Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

Helicoerpa armigera nucleopolyhedrovirus (HearNPV) ha83 is a late expressed gene that encodes a chitin binding protein. Chitin domain truncation studies revealed that the cysteine at the 128 amino acid position probably played an important role in both chitin binding ability and protein transmission of Ha83. In order to study the function of ha83 in the HearNPV infection cycle, an ha83 knockout HearNPV (Ha83KO) was constructed via homologous recombination. Viral growth and viral DNA replication curves showed that fewer budded virions were produced in Ha83KO transfected cells, while viral DNA replication was increased. Electron microscopy revealed that fewer nucleocapsids were transmitted from virogenic stroma in the Ha83KO transfected cell nucleus, and the morphology of occlusion bodies was prominently larger and cube-shaped. Furthermore, DNA quantity in occlusion bodies of Ha83KO was significantly lower than the occlusion bodies of HaWT. The transcription analysis indicated that these changes may be due to the decreased expression level of viral structural associated genes, such as polyhedrin, p10, pif-2, or cg30 in Ha83KO infected cells. Above results demonstrated that the cysteine at the 128 amino acid position in Ha83 might be the key amino acid, and Ha83 plays an important role in BVs production and OBs assembling.

UDP-glucosyltransferase gene (egt) 7 , the late expression factor 2 gene (lef-2) 8 , chitinase 9 , ha81 10 , p10 11 , and the F-protein gene (ha133) 12 have been characterized. HearNPV orf83 (ha83) as a chitin binding domain encoded gene in HearNPV has no homologue that shares over 30% identity in nucleotide sequence when comparing with the genomic sequenced NPVs and GVs except for HezeNPV and SpexNPV. Ha83 protein is a nonstructural protein revealed to contain a conserved chitin binding domain (CBD) in the C-terminal. The protein is first detected in the cytoplasm of infected HzAM1 cells at 12 h post infection (h p.i.) then trafficked into the nucleus at 48 h p.i. 13 . Ha83 is predicted to code for 18.8 kDa protein but its function in the viral life cycle remains unknown.
To determine the role of ha83 in HearNPV replication and BVs production, a HearNPV bacmid was used to generate an ha83 deletion mutant. We found that ha83 is involved in BV production for the deletion mutant and produces fewer BVs than the wild type in one step viral growth curve analysis. However, viral DNA replication was increased by the deletion of ha83 in the quantitative PCR analysis. Electromicroscopy observation showed that ha83 is not required for nucleocapsid formation but it may affect polyhedra morphology. To investigate the functional role of CBD in the Ha83 subcellular transport and chitin binding activity, eukaryotic expression vectors and prokaryotic expression vectors were constructed to truncate the C-terminus region, a cysteine-rich region. The transient eukaryotic expression vectors contained the truncated ha83 fused with egfp and were transfected into HzAM1 cells and confocal microscopic analysis showed that, in late infection, the first N-terminal 141 amino acids were necessary for the traffic of Ha83 from the cytoplasm into the nucleus. The truncated prokaryotic purified proteins also revealed a similar result that the conserved cysteine at the 128 amino acid position was important to Ha83 chitin binding activity.

Results
Transcriptional mapping of the 3′ and 5′ end of ha83 transcripts. The 3′ Rapid Amplification of cDNA Ends (RACE) obtained sequences mapped the 3′ end of the ha83 transcript at 16 nucleotides (nt) downstream of the putative translation stop codon TAA and 13 nt downstream of the last A of the polyadenylation signal sequence AATAAA, which is overlapped with the stop codon TAA (Fig. 1A). Ten clones were sequenced from 5′ RACE and the ha83 mRNA putative start sites were identified at 374 nt upstream of the ATG codon at three contiguous A residues (Fig. 1B). A search of the 374 nt 5′ untranslated regions (UTR) showed that there are two TATA boxes located at positions − 107 and − 293 upstream of the ATG; a late strong promoter ATAAG element was at position − 202; another TAAG late promoter element was found at − 27; five early promoter CATT elements were found at − 67, − 187, − 211, − 255, and − 310 upstream of the ATG (Fig. 1B).

Expression of truncated His-Ha83s and chitin binding assays.
