Abstract
Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group I and II, and Group I appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group I specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group I specific gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus (BV) and occlusion-derived virus (ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies (OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout AcMNPV decreased by about 5–8 and 3–4 fold compared to those of wild type virus, respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.
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Introduction
The baculoviruses are a group of rod-shaped viruses with large DNA genomes that specifically infect insects. Two kinds of virion are produced during a typical baculovirus infection cycle: budded virus (BV) and occlusion-derived virus (ODV), which mediates cell-to-cell and host-to-host infection, respectively (Keddie et al.1989). Baculoviruses are phylogenetically divided into four genera, namely Alpha-, Beta-, Gamma-, and Deltabaculovirus (Jehle et al.2006). The Alphabaculovirus is further divided into Group I and Group II based on phylogenetic analysis (Zanotto et al.1993). Group I and II alphabaculoviruses are also characterized by using GP64 and ancestral F protein as their fusion proteins for BV, respectively (Pearson and Rohrmann 2002). Among all the baculoviruses, Group I alphabaculoviruses are the most recently evolved (Herniou and Jehle 2007), and there are 12 specific genes that are only found in this lineage (Rohrmann 2011). The gp64 gene is one of them and has been suggested to be captured by an ancestral Group I alphabaculovirus relatively late during evolution (Pearson and Rohrmann 2002). The rest 11 Group I specific genes in the prototype baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) are: ac1 (ptp), ac5, ac16 (bv/odv-e26), ac27 (iap-1), ac30, ac72, ac73, ac114, ac124, ac132, and ac151 (ie2). It was proposed that the acquisition of the homologs of gp64 (ac128) and other Group I specific genes may promote virus diversification and host range (Pearson et al.2000; Herniou et al.2001; Jiang et al.2009).
To date, 10 of the 12 Group I specific genes in AcMNPV have been studied. As mentioned above, gp64 encodes the viral major envelope fusion protein that essential for BV entry and infection (Monsma et al.1996). The ac1 deletion can lead to partial defect in occlusion body (OB) formation in Spodoptera frugiperda 21 (Sf21) cells but not in Trichoplusia ni (T. ni) cells (TN-368 cells) (Li and Miller 1995). The ac5 encodes a protein that is an OB protein but not a component of BV or ODV, and is not required for BV production, the oral infectivity, and the formation of per os infectivity factor (PIF) complex (Wang et al.2018). Inactivation of ac16 has no effects on protein synthesis in infected cells and oral infectivity to T. ni or S. frugiperda larvae (O’Reilly et al.1990). ac27 is a gene of inhibiting apoptosis (Zeng et al.2009), and its deletion appears to out-compete wild type virus in a cell-specific way (McLachlin et al.2001). Deletion of ac30 has no obvious effects on BV production (Yu 2015). Though BV and ODV can be normally produced when ac114 or ac124 is deleted, the oral infectivity is significantly reduced or the time to kill infected larvae is increased, respectively (Wei et al.2012; Liang et al.2015). The product of ac132 is a nucleocapsid-associated protein essential for transport of nucleocapsid into nucleus (Fang et al.2016). The ac151 seems to encode a protein that can facilitate DNA replication, virion formation, and infectivity in cell-specific way (Lu and Miller 1995; Prikhod’ko et al.1999). Therefore, among the 10 studied Group I specific genes of AcMNPV, only gp64 and ac132 are essential for virus infection, while others seem to contribute to virus infection in different aspects.
Currently, the function of ac72 and ac73 still remains unclear, although some studies on their homologs have been carried out in Bombyx mori nucleopolyhedrovirus (BmNPV). It was reported that the homolog of ac72 in BmNPV (bm58a) was not required for BV production, ODV assembly, or OB formation, however, it may function in promoting cell lysis and larval liquefaction (Yang et al.2016). The bm59, a homolog to ac73, was first reported as an essential gene for virus infection (Ono et al.2012), but was subsequently demonstrated to be dispensable for the propagation and assembly of BmNPV (Hu et al.2016). Therefore, the reports on the role of bm59 during baculovirus life cycle seem to be controversial and its exact function remains to be clarified.
In this study, we aimed to characterize the function of ac73 during AcMNPV infection. We first detected the transcription and expression of ac73, and then studied the subcellular localization of AC73 during AcMNPV infection and determined whether it is a structure component of BV and ODV. The ac73 knockout and repaired recombinant viruses were constructed and characterized by in vitro and in vivo infection. Results showed that AC73 was expressed at late stage of virus infection and associated with the nucleocapsid fractions of both BV and ODV. Moreover, although ac73 is not an essential gene, it contributed to infectious BV production and viral infectivity in insect larvae to some extent.
Materials and Methods
Cells and Viruses
Sf9 cells were cultured at 27 °C in Grace’s insect medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS). The wild type (WT) AcMNPV E2 strain was obtained from the Microorganisms and Viruses Culture Collection Center, Wuhan Institute of Virology, Chinese Academy of Sciences (storage no. IVCAS1.0315). Ac-egfp and AcBac-egfp-ph were constructed previously (Wang et al.2008; Shang et al.2017). The AcMNPV bacmid (bMON14272) used for the construction of recombinant viruses was derived from the DH10Bac™ Escherichia coli (E. coli) cells in Bac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, CA, USA).
