced into mammalian cells via the interaction of GP64 with the phospholipids on the cell surface (Tani et al., 2001). Consequently, many investigations have focused on exploring and modifying AcMNPV to be an effective tool for gene delivery into mammalian cells (Boyce & Bucher, 1996; Kost & Condreay, 2002). Liang et al. (2005) reported that group II HearNPV failed to transduce mammalian cells, but insertion of the gp64 gene enabled the bivalent virus to transduce mammalian cells.
In this study, the virus infectivity of a recombinant HearNPV, vHaBac-gp64-egfp, carrying the GP64 from AcMNPV and the native F protein from HearNPV (Wang et al., 2005), were characterized both in vivo and in vitro. We found that the incorporation and expression of GP64 resulted in production of BVs that were more infectious than the control vHaBac-egfp. Moreover, by reintroducing the polyhedrin gene (polh), bioassays showed that the efficiency in killing the third-instar larvae of H. armigera was enhanced in a virus expressing both F and GP64, resulting in a shorter median survival time (ST50) and lower median lethal concentration (LC50). This concept of construction of the bivalent virus could improve the use of baculoviruses in different areas, such as gene therapy or biocontrol.
Results
GP64 expression and incorporation into BVs
The schematics of generation of vHaBac-egfp and vHaBac-gp64-egfp are illustrated in Fig. 1 (Wang et al., 2005). Western blot analyses were performed to confirm the correct expression and incorporation of GP64 into the recombinant HearNPV BVs (Fig. 2a). The F1 subunit of HearNPV F (HaF) protein was detected in both viruses (Fig. 2a, left, lanes 1 and 2), while only GP64 could be blotted and detected from vHaBac-gp64-egfp (Fig. 2a, right, lane 2). VP80 was immunoblotted as an internal control of approximately equivalent amounts of BVs in respective lanes (Fig. 2a, middle, lanes 1 and 2).
Schematic representation of recombinant bacmids and construction of recombinant virus vHaBac-gp64-polh. vHaBac-gp64-polh was generated as described in Methods. polh was reintroduced under the control of pPolh, and gp64 was inserted under the control of both early expression promoter pOp166 and late expression promoter pP10. pOp166, Orgyia pseudotsugata NPV (OpMNPV) fusion protein (ORF166) promoter; pPolh, HearNPV polyhedrin promoter; pHsp70, heat shock protein 70 promoter; SV40 poly(A), simian virus 40 polyA; Tn7R and Tn7L, right and left insertion sites, respectively, of mini-AttTn7; Gmr, gentamicin-resistance gene.
Detection of GP64 incorporation into BVs and fusogenic activity. (a) Western blot analyses of envelope proteins in recombinant BVs. Virions were purified from the supernatants of infected HzAM1 cells at 6 days post-infection (p.i.) and subjected to SDS-PAGE. Proteins were separated, blotted and reacted with polyclonal antibodies against HaF1 (left), VP80 (middle) and GP64 (right). Lanes: 1, vHaBac-egfp BV; 2, vHaBac-gp64-egfp BV; M, pre-stained protein markers with indicated molecular mass. (b) Syncytium formation assay of infected HzAM1 cells. HzAM1 cells were incubated with vHaBac-gp64-egfp (left) or vHaBac-egfp (right) at an m.o.i. of 5 TCID50 units per cell. At 48 h p.i., cells were rinsed and incubated with Grace’s insect medium at pH 4.8 for 5 min at room temperature. Syncytium formation was visualized by fluorescence microscopy at 24 h after the pH shift. (c) Quantitative analyses of fusogenic activity induced by envelope fusion proteins. Fixed HzAM1 cells infected by viruses were stained by Hochest 33258 (Biotime) at room temperature. Images under UV light and bright light fields of the same area were taken. The number of cells expressing EGFP and forming syncytia (fused cells containing at least four nuclei) were scored, respectively, within five random fields of view. Fusogenic activity was expressed as the number of EGFP-expressing cells over fused multinuclear cells. Error bars represent sd.