To identify the functional cysteine needed for the chitin binding domain in Ha83, we constructed seven pET-28a(+ ) plasmids containing different parts of HearNPV Ha83 fused with His-tag, as well as an ha81 expression vector to  (Fig. 2A). The His-tag antibody detected specific bands in close agreement with those of target bands in the coomassie blue stained PAGE gel (Fig. 2B). The bound protein (B) and unbound protein in the supernatant (F) was first separated by centrifugation then lyophilized and loaded in each lane of a 10% Tricine-SDS-PAGE system. The gels were stained with Coomassie Blue (Sigma). In order to improve the clarity and conciseness, the figures exhibited here were cropped; the full-length gels or blots are presented in Supplementary Figure S2 The purified His-tag fused proteins were incubated with chitin colloid for 16 h to perform the chitin binding assay. Distinct bound protein bands were found from His-Ha83 and His-TC71 tested lanes in the 10% tricine SDS-PAGE gel (Fig. 2C). For His-TC128, a clear band emerged in the unbound protein loaded lane, as well as in the bounded protein loaded lane (Fig. 2C). The following mutants that lost two or more conserved cysteines showed a weak ability to bind with chitin ( Fig. 2C): The Ha81 did not bind with the chitin colloid as no band was found at the bound lane, but a strong band was found at the unbound lane (Fig. 2C). These results demonstrated that conserved cysteines were related to the Ha83 chitin binding activity.
To further investigate the chitin binding capacity and ability of different mutants, each of the proteins (His-Ha83, His-TC151, His-TC142, His-TC128, His-TC118, His-TC113, and His-TC71) were mixed with a certain quantity of chitin colloid. The chitin binding capacity was first determined by the incubation time (Fig. 2D). After ten hours, the highest binding capacity was ascertained for His-Ha83 (0.044 μ M or 0.97 μ g proteins/mg chitin) followed by His-TC151 (0.038 μ M or 0.81 μ g proteins/mg chitin), His-TC142 (0.032 μ M or 0.63 μ g proteins/mg chitin), His-TC128 (0.024 μ M or 0.43 μ g proteins/mg chitin), His-TC118 (0.023 μ M or 0.38 μ g proteins/mg chitin), His-TC113 (0.023 μ M or 0.37 μ g proteins/ mg chitin), and His-TC71 (0.024 μ M or 0.36 μ g proteins/mg chitin). The chitin binding ability was then determined by equilibration (16 h) of different quantities of tested protein with a chitin colloid sample. The dissociation constants (K d ) were determined by a double-reciprocal plot (Fig. 2E). The slope of the regression lines decreased with the cysteines truncation (Fig. 2E). The highest K d value was obtained for His-TC71 (K d of 7.6 μ M) and the lowest for His-Ha83 (K d of 0.62 μ M), which implied that His-Ha83 binds about 13 times more effectively than His-TC71.
Residue cysteine 128 is important for ha83 traffic from the cytoplasm to the nucleus. To further characterize the function of the Ha83 chitin binding domain in the baculovirus life cycle, the subcellular localization of Ha83 and truncated mutants were analyzed by confocal fluorescence microscopy. All transfected cells showed only background level fluorescence at 6 h p.t. and were observed to contain green fluorescence in the cytoplasm at 12 h p.t. (data not shown). At 24 h p.t., all transfected cells contained green fluorescence evenly in the nucleus and cytoplasm, which indicated that Ha83 began to traffic from the cytoplasm into the nucleus at this point of time without distinction of truncation (Fig. 3A). However, at 48 h p.t., the EGFP fused protein in pGE-Ha83/HaBacHZ8 co-transfected cells were all detected in the nucleus, which indicated that all Ha83 were located in the nucleus (Fig. 3B). Following that, in pGE-TC151 and pGE-TC142 co-transfected cells, target proteins were predominantly localized within the nucleus, with levels of green fluorescence detected in the cytoplasm (Fig. 3B). However, pGE-TC128, pGE-TC118, pGE-TC113, and pGE-TC71 co-transfected HzMA1 cells showed dispersal EGFP fluorescence at the same point of time (Fig. 3B). Whereas, Ha81, which was selected as a negative control, was reported to be located in the cytoplasm from 24 to 48 h.p.t. (10). These results suggested that the cysteine at the 128 amino acid position probably play an important role in the Ha83 protein traffic from cytoplasm to nucleus.