Generation of Polyclonal Antibody against AC73
To generate specific polyclonal antibody against AC73 (anti-AC73), the open reading frame (ORF) of ac73 was amplified with 5′-GCGGAATTCATGAACACGTCCGTGGACG-3′ (EcoRI site underlined) and 5′-GCGCTCGAGTTATTGTACATAATGTTTTATTGTAA-3′ (XhoI site underlined) and inserted into the EcoRI and XhoI sites of pET-28a vector (Novagen, Carlsbad, CA, USA) to generate pET-28a-ac73. Then, the recombinant plasmid was electroporated into E. coli BL21 competent cells. The BL21 cells were induced with isopropyl-β-thiogalactopyranoside (IPTG) at 37 °C for protein expression. The expressed AC73 in BL21 cells was purified using cOmplete His-Tag Purification Resin (Roche Dnostics, Indianapolis, IN, USA) and the purified AC73 protein was used as antigen to generate rabbit polyclonal antiserum as previously described (Zou et al.2016).
Time Course Analysis of ac73 Transcription and Expression
Sf9 cells were infected with WT AcMNPV at a multiplicity of infection (MOI) of 10 and harvested at 0, 3, 6, 12, 18, 24, 36, 48, and 72 h post infection (p.i.). For temporal transcription analysis, the total RNA of the infected cells was isolated by RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instruction. The DNA in RNA samples was eliminated and equal amounts (1 μg) of RNA were reverse transcribed to cDNA using the PrimeScript™ RT reagent with the gDNA Eraser (TaKaRa, Dalian, China) Kit following the manufacturer’s protocol. The total RNA or cDNA was used as the template for PCR amplification of an inner fragment of ac73 (primers: 5′-ATGAACACGTCCGTGGACG-3′ and 5′-ACACCAATTTAAACACATGTTGAT-3′). To detect the expression of AC73 at different time points of infection, Sf9 cells were infected with WT AcMNPV using the same conditions above, and the cells were harvested and treated with protein sample buffer (50 mmol/L Tris-HCl, 2% SDS, 0.1% bromophenol blue, 10% glycerol, 5% 2-mercaptoethanol). Then, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose filter (NC) membrane (Millipore, Billerica, MA, USA) for Western blot analysis. The blots were incubated with anti-VP39 (Wang et al.2010), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Li et al.2018) or anti-AC73 antibody as the primary antibody, and HRP-conjugated goat anti-rabbit antibody (Sigma, St. Louis, MO, USA) as the secondary antibody. The bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).
Immunofluorescence Microscopy of AC73
For immunofluorescence analysis, about 1 × 106 Sf9 cells were infected with Ac-egfp virus at an MOI of 10, and then the cells were fixed at 12, 18, 24, 36, 48, and 72 h p.i. with 5% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min before being incubated with 5% BSA overnight at 4 °C. Cells were then incubated with anti-AC73 antibody as the primary antibody at room temperature for 3 h and subsequently with Alexa 647-conjugated goat anti-rabbit (Abcam, Cambridge, UK) as the secondary antibody. The nuclei of infected cells were stained with Hoechst 33258 dye (Beyotime, Shanghai, China) for 5 min prior to the fluorescence microscopy. The fluorescence signals were observed by fluorescence microscopy (Deltavision softWoRx Imaging Workstation, Applied Precision).
Localization Analysis of Fluorescent Protein Fused AC73
To determine the localization of AC73 in transiently expressed cells, a plasmid containing egfp-fused ac73 was constructed as follows. First, the ORF of egfp was amplified from pEGFP-N1 (Clontech, Mountain View, CA, USA) and subcloned into the BamHI and EcoRI sites of pIZ/V5-His (Invitrogen, Carlsbad, CA, USA) to generate pIZ/V5-egfp. Then, the ORF of ac73 was amplified through PCR with primers 5′-GCGGAATTCATGAACACGTCCGTGGACG-3′ (EcoRI site underlined) and 5′-GCGTCTAGATTATTGTACATAATGTTTTATTGTAA-3′ (XbaI site underlined). Finally, the fragment was inserted into the EcoRI and XbaI sites of pIZ/V5-egfp to produce pIZ/V5-egfp-ac73. The pIZ/V5-egfp-ac73 plasmid was transfected into Sf9 cells using Cellfectin II reagent (Gibco, Carlsbad, CA, USA). To detect the localization of EGFP fused AC73 in infected cells, Sf9 cells were transfected with pIZ/V5-egfp-ac73 plasmid as mentioned above and then infected with WT AcMNPV at an MOI of 5 for 48 h. Cells were then fixed and stained with Hoechst 33258 for observation as described above.