GP64 in HearNPV induced more effective fusogenic activity
A syncytium-formation assay was carried out to determine the fusogenic activity in infected HzAM1 cells induced by vHaBac-gp64-egfp and vHaBac-egfp. Larger syncytia with multinuclear cells were observed in cells infected by vHaBac-gp64-egfp than those infected by vHaBac-egfp 24 h after a pH shift (Fig. 2b). Fusogenic activity was measured as described in Methods. A greater number of cells infected by vHaBac-gp64-egfp (87.0 %±0.9 %) participated in syncytium formation, while the fusiogenic activity induced by vHaBac-egfp was less effective (50.3 %±1.7 %) (Fig. 2c). We conclude that the incorporation of GP64 in HearNPV BVs induces more efficient fusogenic activity.
GP64 produced more infectious progeny BVs
One-step growth-curve analysis was conducted to study the effect of GP64 incorporation on virus growth kinetics of produced BVs with increased infectivity in vitro in comparison to the control. The growth curve showed that both viruses had similar growth kinetics (Fig. 3), but vHaBac-gp64-egfp produced a greater amount of infectious progeny viruses than vHaBac-egfp. At 72 h post-infection (p.i.), the BV titre of vHaBac-gp64-egfp was 2.2±1.2×106 TCID50 ml−1, which was about fivefold higher than that of vHaBac-egfp (3.9±0.6×105 TCID50 ml−1). Statistical analysis revealed that vHaBac-gp64-egfp yielded significantly more infectious BVs than vHaBac-egfp (P<0.05).
One-step growth-curve analysis of recombinant viruses. HzAM1 cells were infected with vHaBac-egfp (⧫) or vHaBac-gp64-egfp (▪) at an m.o.i. of 5 units per cell. Supernatants were harvested and cell debris was clarified at indicated times p.i. BV titres were determined by end-point dilution assay and transformed exponentially. Data were obtained from three separate infections. Error bars represent sd.
To investigate further whether the incorporation of GP64 into HearNPV might facilitate virus production or virus infectivity in vitro, quantitative real-time PCR (qPCR) was performed to determine the number of virus particles released into supernatants of the samples collected at 72 h p.i. from the above one-step growth-curve assay. The result showed that the number of genome copies ml−1 for vHaBac-egfp was 1.7±1.1×1010, while that for vHaBac-gp64-egfp was 3.3±1.5×1010, which was not significantly different (P>0.05) (Table 1). However, one TCID50 unit of vHaBac-egfp is equivalent to 4.2±2.0×104 copies of the viral genome, whereas for vHaBac-gp64-egfp, one TCID50 unit was significantly different (P<0.05) and equivalent to 1.5±0.1×104 copies of the viral genome (Table 1). The difference indicates that vHaBac-gp64-egfp was about three times more infectious than the control.
GP64 facilitated virus entry into cells
Copies of the viral genome DNA in cells at indicated time points post-incubation was measured by qPCR as described in Methods. The efficiency of virus entry into cells was defined as viral DNA copies per cell when the cells were incubated with virus at m.o.i.s of 5, 1 and 0.1, respectively (Fig. 4). The data showed that more copies of viral DNA of vHaBac-gp64-egfp were detected in cells than that of vHaBac-egfp at each indicated time point post-incubation, irrespective of input m.o.i. If we take the m.o.i. of 5 as an example, 1.1±0.3×103 vHaBac-egfp DNA copies per cell were detected at 15 min post-incubation and 1.8±0.2×103 copies per cell at 1 h post-incubation (Fig. 4a). Meanwhile, 1.6±0.3×103 vHaBac-gp64-egfp DNA copies per cell were detected at 15 min post-incubation and 3.0±0.7×103 copies per cell at 1 h post-incubation (Fig. 4a). Therefore, the number of vHaBac-gp64-egfp virus particles doubled inside a cell within the two time periods. At the same time, the number of DNA copies of vHaBac-egfp within a cell did not increase substantially between 15 and 60 min.
Determination of the efficiency of virus entry into HzAM1 cells. HzAM1 cells were incubated with vHaBac-egfp or vHaBac-gp64-egfp at an m.o.i. of 5 (a), 1.0 (b) or 0.1 (c) units per cell at 27 °C. The cells were rinsed and harvested at the indicated time points. Cellular genomic DNA was isolated for qPCR to measure the number of viral genome DNA copies in infected cells. The viral DNA copies per cell were calculated. Each analysis was done in triplicate. Error bars represent sd.