Deletion of ha83 affects BV production. DNA  To determine infectious budded virus production, supernantant from Ha83KO, Ha83Rep, and HaWT DNA transfected cells were harvested at 96 h p.t. and used to treat uninfected HzAM1 cells. By 72 h p.i., the majority of the Ha83Rep or HaWT infected cells expressed EGFP, whereas in Ha83KO DNA infected cells, only 75% expressed EGFP, which confirmed that ha83 deletion affected infectious BV production (Fig. 4A).
Microscopy analyses revealed OBs in infected cells when all of the three constructs were used, and no differences in the numbers of cells containing OBs were observed at 48 h p.i. (data not shown). By 96 h p.i., most of the Ha83Rep and HaWT infected cells contained OBs, whereas fewer of the Ha83KO infected cells contained OBs (Fig. 4A). The infection experiments showed that deletion of ha83 led to a decrease in the production of infectious BVs but did not affect OB formation.
To further assess the effect of ha83 deletion on BVs production, a one step viral growth curve was performed. As expected, the supernatants from HzAM1 cells transfected with Ha83Rep and HaWT revealed a steady increase in virus production, and the ha83 repaired virus showed similar growth kinetics in titer to the WT virus (   Ha83 is critical for polyhedra formation. To further investigate the lower BV production in the ha83 knockout virus and to analyze whether the deletion of ha83 has any effect of virus morphogenesis, electron microscopy analyses were performed on Ha83KO, Ha83Rep, and HaWT transfected HzAM1 cells. As expected, cells transfected with the control bacmid, Ha83Rep, showed typical characteristics of a HearNPV infection at 72 h p.i., including a net-shaped virogenic stroma (VS) inundated with electron-dense rod-shaped nucleocapsids (Fig. 5Ae,f), numerous nucleocapsids transported from the VS to nuclear membrane (Fig. 5Ae), and polyhedra containing mature virions in the ring zone (Fig. 5Af). Cells transfected with HaWT showed characteristics similar to those transfected with Ha83Rep (data not shown).
In Ha83KO bacmid transfected cells, a well-defined VS and abundant electron-dense rod-shaped nucleocapsids were observed (Fig. 5Ab) and the nucleocapsids were morphologically indistinguishable from those observed in HaWT (data not shown) and Ha83Rep transfected cells (Fig. 5Ae). However, most of the nucleocapsids were observed in the VS, and seldom were transported from the VS to nuclear membrane (Fig. 5Aa,b,c). Consequently, even though the shape and size of the polyhedra were comparable to those in WT bacmid transfected cells (data not shown) and repair bacmid transfected cells (Fig. 5Ad,e,f), the shape of polyhedra observed in the Ha83KO bacmid transfected cells turned into a square and appeared to contain fewer ODVs (Fig. 5Aa,c).
Additional TEM observation of polyhedra purified from Ha83KO and HaWT infected larvae showed similar morphology changes and ODV quantity of OBs as described above (Fig. 5B). The shape of polyhedra was further verified by scanning electron microscopy (Fig. 5C). Ha83KO OBs had diameters between 0.71 and 1.62 μ m with an average (± SE) value of 1.11 ± 0.19 μ m (N = 80), which was significantly larger than the diameter of HaWT OBs that ranged from 0.64 to 1.24 μ m with an average value of 0.89 ± 0.13 μ m (N = 80) (t = 24.39, df = 79, P < 0.0001). Ha83KO OBs were 25% larger than those of HaWT OBs on average diameter . In addition, the morphological form of Ha83KO OBs had a cubic shape compared to the polyhedral form of wild type OBs (Fig. 5).