Detection of AC73 in BV and ODV
BVs from WT AcMNPV infected Sf9 cells and ODVs embedded in occlusion bodies (OBs) from virus infected larvae were purified as previously described (Braunagel and Summers 1994; Wang et al.2010). The purified BVs and ODVs were further separated into envelope (E) and nucleocapsid (NC) fractions as previously described (Hou et al.2013). Proteins in purified BVs, ODVs, and their E and NC fractions were then detected with anti-GP64, anti-VP39, anti-ODV-E66 (Wang et al.2010), anti-PIF5 (Wang et al.2018), or anti-AC73 antibody by Western blots as described above.
Construction of ac73 Knockout and Repaired Recombinant Bacmids
A 326-base pair (bp) fragment upstream and a 220-bp fragment downstream of ac73 were PCR amplified with primers: AC73KO-UP-F (5′-TAAGGTACCCACGTTAGGCAGACAGTTG-3′, KpnI site underlined) and AC73KO-UP-R (5′-GCGCTCGAGATATTTATTATTCCACGGACGTGTTCATG-3′, XhoI site underlined) or AC73KO-Down-F (5′-GGGGATATCGCAACGCCATAGTGTTTGAC-3′, EcoRV site underlined) and AC73KO-Down-R (5′-GGGTCTAGAGTGTCGCATCTAAGCGACG-3′, XbaI site underlined). The two fragments were inserted into pKS-egfp-Cmr plasmid (provided by Dr. Just M. Vlak, Wageningen University and Research, the Netherlands) to generate pKS-ac73up-egfp-Cmr-ac73down plasmid. Then, the ac73up-egfp-Cmr-ac73down cassette was amplified through PCR using AC73KO-UP-F and AC73KO-Down-R primers. The purified linear fragment was electroporated into E. coli BW25113 competent cells containing AcMNPV bacmid (bMON14272) and λ Red recombinase-encoding plasmid pKD46 to generate Ac∆73 bacmid as described previously (Hou et al.2002). A fragment, nucleotides (nt) 62757-62449, containing the promoter of ac73 was amplified from WT AcMNPV DNA with 5′-CAGCCCGGGCACGTTAGGCAGACAGTTG-3′ (SmaI site underlined) and 5′-GGGCTCGAGGTTTCTTTTTTGAAAACTAAATTG-3′ (XhoI site underlined). Then, the fragment was ligated into pFBD-ph (Li et al.2018) to construct pFBD-Pac73-ph. The ORF of ac73 and the poly(A) signal sequence of ac73 were amplified with 5′-CGCCTCGAGATGTACCCATACGACGTCCCAGACTACGCTATGAACACGTCCGTGGACG-3′ (XhoI site underlined; HAtag sequence in bold) and 5′-GGGGCATGCGTGTCGCATCTAAGCGACG-3′ (SphI site underlined) and further inserted into pFBD-Pac73-ph to generate the donor plasmid pFBD-Pac73-HAtag-ac73-ph. The ac73 knockout (Ac∆73-ph) and repaired (Ac∆73-ac73R-ph) recombinant bacmids were generated by transposition of pFBD-ph or pFBD-Pac73-HAtag-ac73-ph into the LacZ mini-attTn7 locus of Ac∆73 using Bac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, CA, USA).
Production and Identification of Recombinant BVs
About 1 × 106 Sf9 cells were transfected with recombinant bacmid DNA of Ac∆73-ph or Ac∆73-ac73R-ph with Cellfectin II reagent, and fluorescence was observed at 48 and 96 h post transfection (p.t.) to determine the production of infectious BVs. At 120 h p.t., the supernatants were collected and used to infect healthy Sf9 cells for 48 h before fluorescence microscopy. To verify the correctness of the obtained Ac∆73-ph or Ac∆73-ac73R-ph virus, Sf9 cells were infected with AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph, then AC73 protein in the infected cells was detected through Western blot as described above. VP39 and GAPDH were also detected to serve as controls.
One-Step Growth Curve
Cells were infected in triplicate with AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph virus at an MOI of 10, and 50 μL supernatant of each infection was collected at different time points post infection. The BV titers were determined by endpoint dilution assay (EPDA), and the averages of titers from three independent infections at each time point were calculated to plot one-step growth curves of these viruses. The statistical analysis was performed by one-way analysis of variance (ANOVA) method with SPSS software (IBM, Armonk, NY, USA).
Electron Microscopy (EM)
For transmission electron microscopy (TEM) analysis, cells (1 × 106) were infected with AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph virus at an MOI of 10. At 24, 48, and 72 h p.i., cells were fixed with 2.5% glutaraldehyde. The samples were processed for TEM analysis as previously described (Li et al.2018). The TEM images were taken using Tecnai G2 20 TWIN TEM (FEI, Hillsboro, OR, USA) at an accelerating voltage of 200 kV.
