Introduction Baculoviruses are insect-specific large double-stranded DNA viruses, which have a wide range of applications in biological control, foreign genes expression and gene delivery. As eukaryotic expression vectors, baculoviruses have advantages in expression of high level of proteins, insertion of large size foreign DNAs, as well as posttranscriptional modifications (Kost and Kemp 2016; Chambers et al. 2018). A huge number of exogenous proteins have been successfully expressed by baculovirus/ insect expression system since it was first established in 1983 (Smith et al. 1983). In addition, baculovirus-mediated gene transduction of mammalian cells is emerging as an avenue for gene therapy and drug discovery (Thimiri Govinda Raj et al. 2020).
During hundreds of million-year co-evolution with insect hosts (Théxzé x et al. 2011), most baculoviruses have developed a unique biphasic life cycle, which is characterized by the production of two morphologically distinct virion phenotypes, budded viruses (BVs) and occlusionderived viruses (ODVs). ODVs are embedded in occlusion bodies (OBs), which provide stability and environmental protection to ODVs. After being ingested by insect hosts, ODVs are released from OBs due to the alkaline environment in insect midgut and an endogenous alkaline protease, leading to a primary infection of epithelial cells. Subsequently, progeny BVs are produced, bud from infected cells and disseminate infection to permissive cells and tissues resulting in a systemic infection and death of the infected hosts (Blissard and Theilmann 2018).
When baculoviruses are used for biological control of pests, the infectivity of both ODVs and BVs is required for the primary and the secondary infection, respectively. In contrast, when baculoviruses are used as expression or gene delivery vectors in vitro, only the infectivity of BVs is required. To date, total genome sequencing of more than 172 baculoviruses has been accomplished (Wennmann et al. 2018), revealing a genome size range between 80 and 180 kb and encoding approximately 90–180 genes. Studies on comparative genomics revealed 38 core genes conserved in all of the baculoviruses (Garavaglia et al. 2012; Javed et al. 2017). These core genes are indispensable for the complete life cycle of baculoviruses. Among these core genes, some are not required for BV production, such as the per os infectivity factors (PIFs), which are only essential for oral infection mediated by ODVs (Wang et al. 2017). So far, 10 PIFs have been identified, and deletion of any had no impact on BV productions (Wang et al. 2019). As for the whole baculovirus genome, there are likely more genes which are not required for infectious BV production. Removing genes dispensable for BV production could generate a smaller baculovirus genome that theoretically should be a better vector for the insertion and expression of exogenous genes.
Synthetic biotechnology provides a powerful tool for genome wide functional studies, and the success of synthesizing a minimal bacterial genome was a hall mark (Hutchison et al. 2016). Recently, a synthetic baculovirus was made in our lab, which exhibited similar biological properties as its parental Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculovirus (Shang et al. 2017). AcMNPV, the best-studied baculovirus, encodes 155 open reading frames (ORFs) and the functions of most genes have been successively uncovered. Review of the literatures identified 42 ORFs in AcMNPV had not been functionally verified by geneknockout assay before we initiated this study. Therefore, we systematically analyzed the impacts of these ORFs on infectious BV production by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. In addition, we determined the one-step growth curves of the knock-out viruses, aiming for future construction of an AcMNPV vector with a reduced genome size while retaining the properties of proper BV production.
AcMNPV encodes 155 ORFs, the functions of most have already been elucidated but prior to our study, 42 ORFs were still functionally unverified by gene-knockout assays (Table 1). To cast light on their roles in BV production, we constructed gene-knockout bacmids (Fig. 1A).