Discussion
HearNPV ha83 has been identified as one of the 20 unique genes 6 , which share no more than 30% amino acid sequence identity with other baculovirus 13 . Our studies have shown that ha83 is important for BV production and the assembling of normal OBs; and that the cysteine at 128 amino acid position in Ha83 chitin binding domain plays an important role in chitin binding activity and protein transmission. 5′ RACE analysis showed that the transcription initiation site of ha83 is at 374 nt upstream of ATG, and this long promoter contains many well characterized promoter elements. Although ha83 has been reported as a late expression gene, there are five CATT early promoter elements within the 5′ UTR of ha83 13 , whereas a similar inconsistent early gene ac78 with two consensus baculovirus late gene promoter motif TAAG has been reported in AucaMNPV 14 . The products of baculovirus early genes are often involved in DNA replication, regulation of late gene expression and host-modification processes 15 . Ha83 contains a predicted type Ⅱ chitin binding domain at the C terminal which is characterized by the presence of a 6-cysteine motif with the consensus sequence C-X 13-20 -C-X 5-6 -C-X 9-19 -C-X 10-14 -C-X 4-14 -C (where X is any amino acid other than cysteine) 16 . Detailed analysis of the six truncated mutants of Ha83 suggested that the cysteine at the 128 aa position might be the key amino acid in the Ha83 CBD, for the truncated mutant without the cysteine128 showed weak ability to bind with chitin colloid or protein subcellular traffic (Fig. 2, Fig. 3). The type Ⅱ CBD is referred to as the peritrophin-A domain because it is found in peritrophin-A chitin-binding proteins, particularly in the peritrophins of insect peritrophic membranes (PM) 16 , such as Ac83, which is identified as a per os infectivity infector 17,18 . However, as Ha83 contains only type Ⅱ CBD, the ha83 deleted HearNPV would lead to lower oral infectivity compared with wild type OBs (data not shown). This result is quite different from the function of Ac83 which would lead to no oral infectivity at all when the worms were fed with the knockout virus 17 . GP37, another typical CBD contained a protein encoded by Spodoptera litura multicapsid nucleopolyhedrovirus (SpltNPV) orf32, was reported to have chitin binding ability with a dissociation constant (K d ) for 0.28 μM 19 which gives it an approximately two times higher efficiency to bind with chitin than Ha83 with a K d value for 0.62 μM (Fig. 2E). This GP37 has been shown to have pancellular dispersal in SpltNPV infected Sl-zsu-1 cells, which is very different from Ha83, for Ha83 was transported into nucleus in transfected HzAM1 cells at 48 h p.t. (Fig. 3). Interestingly, the localization of Ha83 became even in transfected cells with the sequential deletion of cysteine at the C terminal of Ha83 (Fig. 3). This is the first time to verify the CBD involved in protein subcellular localization. These results suggested that CBD encoded by ha83 plays a different role with other reported CBD containing proteins, for Ha83 is not involved in PIF but it affects the protein transmission. And the cysteine at the 128 aa position in Ha83 CBD is probably the key conserved amino acid for CBD function.
The homologous GP37 encoded by ac64 resembles the entomopoxvirus protein spheroidin, and has been shown to be associated with polyhedra but not with ODVs and is not essential for baculovirus replication in vivo or in vitro 20,21 . To investigate the functional role of ha83 in the baculovirus life cycle, we generated a ha83-knockout virus. Similar to ac64, ha83 was not essential for BV production (Fig. 4) but it was associated with polyhedra assembly (Fig. 5). Most of the structural gene deletion mutants, such as ha81, ac93, and bm95 thoroughly block the BV production without disrupting viral DNA replication 10,22,23 ; some reported baculovirus-host interaction genes, such as ha132, ac68, sf32, or bm79, do not affect the BV production and viral DNA replication simultaneously after deletion [24][25][26][27] ; besides, some special gene knockout mutants would increase the BV production, for example, fp25k and ha107 28,29 . However, deletion of ha83 resulted in an 84% decrease in BV infectivity of the bacmid virus and a 134 fold increase of DNA replicates, this peculiar change is different from any other previously reported characterized baculovirus genes, and the reasons remain unclear.
Interestingly, Ha83KO OBs were 25% larger in diameter than those of the complete virus and the shape of the occlusion bodies changed into a cuboid, whereas a 100 fold decrease in the average amount of DNA or ODVs per OB was observed in the larger occlusion bodies of ha83 deleted HearNPV mutants. A similar diameter increase happened in sf32 deleted SpfrMNPV, with enlargement of OBs and the nucleocapsids content reported to be 17% more in the larger sf32 knockout SpfrMNPV OBs 26 . Other genes, such as ac23, modify the number of nucleocapsids per OB without affecting the number of occluded ODVs 30,31 . In this case, it can be assumed that the role of ha83 in the HearNPV virus＜ therefore＜ differs from that of ac23 in AucaMNPV and sf32 in the SpfrMNPV. The decreased amount of ODVs in the larger Ha83KO OBs suggested that ha83 deletion may interrupt some of the ODV associated genes expression. Not only are the OBs larger in diameter, but the morphology of Ha83KO OBs had changed into a cuboid. An abnormal morphology change of OBs was also found in the p10 and pep deleted OrpsNPV mutants, as the polyhedra would become irregular in shape with pitted surfaces 32 . A fp25k mutant AucaMNPV isolate would assemble an abnormally large cubic polyhedron without any occluded virions inside, but this change had been verified by a single point mutation resulting in an amino acid change of Gly25 to Asp in the polyhedrin gene 33 . All those genes (p10, pep, and polyhedrin) are polyhedron associated structural genes, which directly influence the OBs morphology with any changes in expression. However, ha83 is absolutely not an OBs structural gene, and the ha83 deletion appears to affect the polyhedron generation in an indirect way.