Bioassay
The bioassay was conducted using a droplet method (Hughes et al.1986). Briefly, 48 early third-instar S. exigua larvae were fed with the OBs of AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph using droplet method at the concentration of 1 × 104, 3 × 104, 1 × 105, 3 × 105, 1 × 106, 3 × 106, 1 × 107, or 3 × 107 OBs/mL. Bioassays were performed twice and the infected larvae were monitored daily until all larvae had either pupated or died. The calculation of median lethal concentration (LC50) and the 95% confidence limits (CL) or the comparison of LC50 values among viruses were carried out using Probit regression method or the potency ratio test in SPSS software.
Results
Ac73 Is a Late Viral Gene
To study the transcription of ac73 in AcMNPV infected Sf9 cells, we first predicted the promoter of ac73. A TTAAG motif which is the typical feature of baculovirus late promoter (Morris and Miller 1994), was found from nt 61–57 upstream of ATG of ac73 (Fig. 1A), indicating that ac73 may be a late gene. This was consistent with a report that in AcMNPV infected T. ni cells, ac73 was mainly transcribed during late infection at nt 57 upstream of ATG of ac73 (Chen et al.2013). To further determine whether ac73 is really a late gene, the transcripts of ac73 at different time points of infection were detected through PCR amplification of an inner fragment (~ 270 bp) within ac73. Result showed that the transcripts of ac73 could be detected from 12 to 72 h p.i. (Fig. 1B), indicating that ac73 was expressed at the late stage of infection. In addition, Western blot analysis was performed to detect AC73 protein levels in infected cells. The AC73 protein was under the detectable level before 18 h p.i., but was clearly detected since 24 h p.i. (Fig. 1C). For reference, VP39, the well-known viral late protein (Thiem and Miller 1989), could also be clearly detected since 18 h p.i. (Fig. 1C). With the above results, we can conclude that ac73 is a late gene of AcMNPV.
Cellular Localization of AC73
Next, the subcellular localization of AC73 in infected cells was determined by immunofluorescence microscopy. In Ac-egfp infected cells, AC73 could be clearly detected in the nucleus of infected cells since 18 h p.i., and it was mainly localized in the ring zone region peripheral to the nuclear membrane during virus infection (Fig. 2A). To further determine whether AC73 can enter the nucleus independently, EGFP fused AC73 or the control EGFP alone was transiently expressed by transfection of the corresponding plasmid into Sf9 cells. The result showed that both EGFP and EGFP fused AC73 were evenly distributed in the cytoplasm and nucleus (Fig. 2B), suggesting that AC73 alone could not enter the nucleus completely. This is consistent with the fact that no nuclear localization signal could be predicted in AC73 (data not shown). To exclude the possibility of effect of EGFP on the localization of AC73, Sf9 cells were first transfected with plasmid encoding EGFP or EGFP fused AC73 and then infected with WT AcMNPV. Compared to the result of transient expression, EGFP fused AC73 but not EGFP in the super-infected cells showed clear nuclear localization and was embedded into OBs (Fig. 2B). Thus, the results suggested that AC73 could enter the nucleus in an infection-dependent way and it seemed to be assembled into OBs.
AC73 is a Nucleocapsid Protein of Both BV and ODV
Previous proteomics data revealed that AC73 was associated with BV (Wang et al.2010), but not with ODV (Braunagel et al.2003). However, our result found that EGFP fused AC73 could be assembled into OBs, suggesting that AC73 may also be ODV-associated. To test this possibility, BVs and ODVs were prepared from the supernatant of WT AcMNPV infected cells and larvae, respectively, and then analyzed by Western blot with anti-AC73 antibody. As shown in Fig. 3A, AC73 could be probed in both BV and ODV samples. To further confirm this result and to determine the localization of AC73 in virion more accurately, the BVs and ODVs were fractionated into envelope and nucleocapsid components. As the Western blot result showed, AC73 could be detected in nucleocapsid samples of BV and ODV, but not in the envelope samples (Fig. 3B). Thus, AC73 is a nucleocapsid component of both BV and ODV.
Ac73 is Not Essential for Infectious BV Production
To determine the function of AC73 in virus infection, ac73 knockout and repaired bacmids were constructed. A 168 bp of ac73 in AcMNPV bacmid bMON14272 was replaced with egfp and Cmr genes to generate Ac∆73 bacmid, and then ph alone or both ph and HAtag-fused ac73 genes were inserted into the Ac∆73 bacmid to produce Ac∆73-ph or Ac∆73-ac73R-ph bacmid (Fig. 4A). Then, Ac∆73-ph or Ac∆73-ac73R-ph bacmid was transfected into Sf9 cells. The result showed that the number of fluorescent cells increased obviously from 48 to 96 h p.t. in both Ac∆73-ph and Ac∆73-ac73R-ph transfected cells (Fig. 4B, left two panels), indicating that infectious BVs could be produced when ac73 was deleted. To further confirm this, the supernatants from transfected cells were collected and then used to infect healthy Sf9 cells. Cells were successfully infected with Ac∆73-ph or Ac∆73-ac73R-ph virus as indicated by the occurrence of EGFP fluorescence (Fig. 4B, right panel). To confirm the correctness of recombinant viruses, AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph virus infected Sf9 cells were analyzed by Western blot. The result showed that the WT AC73 and HAtag-AC73 protein could be detected in AcBac-egfp-ph and Ac∆73-ac73R-ph infected cells, respectively, but no signal could be detected in Ac∆73-ph infected cells (Fig. 4C), suggesting Ac∆73-ph and Ac∆73-ac73R-ph were correctly constructed and produced. Taking together, these results indicated that ac73 is non-essential for BV propagation in cultured cells.