When the cells were infected with viruses at lower m.o.i. (1.0 or 0.1), the number of vHaBac-gp64-egfp DNA copies per cell at 1 h post-incubation increased more than twice between 15 and 60 min post-incubation. The vHaBac-egfp DNA copies hardly increased between 15 and 60 min post-incubation (Fig. 4b, c). Therefore, we concluded that HearNPV BVs invasion into HzAM1 cells benefited from the presence of GP64.
GP64 accelerated mortality of infected insects and reduced the LC50
A recombinant virus vHaBac-gp64-polh was generated (Fig. 1a). Occlusion bodies (OBs) were purified from vHaBac-gp64-polh-infected larvae, and so were OBs of vHaBac-egfp-polh (Song et al., 2008). The latter virus was used as a control in bioassays. Third-instar H. armigera larvae were infected with different OB concentrations of vHaBac-gp64-polh or vHaBac-egfp-polh (Table 2). The LC50 of the two recombinant viruses in third-instar larvae showed a significant difference. The mean LC50 of vHaBac-gp64-polh is 1.6×104 polyhedral inclusion bodies (PIBs) ml−1, approximately 20 % of that of the control virus vHaBac-egfp-polh. The ST50 was calculated using an OB concentration of 3×106 PIBs ml−1. When larvae were infected orally with viruses, the ST50 of vHaBac-egfp-polh was 98 h p.i., while that of vHaBac-gp64-polh was 90 h p.i. (Table 3). Statistical analysis indicated that the ST50 values of the two viruses were significantly different (P<0.01) (Table 3). Therefore, insertion of gp64 helped not only to reduce the LC50, but also to shorten the ST50 so as to accelerate the mortality of infected larvae.
Discussion
It is known that both GP64 and the F protein are baculovirus envelope fusion proteins responsible for low-pH-dependent fusogenic activity, which is believed to be important for baculovirus entry into cells via the endocytic pathway. In this study, vHaBac-gp64-egfp is a bivalent virus carrying the two envelope fusion proteins, GP64 and the native F protein. We investigated the value added to the virus biology from the presence of GP64 in BVs. The bivalent virus generated larger-scale and more abundant syncytia of infected HzAM1 cells (Fig. 2b, c). This confirmed the result that the membrane-fusion capability of GP64 is higher than that of the F protein in HzAM1 cells (Wang et al., 2010). If GP64 could induce more efficient fusion, the infection process or alternatively virus entry into cells should benefit from the incorporation of this protein into BVs. One-step growth-curve analysis revealed that vHaBac-gp64-egfp produced more infectious progeny virus particles than the control virus, vHaBac-egfp (Fig. 3). However, the number of viral DNA genome copies ml−1 of vHaBac-gp64-egfp was not significantly higher than that of vHaBac-egfp (Table 1). These data showed that the insertion of GP64 did not have a significant effect on the number of BV particles released from cells, but more virus particles of vHaBac-gp64-egfp than vHaBac-egfp were able to infect HzAM1 cells.
Virus entry into cells proceeds in a stepwise fashion, such as recognition and association with cell receptors (binding), internalization via endocytic vesicles (endocytosis) and escaping from endosomes (fusion). In this research, the efficiency of virus entry into HzAM1 cells was examined by qPCR. We found that the longer the time the cells were incubated with viruses, the more viral DNA copies per cell were detected. However, more viral DNA copies of vHaBac-gp64-egfp accumulated in cells with greater efficiency than that of the control virus (Fig. 4). It was suggested that GP64 binds to the same virus-binding sites in HzAM1 cells where HearNPV also binds to gain entry into cells (Wang et al., 2010). It still remains to be investigated whether GP64 incorporation into HearNPV BVs facilitates binding to cell receptors (virus association with cells) or escaping from endosomes via membrane fusion or both.
When the polyhedrin gene (polh) was reintroduced into these viruses, the OBs of vHaBac-gp64-polh not only killed the third-instar larvae of H. armigera more rapidly (shortening the ST50), but also reduced the concentration sufficient to kill insects (decreased the LC50) (Tables 2 and 3). ODVs of baculoviruses initiate primary infection by fusing with the insect midgut epithelial cells. The BVs are responsible for cell-to-cell spread and transmission through susceptible cells and tissues in the body of insects and, finally, insect mortality. It is suggested that the insertion of gp64 appeared to accelerate the spread of vHaBac-gp64-polh BVs throughout the insect body and thus enhanced the capability of the virus to kill the insect. It would be very interesting to test a recombinant HearNPV with GP64 under field conditions to assess whether its bioinsecticidal activity has actually been enhanced.