Transcription qPCR analysis was performed as the ha83 deletion affected HearNPV BV production, viral DNA replication, and OBs structure in a totally different way. Twenty ODVs and OBs structure associated genes were selected for analysis. The polyhedrin and p10 transcripts were greatly reduced in Ha83KO transfected cells compared to HaWT transfected cells, which indicated that the OBs morphology change was directly caused by the low transcription level of polyhedrin and p10 gene in the ha83 knockout HearNPV. The mechanism from ha83 that affects the polyhedrin and p10 gene expression is still unknown. ODV envelope proteins are much more complex in composition than those of OBs envelopes. ODV envelope proteins are composed of Ha96 (ODV-E66) 34 , Ha73 (GP41) [35][36][37] , Ha15 (ODV-E56) 38 , Ha94 (ODV-E43) 39 , Ha44, Ha100 40 , LEF-3 (Ha65) 41 , Ha9, Helicase, Ha84 42 , VP80 (Ha92) 43,44 , and a group of PIFs [45][46][47] , including Ha132 (PIF2) 24,48 and Ha98 (PIF3) 49 . Most of these ODV associated envelope encoding genes were decreased in transcription level with the deletion of ha83 (Fig. 6). These results supported our hypothesis that the reduced appearance of ODVs in the enlarged ha83 deleted OBs is partly due to the deceasing expression level of most of these ODVs associated protein encoding genes. GP41 is a tegument protein modified with O-linked N-acetylglucosamine located between the virion envelope and capsid 43,44 that is required for the egress of nucleocapsids from the nucleus 50 . It was found to be associated with ODV by mass spectrometry 40,51 , and homologs are present in all baculovirus genomes. The increasing ha73 (gp41) transcriptional level was contrary to the other selected genes (Fig. 6Bd). Even with more gp41 transcripts, fewer nucleocapsids were found in the cytoplasm in ha83 deleted bacmid transfected HzAM1 cells (Fig. 5Ab). Therefore, we doubt that the Ha83 was involved in nucleocapsids egression from nucleus, and might be somewhat related to GP41.
In conclusion, the Ha83 protein is a nonessential protein, as BV production was not blocked by gene deletion. This gene might be indirectly involved in mediating nucleocapsid organization during ODV and polyhedra assembly and occlusion. A less essential structural protein transmitted into the nucleus without the help of Ha83 might be the reason for reduced nucleocapsid production after the deletion of ha83. The 3′ and 5′ RACE procedures were performed using 3′ and 5′ -Full RACE Kits (TAKARA) with 1.0 μ g of purified total RNA isolated from HearNPV infected HzAM1 cells at 48 h p.i. Ha83 specific primers Ha835O and Ha833O (Supplementary Table S1), and nested primers, Ha835I and Ha833I (Supplementary Table S1), were used for PCR amplification. The obtained PCR products were purified and cloned into a pGEM-T easy vector (Promega, USA). Each end ten clones were selected for sequencing.

Methods
Chitin binding assay. In order to determine the key cysteine in the CBD of Ha83, a series of different lengths of purified His-tag fused truncated Ha83 protein were generated. Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into BamHI and Xho I sites of the pET-28a(+ ) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) (Supplementary Table S1). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83.