The ac73 Deletion Resulted in Decreased Production of Infectious BVs
To quantify whether ac73 contributes to BV production, one-step growth curve analysis of AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph was performed. Sf9 cells were infected with these viruses at an MOI of 10, and BV titers at 0, 24, 48, 72, and 96 h p.i. were determined. In contrast to the result of bm59 deletion which did not affect BV production (Hu et al.2016), one-step growth curve assay revealed the BV titers of Ac∆73-ph decreased by approximately 8- and 5-fold compared to that of AcBac-egfp-ph virus at 72 and 96 h p.i. respectively (P < 0.05) (Fig. 5). By comparison, at all the time points of the infection, the BV titers of Ac∆73-ac73R-ph showed no significant difference with those of AcBac-egfp-ph (P > 0.05) (Fig. 5). Thus, though ac73 is non-essential for BV production, it does play a role in optimal production of infectious BVs.
Electron Microscopy of AcBac-egfp-ph, Ac∆73-ph, and Ac∆73-ac73R-ph infected cells
Next, we determined whether ac73 is essential for the morphogenesis of ODV and OB. To this end, AcBac-egfp-ph, Ac∆73-ph, and Ac∆73-ac73R-ph infected cells at 24, 48, and 72 h p.i. were subjected to electron microscopy. At 24 h p.i., the nucleocapsids and ODVs could be detected in the nucleus of infected cells for AcBac-egfp-ph and Ac∆73-ac73R-ph, as well as Ac∆73-ph (Fig. 6, upper panel), suggesting the ac73 was neither essential for the nucleocapsid assembly, nor for the envelopment of nucleocapsids to form ODV. At 48 and 72 h p.i., the OBs that embedded with ODVs were formed in AcBac-egfp-ph and Ac∆73-ac73R-ph, as well as in Ac∆73-ph infected cells (Fig. 6, lower two panels). These results showed that ac73 was not required for ODV or OB formation in infected cells.
The Effects of ac73 Deletion on Viral Infectivity in Insect Larvae
To further investigate the function of AC73 in vivo, bioassay was performed to determine the effects of ac73 deletion on viral infectivity in host level. The early third-instar S. exigua larvae were orally infected with OBs of AcBac-egfp-ph, Ac∆73-ph, or Ac∆73-ac73R-ph at different concentrations using droplet method (Hughes et al.1986). Liquefaction of the infected larvae after death was observed for AcBac-egfp-ph, Ac∆73-ph, and Ac∆73-ac73R-ph viruses (data not shown), indicating that ac73 was not essential for oral infection and liquefaction of infected larvae. In two independent experiments, the potency ratio test showed that there was no significant difference between the AcBac-egfp-ph and Ac∆73-ac73R-ph viruses as evidenced by the including of the value 1.0 for 95% CL (Robertson et al.2007), but the LC50 of Ac∆73-ph virus was 3–4 fold higher than that of AcBac-egfp-ph virus (the potency ratio didn’t include 1.0) (Table 1), suggesting that the deletion of ac73 reduced the viral infectivity of AcMNPV in S. exigua larvae. Thus, ac73 is a virulent gene that contributes to virus infection in vivo.
Discussion
The ac73 is one of the 12 specific genes of Group I alphabaculoviruses, but its function during AcMNPV infection was unknown. In this study, we showed that ac73 was a late gene (Fig. 1) and its product, AC73, was associated with the nucleocapsid fractions of both BV and ODV (Fig. 3). In addition, AC73 appeared to be assembled into the OBs (Fig. 2B). Deletion of ac73 resulted in about 5–8 fold decrease of BV production at late stages of viral infection (Fig. 5) and about 3–4 fold decrease of viral infectivity in host level (Table 1). Therefore, like many of other Group I specific proteins which are not essential but may benefit for virus infection, such as AC1 (Li and Miller 1995), AC114 (Wei et al.2012), and AC124 (Liang et al.2015), AC73 is also a luxury protein that remained/captured during evolution to contribute to virus infection.
Some of our results of ac73 were different from the studies of its homologue bm59 in BmNPV. First, ac73 was found as a late gene and this was consistent with the present of a late transcription motif TTAAG in its promoter region (Fig. 1A) as well as the transcriptome result of AcMNPV in T. ni cells (Morris and Miller 1994; Chen et al.2013). However, bm59 was characterized as an early gene with an atypical transcriptional start motif, CAAC motif (Hu et al.2016). We found that a TTAAG motif is also present at nt 61-57 upstream of the ATG of bm59. It remains unknown why bm59 does not use the conserved TTAAG motif for late gene transcription. Second, deletion of ac73 resulted in the reduction of infectious BV production, however, bm59 deletion was initially reported to be essential for BV production (Ono et al.2012), but later showed no impact on infectious BV production (Hu et al.2016). Considering AC73 and Bm59 share high sequence similarity (~ 90% aa identity), these differences may reflect that ac73 and bm59 are diverged at relatively late stage of baculovirus evolution and are adapting to their different hosts.