GP64 and the F protein are functional analogues. GP64-containing baculoviruses, such as AcMNPV and Orgyia pseudotsugata NPV (OpMNPV), still encode homologues of the F protein (F-like protein), such as Ac23 (Lung et al., 2003) and Op21 (Pearson et al., 2001), which lack fusogenic ability but are virulence factors in vivo and in vitro (Lung et al., 2003; Wang et al., 2008a). GP64 could not rescue the activity of the F protein in f-null SeMNPV (Westenberg & Vlak, 2008), but could partially rescue the function of the F protein in f-null HearNPV, resulting in much less infectious recombinant viruses (Wang et al., 2010). This indicates that, unlike GP64, the F protein has more than one function (Wang et al., 2010). In this study, we found that by having both the F protein and GP64, the bivalent virus vHaBac-gp64-egfp is more infectious than the control. The virus vHaBac-gp64-egfp can be considered a hybrid of group I and group II NPVs with a native F protein and a fully functional GP64. So vHaBac-gp64-egfp may take great advantage of the functional combination of GP64 and the F protein to become more infectious. Furthermore, it is suggested that the bivalent virus vHaBac-gp64-egfp would be a suitable model to investigate the evolutionary pressure on the F protein.
In summary, our research points to the advantages of having GP64 in HearNPV and its implications on the infection process. By incorporation of GP64 into HearNPV, the virus became more infectious not only in vitro but also in vivo. Moreover, this concept of construction of the bivalent virus could improve the use of baculoviruses for gene therapy, and the bivalent virus vHaBac-gp64-polh could be developed into more effective biopesticides.
Methods
Cells and viruses.
Helicoverpa zea (HzAM1) cells were cultured at 27 °C in Grace’s insect medium (Gibco-BRL), pH 6.0, supplemented with 10 % (v/v) FBS (Gibco). Viruses used in this study were generated previously, including vHaBac-egfp (Wang et al., 2005), vHaBac-gp64-egfp (Wang et al., 2005) and vHaBac-egfp-polh (Song et al., 2008); schematics are illustrated in Fig. 1.
Generation of recombinant bacmid HaBac-gp64-polh.
Since HearNPV bacmid (HaBacHz8) does not contain polh (Wang et al., 2003), polh was reintroduced into HaBacHz8. Competent DH10Bac cells containing the HearNPV bacmid (HaBacHz8) and a helper plasmid were transformed with the donor plasmid pFB-Op166-gp64-polh (Wang et al., 2010) (Bac-to-Bac Baculovirus Expression System user manual; Invitrogen). The recombinant bacmid, referred to as HaBac-gp64-polh (Fig. 1), was selected by gentamicin resistance and blue/white screening and verified by PCR.
Transfection and infection assay.
HzAM1 cells were seeded into 35 mm diameter tissue-culture dishes with 3×105 cells per dish. Transfection was performed by usingapproximately 1 µg bacmid DNA and 12 µl Lipofectin reagent (Invitrogen) according to the Bac-to-Bac Baculovirus Expression System’s manual. At 6 days post-transfection, supernatants were harvested and clarified by centrifugation (3000 g, 5 min) and then used to infect fresh HzAM1 cells.
Western blots analyses of GP64 incorporation into BVs.
Supernatants from infected HzAM1 cells were harvested and centrifuged to remove cell debris. BVs in the supernatants were spun down by centrifugation (4 °C, 14 000 g, 45 min), disrupted under reducing condition (4×SDS-PAGE sample buffer) and separated by SDS-PAGE. The proteins in the gel were transferred onto a PVDF membrane (Millipore Corporation) and immunoblotted first with polyclonal anti-F1, anti-GP64 and anti-VP80 antibodies (generated in our laboratory), respectively, then with alkaline phosphatase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) as secondary antibody. The signals were detected with NBT/BCIP reagents (Sino-American Biotechnology Company).
Low-pH-induced envelope fusion assay.