The recombinant Ha83, truncated proteins (TC151, TC142, TC128, TC118, TC113, and TC71), as well as the negative control protein Ha81 were then induced by 1 mM isopropyl-β -d-thiogalactoside (IPTG) (Sigma, USA) and purified using affinity chromatography of cOmplete His-Tag Purification Resin (Roche, SUI). In the same way, all of these protein samples were investigated by 15% SDS-PAGE and stained with Coomassie Blue (Sigma). Western Blot was used for further investigation with standard protocols with the protein samples primitively separated by 10% Tricine-SDS-PAGE system 54 and transferred to a nitrocellulose membrane. A monoclonal anti-His tag antibody (1:4000) (Abcam, GBR) was used as the primary antibodies. The horseradish peroxidase (HRP)-conjugated goat anti-mouse lgG (1:5000) (Abcam, GBR) was used as the secondary antibodies. The proteins were visualized with Enhanced HRP-DAB Chromogenic Substrate Kit (TIANGEN, CHN), according to the manufacturer's instructions.
In order to optimize the binding conditions for the CBD proteins, the chitin powder (Sigma) was first manufactured into a chitin colloid by the following procedures: 1 g chitin powder was mixed with 40 ml HCl in a mortar and ground into a yellow homogeneous paste at 4 °C. The white chitin colloid was generated by adding the yellow mixture into 200 ml perchilled 50% ethyl alcohol and vigorously stirred for two hours. The colloid was collected by centrifugation (5000 × g, 5 min), and washed several times with double distilled water (ddH 2 O) until the pH value of the colloid increased to 7.1. The chitin binding assays were performed as described previously 55,56 . The purified protein samples were incubated with the chitin colloid at 4 °C for 16 hours. The unbounded proteins and chitin bounded proteins were separated by centrifugation (5000 × g, 5 min). Several wash steps were needed to reduce the unbounded proteins in the pellets. Identical volumes of the supernatant and the suspended pellet were mixed with 5 × protein Scientific RepoRts | 5:11088 | DOi: 10.1038/srep11088 loading buffer, heated for 10 min at 100 °C, and loaded onto a 10% tricine-SDS-PAGE gel. Proteins were detected by Coomassie blue staining.
In order to quantify the chitin binding activity of each protein sample, the dissociation constants detection was performed according to Kolbe et al. 55,56 55,56 . The quantity of free protein was determined by enzyme-linked immuno sorbent assay (ELISA) with monoclonal anti-His (1:4000 dilution) used as the primary antibodies and anti-(mouse IgG)-HRP used as secondary antibodies.

Subcellular localization assays of Ha83 truncated proteins in HzAM1 cells.
To determine the effect of truncated CBD on the subcellular traffic of Ha83, we generated a series of eukaryotic expression vectors in which the truncated protein was fused with EGFP. First, the 720-bp fragment containing the egfp gene was PCR amplified from pEGFP-N1 (Clontech, JPN) using the primers Egfp-F and Egfp-R (Hind III and Xba I sites, Supplementary Table S1). The resulting product was digested with Hind III III and Xba I, then ligated into plasmid pGL3-Basic vector which was also digested with the same mixture to generate the recombinant plasmid pGE. The ha83 with its native promoter was PCR amplified from HaBac-HZ8 bacmid using forward primer pHa83-F and reverse primer Ha83-R and digested with Kpn I and Hind III and inserted into pGE, generating the eukaryotic expression plasmid pGE-Ha83. In a similar way, plasmids pGE-TC151, pGE-TC142, pGE-TC128, pGE-TC118, pGE-TC113, and pGE-TC71 were constructed.
Generation of ha83 knockout HearNPV bacmid. The ha83 knockout HearNPV bacmid was generated through λ -Red recombinant system reported by Hou 57 . The fragment of chloramphenicol resistant gene (CmR) and enhanced green fluorescent protein gene (egfp) were amplified with primers HaPD83F and HaPD83R (Supplementary Table S1) from PKSE vector. Homologous recombination between the 2200 bp PCR products and ha83 was performed as previously described 10 .
Three different primers were used to confirm that ha83 had been deleted from the ha83 locus of the HearNPV bacmid genome. Primers Id83F and Id83R1 (Supplementary Table S1) were used to detect the correct insertion of the chloramphenicol resistant gene and egfp. Primers Id83F and Id83R2 (Supplementary Table S1) were used to examine the recombination junctions of the upstream and downstream flanking regions. The recombinant bacmid with all of the correct PCR confirmations was then digested by Hind III restriction enzyme to verify the Hind III-fragments changes toward the HaBacHZ8 Hind III digestion map. The resulting ha83 knockout HearNPV was designated HaBac-ha83KO.