Actually, it is not surprising to find that the Group I specific gene homologues function differently in AcMNPV and BmNPV. For example, ac5 had no obvious effects on OB formation when deleted (Wang et al.2018), but its homolog, bm134, was found to be important for the embedding of ODVs into OBs in BmNPV (Shen et al.2018). In addition, though ac16 deleted virus could produce infectious BVs (O’Reilly et al.1990), its homolog, bm8, is essential for infectious BV production (Imai et al.2004). Similarly, BVs could be normally produced when ac124 was deleted (Liang et al.2015), but bm101 (homolog of ac124) was found to be essential for BV production in BmNPV (Chen et al.2014). Although AcMNPV and BmNPV are two closely related viruses with an average ~ 90% amino acid sequence identity between homologous ORFs (Gomi et al.1999), they show a striking difference in host range. BmNPV is host specific that it only infects B. mori or B. mandarina (Shirata et al.1999; Iwanaga et al.2009), but AcMNPV shows a wide host range of at least 32 lepidopteran insect species (Granados and Williams 1986), yet it is unable to complete a productive replication in B. mori cells or kill B. mori larvae (Morris and Miller 1993; Grasela et al.2000). In contrast to the functional diversity between the Group I specific genes of the two viruses, the function of most other genes appeared to be relatively consistent between AcMNPV and BmNPV (data not shown). Therefore, our study and previous studies highlighted the uniqueness of Group I specific genes in the evolution of alphabaculoviruses.
When searched against non-redundant protein database at National Center for Biotechnology Information (NCBI) using Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST), AC73 was found to share high similarity with Bcl-2-associated athanogene (BAG) domains of some proteins, for example, the BAG domain of Starvin protein from Drosophila melanogaster which is required for larval food uptake and involved in the recovery from cold stress (Coulson et al.2005; Colinet and Hoffmann 2010), the BAG domain of Samui protein from B. mori which is cold-inducible and can protect against cold-injures or transmit cold signal for gene expression (Moribe et al.2001), and the BAG domain of BAG-4 (also known as silencer of death domains (SODD)) from Homo sapiens which interacts with Hsp70 or tumor necrosis factor receptor type 1 (TNFR1) to affect cell death (Miki and Eddy 2002). A BAG domain can bind to the ATPase domain of Hsc70/Hsp70 to regulate its activity (Bimston et al.1998; Terada and Mori 2000; Gassler et al.2001). Therefore, ac73 may be acquired from a host during evolution and produce a protein to mimic the functions of host BAG domain-containing proteins to facilitate virus infection under certain conditions. But further investigations are required to identify whether AC73 functions as a BAG domain-like protein and to unravel the detailed role and the function mode of ac73 in virus life cycle.
References
Bimston D, Song J, Winchester D, Takayama S, Reed JC, Morimoto RI (1998) Bag-1, a negative regulator of hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release. EMBO J 17:6871–6878
Braunagel SC, Summers MD (1994) Autographa californica nuclear polyhedrosis virus, PDV, and ECV viral envelopes and nucleocapsids: Structural proteins, antigens, lipid and fatty acid profiles. Virology 202:315–328
Braunagel SC, Russell WK, Rosas-Acosta G, Russell DH, Summers MD (2003) Determination of the protein composition of the occlusion-derived virus of Autographa californica nucleopolyhedrovirus. Proc Natl Acad Sci USA 100:9797–9802
Chen H, Li M, Mai W, Tang Q, Li G, Chen K, Zhou Y (2014) Analysis of BmNPV orf101 disruption: Orf101 is essential for mediating budded virus production. Cytotechnology 66:1021–1029
Chen YR, Zhong S, Fei Z, Hashimoto Y, Xiang JZ, Zhang S, Blissard GW (2013) The transcriptome of the baculovirus Autographa californica multiple nucleopolyhedrovirus in Trichoplusia ni cells. J Virol 87:6391–6405
Colinet H, Hoffmann A (2010) Gene and protein expression of Drosophila starvin during cold stress and recovery from chill coma. Insect Biochem Mol Biol 40:425–428
Coulson M, Robert S, Saint R (2005) Drosophila starvin encodes a tissue-specific bag-domain protein required for larval food uptake. Genetics 171:1799–1812
Fang Z, Li C, Wu W, Yuan M, Yang K (2016) The Autographa californica multiple nucleopolyhedrovirus ac132 plays a role in nuclear entry. J Gen Virol 97:3030–3038