HzAM1 cells were infected with recombinant viruses at an m.o.i. of 5. At 48 h p.i., cells were rinsed three times with Grace’s insect medium (pH 6.0) and incubated with 1 ml Grace’s insect medium at pH 4.8 for 5 min. The supernatants were then replaced with normal Grace’s insect medium (pH 6.0) with 10 % FBS. Twenty-four hours after the pH shift, syncytium formation was visualized by fluorescence microscopy using a Nikon ECLIPSE TE2000-5 microscope. To quantify the syncytium formation induced by recombinant viruses, the cells were fixed with 4 % paraformaldehyde (Sigma-Aldrich) and incubated with Hochest 33258 (Biotime) at room temperature. Fields of multinuclear cells were photographed. The number of cells expressing EGFP and forming syncytia was scored within five fields that were chosen at random. Fusogenic activity was expressed as the ratio of fused cells to infected cells. Syncytial masses were defined as fused cells containing at least four nuclei.
One-step viral growth-curve analysis.
One-step viral growth-curve analysis was performed as described previously with a slight modification (Wang et al., 2008b). HzAM1 cells (8×105 cells per well) were infected at an m.o.i. of 5 TCID50. Supernatants were harvested at 0, 12, 24, 36, 48, 60 and 72 h p.i. and titrated by an end-point dilution assay. The experiment was done in triplicate. BV titres were log-transformed and sd was determined by Microsoft Excel Software (version 2003) based on three replicates.
qPCR analysis of BV infectivity and virus entry into HzAM1 cells.
To isolate BV DNA, supernatants (100 µl) harvested at 72 h p.i. were incubated with 100 µl 20 % PEG8000 (Sigma-Aldrich) in 1 M NaCl for 30 min at room temperature. Virions were spun down (4 °C, 12 000 g, 15 min) and then resuspended in 20 µl H2O, 80 µl virus disruption buffer (10 mM Tris/HCl pH 7.6, 10 mM EDTA, 0.25 % SDS) and 5 µl proteinase K (20 mg ml−1: Merck). Virions were lysed at 50 °C for 1 h, and viral DNA was extracted with phenol and then precipitated with ethanol (Sinopharm Chemical Reagent Co. Ltd) and dissolved in 40 µl H2O.
To determine the efficiency of virus entry into cells, HzAM1 cells (1×105 cells per well) were incubated with viruses at different m.o.i.s at 27 °C. At 15 min, 35 min, 1 h and 3 h post-incubation, the cells were rinsed three times with PBS and harvested for genomic DNA isolation using a commercial kit (Genomic DNA rapid isolation kit; BioDev). Templates comprising 5 µl BV DNA and 1 µl total cellular DNA were used for qPCR analysis as described previously (Wang et al., 2008a). The data were analysed by spss (SPSS Inc.) using a Compare Means t-test.
Bioassays.
Oral infectivity of the recombinant viruses was measured by the droplet-feeding method in early-third-instar H. armigera larvae. OBs were harvested and purified from diseased larvae as described earlier (Sun et al., 1998). Larvae were starved overnight and exposed to concentrations of 3×106, 1×106, 3×105, 1×105, 3×104, 1×104, 3×103 and 1×103 OBs ml−1. In each analysis, 48 insects were tested per concentration. The experiments were done in duplicate. LC50 values (OBs ml−1) were determined by polo probit analysis and compared by standard lethal dose ratio comparison (SPSS Inc.). ST50 of the viruses was determined with third-instar larvae using a food-contamination method. Ten microlitres of OBs (3×105 OBs ml−1 or 3×106 OBs ml−1) of the viruses were applied to small pieces of diet (3 mm3) and exposed to early-third-instar H. armigera larvae. The intake was sufficient to kill 100 % of the tested larvae. Time 0 was defined as the point at which larvae were transferred onto fresh diet. For each virus, 48 insects were used and the mortality was checked every 6 h. The experiments were done in duplicate. ST50 values were calculated using the Kaplan–Meier estimator and further compared using the log-rank test (Mantel–Cox).
Acknowledgements
This work was supported by a 973 project (2009CB118903), grants from the National Natural Science Foundation of China (31130058, 31125003 and 31100120), the PSA project from MOST and KNAW (2008DFB30220) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-G-16). We thank Dr Xiulian Sun (Wuhan Institute of Virology, Chinese Academy of Sciences) for statistical analysis.