Construction of knockout, repair, and wt HearNPV bacmids containing polyhedron. In order to determine if ha83 deletion has any effect on occlusion morphogenesis, ph was inserted into the polyhedrin locus by site-specific transposition as previously described by Li 10 . Accordingly, the pMON7124 helper plasmid was isolated, purified, and transformed into BW25113 cells containing the HaBacHZ8 or into BW25113 cells containing the HaHZ8-ha83KO. The HTb-polh vector containing HearNPV polyhedrin with its own promoter 10 was transformed into electrocompetent BW25113 cells containing the pMON7124 helper plasmid and HaHZ8-ha83KO to generate the ha83 knockout bacmid (Ha83KO). The HTb-polh-egfp donor vector containing HearNPV polyhedrin and egfp 10 was transformed into electrocompetent BW25113 cells containing the pMON7124 helper plasmid and HaHZ8 bacmid to generate the positive control bacmid (HaWT).
To generate a ha83 repair bacmid, a donor plasmid (HTb-polh-ha83) was constructed. The ha83 with its native promoter region was amplified from HearNPV with primers pha83F and ha83R (Supplementary  Table S1). This fragment was introduced into the corresponding restriction sites (Bst1107 I and Xho I) of the pFastBac HTb vector to generate HTb-ha83. Another fragment containing the coding region of the ph gene with its own promoter and the SV40 poly(A) signal was amplified with primers phS-F and phS-R (Supplementary Table S1) from the HTb-polh vector 10 . The PCR product was digested with Bcl I and inserted into the Bcl I site in HTb-ha83 to generate the final donor plasmid HTb-ha83-ph. The donor plasmid was then transformed into electrocompetent BW25113 cells harboring the pMON7124 helper plasmid and HaBac-ha83KO to generate the ha83 repair bacmid, Ha83Rep. The correctness of transpositions were verified by PCR with primers puc/M13F and puc/M13R (Supplementary Table S1), and followed by PCR product sequencing.
Scientific RepoRts | 5:11088 | DOi: 10.1038/srep11088 One step viral growth curve and viral DNA replication analysis. To analyze BV production, a viral growth curve was constructed as previously described 10 . Titers of BV were determined by a TCID 50 assay and compared by F-test in SPSS 15.0 (SPSS Inc, Chicago, IL). To evaluate any effect of ha83 deletion on viral DNA replication, qPCR analysis was performed as previously described 10 . Electron microscopy. HzAM1 cells (10 6 cells per 35-mm-diameter well) were transfected with 1.0 μg of the constructed bacmid HaWT, Ha83KO, or Ha83Rep. At 72 h p.i., cells were dislodged with a rubber policeman and centrifuged at 2000 × g for 10 min. Occlusion bodies HaWT and Ha83KO were washed and resuspended in 100 μ l of 2% agarose. The OBs/agarose pellets were washed in double sterile water and sliced into 2 mm cubes. Both the cells and OBs were fixed in 2.5% glutaraldehyde over night, and then dehydrated, embedded, sectioned, and stained as described previously 10 . Samples were examined with a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 80 kV. For analysis by scanning electron microscopy, occlusion bodies were coated with gold and examined with a Hitachi 5X-300000X scanning electron microscope. OB measurements were normalized by square root transformation and compared by t-test using SPSS 15.0.
Transcriptional analysis of HearNPV occlusion body and ODV associated genes. To further examine the effects of ha83 knockout, transcripts from several well-characterized polyhedron and ODV associated structural genes were analyzed. HzAM1 cells (106 cells per 35-mm-diameter well) were transfected with 1.0 μg of the constructed bacmid DNA ha83KO and HaWT as described above. Total RNA extraction and cDNA synthesis from transfected cells at 24, 48, and 72 h p.t. was performed as described above. As target genes, we selected polyhedrin and p10 as representative OBs structural genes. pif2, ha9, an, ha100, odv-e66, p24, cg30, odv-e56,lef-3, vp80, ha66, ha40, helicase, odv-e43, ha90, ie-1, 38k, and gp41 were selected as representative ODV associated structural genes. qPCR was then performed as described above, using the primers listed in Supplementary Table S1. The relative expression level was compared by F-test using SPSS 15.0.