Gassler CS, Wiederkehr T, Brehmer D, Bukau B, Mayer MP (2001) Bag-1 m accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor. J Biol Chem 276:32538–32544
Gomi S, Majima K, Maeda S (1999) Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80:1323–1337
Granados RR, Williams KA (1986) In vivo infection and replication of baculoviruses. In: Granados RR, Federici BA (eds) The biology of baculoviruses, vol 1. CRC Press, Boca Raton, pp 89–108
Grasela JJ, McIntosh AH, Goodman CL, Wilson LE, King LA (2000) Expression of the green fluorescent protein carried by Autographa californica multiple nucleopolyhedrovirus in insect cell lines. Vitro Cell Dev Biol Anim 36:205–210
Herniou EA, Jehle JA (2007) Baculovirus phylogeny and evolution. Curr Drug Targets 8:1043–1050
Herniou EA, Luque T, Chen X, Vlak JM, Winstanley D, Cory JS, O’Reilly DR (2001) Use of whole genome sequence data to infer baculovirus phylogeny. J Virol 75:8117–8126
Hou D, Zhang L, Deng F, Fang W, Wang R, Liu X, Guo L, Rayner S, Chen X, Wang H, Hu Z (2013) Comparative proteomics reveal fundamental structural and functional differences between the two progeny phenotypes of a baculovirus. J Virol 87:829–839
Hou S, Chen X, Wang H, Tao M, Hu Z (2002) Efficient method to generate homologous recombinant baculovirus genomes in E. coli. Biotechniques 32:783–788
Hu X, Shen Y, Zheng Q, Wang G, Wu X, Gong C (2016) Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus. Mol Genet Genomics 291:145–154
Hughes PR, Vanbeek NAM, Wood HA (1986) A modified droplet feeding method for rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae. J Invertebr Pathol 48:187–192
Imai N, Kurihara M, Matsumoto S, Kang WK (2004) Bombyx mori nucleopolyhedrovirus orf8 encodes a nucleic acid binding protein that colocalizes with ie1 during infection. Arch Virol 149:1581–1594
Iwanaga M, Arai R, Shibano Y, Kawasaki H, Imanishi S (2009) Establishment and characterization of the Bombyx mandarina cell line. J Invertebr Pathol 101:124–129
Jehle JA, Blissard GW, Bonning BC, Cory JS, Herniou EA, Rohrmann GF, Theilmann DA, Thiem SM, Vlak JM (2006) On the classification and nomenclature of baculoviruses: A proposal for revision. Arch Virol 151:1257–1266
Jiang Y, Deng F, Rayner S, Wang H, Hu Z (2009) Evidence of a major role of gp64 in group I alphabaculovirus evolution. Virus Res 142:85–91
Keddie BA, Aponte GW, Volkman LE (1989) The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243:1728–1730
Li Y, Miller LK (1995) Properties of a baculovirus mutant defective in the protein phosphatase gene. J Virol 69:4533–4537
Li Y, Shen S, Hu L, Deng F, Vlak JM, Hu Z, Wang H, Wang M (2018) The functional oligomeric state of tegument protein gp41 is essential for baculovirus budded virion and occlusion-derived virion assembly. J Virol 92:e02083-17
Liang C, Lan D, Zhao S, Liu L, Xue Y, Zhang Y, Wang Y, Chen X (2015) The ac124 protein is not essential for the propagation of Autographa californica multiple nucleopolyhedrovirus, but it is a viral pathogenicity factor. Arch Virol 160:275–284
Lu A, Miller LK (1995) Differential requirements for baculovirus late expression factor genes in two cell lines. J Virol 69:6265–6272
McLachlin JR, Escobar JC, Harrelson JA, Clem RJ, Miller LK (2001) Deletions in the ac-iap1 gene of the baculovirus AcMNPV occur spontaneously during serial passage and confer a cell line-specific replication advantage. Virus Res 81:77–91
Miki K, Eddy EM (2002) Tumor necrosis factor receptor 1 is an atpase regulated by silencer of death domain. Mol Cell Biol 22:2536–2543
Monsma SA, Oomens AG, Blissard GW (1996) The gp64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70:4607–4616
Moribe Y, Niimi T, Yamashita O, Yaginuma T (2001) Samui, a novel cold-inducible gene, encoding a protein with a bag domain similar to silencer of death domains (sodd/bag-4), isolated from Bombyx diapause eggs. Eur J Biochem 268:3432–3442
Morris TD, Miller LK (1993) Characterization of productive and nonproductive AcMNPV infection in selected insect-cell lines. Virology 197:339–348
Morris TD, Miller LK (1994) Mutational analysis of a baculovirus major late promoter. Gene 140:147–153
O’Reilly DR, Passarelli AL, Goldman IF, Miller LK (1990) Characterization of the DA26 gene in a hypervariable region of the Autographa californica nuclear polyhedrosis virus genome. J Gen Virol 71:1029–1037
Ono C, Kamagata T, Taka H, Sahara K, Asano S, Bando H (2012) Phenotypic grouping of 141 BmNPVs lacking viral gene sequences. Virus Res 165:197–206