Gene Knockout bacmid BV production (P value*) Category ac12 bAc△12 P > 0.05 Dispensable ac19 bAc△19 P > 0.05 Dispensable ac26 bAc△26 P > 0.05 Dispensable ac29 bAc△29 P > 0.05 Dispensable ac33 bAc△33 P > 0.05 Dispensable ac42 bAc△42 P > 0.05 Dispensable ac44# bAc△44-45 P > 0.05 Dispensable ac45# bAc4△4-45 P > 0.05 Dispensable ac47 bAc△47 P > 0.05 Dispensable ac48 bAc△48 P > 0.05 Dispensable ac52 bAc△52 P > 0.05 Dispensable ac55 bAc△55 P > 0.05 Dispensable ac56 bAc△56 P > 0.05 Dispensable ac57 bAc△57 P > 0.05 Dispensable ac58# bAc△58-59 P > 0.05 Dispensable ac59# bAc△58-59 P > 0.05 Dispensable ac60 bAc△60 P > 0.05 Dispensable ac63 bAc△63 P > 0.05 Dispensable ac72 bAc△72 P > 0.05 Dispensable ac74 bAc△74 P > 0.05 Dispensable ac84 bAc△84 P > 0.05 Dispensable ac85 bAc△85 P > 0.05 Dispensable ac87 bAc△87 P > 0.05 Dispensable ac91 bAc△91 P > 0.05 Dispensable ac97 bAc△97 P > 0.05 Dispensable ac111 bAc△111 P > 0.05 Dispensable ac112# bAc△112-113 P > 0.05 Dispensable ac113# bAc△112-113 P > 0.05 Dispensable ac116# bAc△116-117 P > 0.05 Dispensable ac117# bAc△116-117 P > 0.05 Dispensable ac118 bAc△118 P > 0.05 Dispensable ac121# bAc△120-122-REP120 P > 0.05 Dispensable ac122# bAc△120-122-REP120 P > 0.05 Dispensable ac140 bAc△140 P > 0.05 Dispensable ac149 bAc△149 P > 0.05 Dispensable ac154 bAc△154 P > 0.05 Dispensable ac13 bAc△13 0.01 < P < 0.05 Important ac51 bAc△51 P < 0.01 Important ac120# bAc△120-122 0.01 < P < 0.05 Important ac62 bAc△62 NA Essential ac82 bAc△82 NA Essential ac106/107 bAc△106/107 NA Essential *P value was the statistical analysis result of the one-step growth curve of the knockout virus in comparison with that of the control virus AcMNPV-egfp and the statistical analyses were analyzed by one-way analysis of variance (one-way ANOVA) using BV titers through 0 h to 96 h p.i.
#Those genes were analyzed using a jointly-deleted bacmid
Table 1. Summary of the impact to BV production by the knockout of the 42 genes.
Figure 1. Construction and characterization of knockout and repaired AcMNPV bacmids. A Schematic diagram of the parental and recombinant bacmids. Knockout bacmids were constructed by replacing either the entire or partial ORF with a Zeocin cassette via homologous recombination in E. coli. For constructing repaired bacmids, each ORF driven by its native promoter was cloned into the pFastBacDual transfer vector and inserted into the attTn7 locus of the knockout bacmid by transportation. B Fluorescence microscopy of transfection of the 36 knockout bacmids. Sf9 cells were individually transfected with the knockout bacmids and the images were taken at 72 h post transfection (p.t.). Bar, 400 μm. C Fluorescence microscopy of infection of the 36 knockout bacmids. At 96 h p.t., the supernatants were used to infect Sf9 cells and the images were captured at 72 h post infection (p.i.). Bar, 400 μm. D Fluorescence microscopy of transfection and infection results of the repaired recombinants. The images were taken at 72 h post transfection (upper panel) or postinfection (lower panel). Bar, 400 μm.
As orf44 and orf45 overlap with each other, they were deleted together in the bacmid bAc△44-45; similarly, orf58 and orf59 were jointly deleted in bAc△58-59, orf112 and orf113 were jointly-deleted in bAc△112-113, orf116 and orf117 were jointly-deleted in bAc△116-117, and orf120, orf121 and orf122 were jointly-deleted in bAc△120-122. Ac106 and ac107 were initially identified as two separate ORFs but later found to be a single gene (Harrison and Bonning 2003), consequently, ac106/107 was regarded as a single gene in our study. Therefore, a total of 36 knockout bacmids were generated. All the knockout bacmids were confirmed by PCR (data not shown).
To determine if the deletion of the target genes were essential for BV production, the knockout bacmids and the parental bacmid bAcMNPV-egfp were analyzed by transfection and infection assays.
Fluorescence microscopy showed that at 72 h, the cells transfected with bacmids of bAc△13, bAc△51, bAc△62, bAc△82, bAc△106/107, and bAc△120-122 exhibited much less fluorescence, while the rest of the bacmids did not have an obvious effect on fluorescence expression when compared to the control bacmid (Fig. 1B). The results of subsequent infection showed that knockout of 3 genes (ac62, ac82 and ac106/107) abolished the production of infectious BVs, indicating these genes are essential for infectious BV production. For the rest of the bacmids, infectious BVs were produced, although the deletion of ac51 appeared to significantly affect the efficiency of BV production (Fig. 1C).