Pearson MN, Rohrmann GF (2002) Transfer, incorporation, and substitution of envelope fusion proteins among members of the Baculoviridae, Orthomyxoviridae, and Metaviridae (insect retrovirus) families. J Virol 76:5301–5304
Pearson MN, Groten C, Rohrmann GF (2000) Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the baculoviridae. J Virol 74:6126–6131
Prikhod’ko EA, Lu A, Wilson JA, Miller LK (1999) In vivo and in vitro analysis of baculovirus ie-2 mutants. J Virol 73:2460–2468
Robertson JL, Russell RM, Preisler HK, Savin NE (2007) Bioassays with arthropods, 2nd edn. CRC Press, Boca Raton, p 224
Rohrmann GF (2011) Baculovirus molecular biology. National Center for Biotechnology Information, Bethesda
Shang Y, Wang M, Xiao G, Wang X, Hou D, Pan K, Liu S, Li J, Wang J, Arif BM, Vlak JM, Chen X, Wang H, Deng F, Hu Z (2017) Construction and rescue of a functional synthetic baculovirus. ACS Synth Biol 6:1393–1402
Shen YW, Wang HP, Xu WF, Wu XF (2018) Bombyx mori nucleopolyhedrovirus orf133 and orf134 are involved in the embedding of occlusion-derived viruses into polyhedra. J Gen Virol 99:717–729
Shirata N, Ikeda M, Kamiya K, Kawamura S, Kunimi Y, Kobayashi M (1999) Replication of nucleopolyhedroviruses of Autographa californica (Lepidoptera: Noctuidae), Bombyx mori (Lepidoptera: Bombycidae), Hyphantria cunea (Lepidoptera: Arctiidae), and Spodoptera exigua (lepidoptera: Noctuidae) in four lepidopteran cell lines. Appl Entomol Zool 34:507–516
Terada K, Mori M (2000) Human Dnaj homologs dj2 and dj3, and bag-1 are positive cochaperones of hsc70. J Biol Chem 275:24728–24734
Thiem SM, Miller LK (1989) Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 63:2008–2018
Wang M, Tan Y, Yin F, Deng F, Vlak JM, Hu Z, Wang H (2008) The F-like protein ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein f. J Virol 82:9800–9804
Wang R, Deng F, Hou D, Zhao Y, Guo L, Wang H, Hu Z (2010) Proteomics of the Autographa californica nucleopolyhedrovirus budded virions. J Virol 84:7233–7242
Wang X, Chen C, Zhang N, Li J, Deng F, Wang HL, Vlak JM, Hu ZH, Wang ML (2018) The group I alphabaculovirus-specific protein, ac5, is a novel component of the occlusion body but is not associated with odvs or the pif complex. J Gen Virol 99:585–595
Wei W, Zhou Y, Lei C, Sun X (2012) Autographa californica multiple nucleopolyhedrovirus orf114 is not essential for virus replication in vitro, but its knockout reduces per os infectivity in vivo. Virus Genes 45:360–369
Yang R, Zhang J, Feng M, Wu X (2016) Identification of Bombyx mori nucleopolyhedrovirus bm58a as an auxiliary gene and its requirement for cell lysis and larval liquefaction. J Gen Virol 97:3039–3050
Yu Q (2015) Sequence analysis and functional study on ac30 gene. J Huazhong Agric Univ 34:13–19
Zanotto PM, Kessing BD, Maruniak JE (1993) Phylogenetic interrelationships among baculoviruses: Evolutionary rates and host associations. J Invertebr Pathol 62:147–164
Zeng X, Nan F, Liang C, Song J, Wang Q, Vlak JM, Chen X (2009) Functional analysis of the Autographa californica nucleopolyhedrovirus iap1 and iap2. Sci China C Life Sci 52:761–770
Zou Z, Liu J, Wang Z, Deng F, Wang H, Hu Z, Wang M, Zhang T (2016) Characterization of two monoclonal antibodies, 38f10 and 44d11, against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus. Virol Sin 31:490–499
Acknowledgements
This research was supported by the grants from the Key Research Project of Frontier Science (QYZDJ-SSW-SMC021), the Strategic Priority Research Program (grant No. XDB11030400) from the Chinese Academy of Sciences, and the grants (No. 31621061) from the National Natural Science Foundation of China. We would like to thank Mr. Ding Gao, Ms. Pei Zhang and Ms. An-na Du, Mr. He Zhao, and Ms. Li Li from The Core Facility and Technical Support of Wuhan Institute of Virology for their technical assistance.
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WS, ZH and MW designed the experiments. WS, LH and QC performed the experiments and analyzed the data. WS, ZH and MW wrote the manuscript. JL, FD, HW, ZH and MW edited and commented on the manuscript.
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Shao, W., He, L., Chen, Q. et al. Functional Characterization of the Group I Alphabaculovirus Specific Gene ac73. Virol. Sin. 34, 701–711 (2019). https://doi.org/10.1007/s12250-019-00146-9
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DOI: https://doi.org/10.1007/s12250-019-00146-9