To confirm the impacts of the deleted genes on BV production, repaired bacmids containing ac13, ac51, ac62, ac82, ac106/107, or ac120-122 were constructed on the backbone of the relevant deletion bacmids (Material and Methods, Fig. 1A). In addition, ac120, ac121, and ac122 were individually repaired on the backbone of bAc△120- 122. Finally, 9 repaired bacmids were generated and named as bAc△13-REP, bAc△51-REP, bAc△62-REP, bAc△82- REP, bAc△106/107-REP, bAc△120-122-REP, bAc△120- 122-REP120, bAc△120-122-REP121 and bAc△120-122- REP122.
The cells transfected with bAc△13-REP, bAc△51-REP, bAc△62-REP, bAc△82-REP, bAc△106/107-REP, bAc△120-122-REP and bAc△120-122-REP120 exhibited similar fluorescence expression to that of the control bacmid at 72 h, while bAc△120-122-REP121, bAc△120-122- REP122 exhibited less fluorescence expression in comparison to that of the control virus (Fig. 1D) and subsequent infection assays showed all the repaired recombinant viruses could produce progeny viruses (Fig. 1D). The results suggest that the impact to the BV production were indeed caused by the knockout of the genes, and ac120, appears to be the key factor among ac120-122.
The kinetics of the knockout viruses were elucidated by one-step growth curve analyses performed with each recombinant virus except those could not produce any infectious BVs. As shown in Fig. 2A, the growth curves of vAc∆13, and vAc∆120-122 produced obviously lower yields of progeny virions from 24 h p.i. to 96 h p.i. that were significantly different from that of the control virus (0.01 < P < 0.05). In addition, vAc∆51 produced significantly lower levels of infectious BVs than those obtained with the control virus (P < 0.01), and the growth curve was performed with a low MOI of 0.2 (Fig. 2B). The rest of the knockout viruses produced levels of BV comparable to that of the WT virus (P > 0.05) (Supplementary Fig. S1, Table 1).
Figure 2. One-step growth curves. A One-step growth curves of vAc413, vAc4120–122 and vAcMNPV-egfp (vWT). Sf9 Cells were infected at an MOI of 5. B Growth curves of vAc451 and vAcMNPV-egfp at an MOI of 0.2. C One-step growth curves of the 9 repaired viruses and vAcMNPV-egfp. The supernatants from infected cells were collected at the indicated time points and BV titers were determined by an endpoint dilution assay in triplicates. The growth curves with significant difference to that of the vWT were indicated: ★: 0.01 < P < 0.05. ★★: P < 0.01. The results of one-step growth curves of other knockout viruses were shown in Supplementary Fig. S1, and were statistically insignificant (P > 0.05) in comparison with that of vAcMNPV-egfp (Table 1).
The kinetics of the repaired viruses were also conducted and the growth curves of vAc∆13-REP, vAc∆51-REP, vAc∆62-REP, vAc∆82-REP, vAc∆106/107-REP, vAc∆120-122-REP and vAc∆120-122-REP120 exhibited no significant difference from that of the control virus (P[ 0.05) (Fig. 2C). However, vAc∆120-122-REP121 and vAc∆120-122-REP122 produced significantly lower levels of progeny BVs than that of the control virus (0.01 < P < 0.05) (Fig. 2C).
Based on the results of transfection and one-step growth curves, we classified the target genes into three categories: (1) Dispensable: the BV production of the knockout virus is not significantly different from that of the control virus (P > 0.05); (2) Essential: the knockout bacmid was totally impaired in infectious BV production; and (3) Important: the BV production of the knockout virus is significantly different from that of the control virus (P < 0.05). According to the delineated criteria, among the 42 genes investigated, 3 genes, ac62, ac82 and ac106/107 are essential; 3 genes, ac13, ac51, ac120 are important, while the rests are dispensable for infectious BV production (Table 1, Fig. 2).
Following the above definition, we classify all the AcMNPV ORFs based on their impacts on infectious BV production by combining our results to previous publications. Among the 155 AcMNPV ORFs, 99 are dispensable, 46 are essential, and 10 are important for BV production. The AcMNPV genes, which are essential or important for infectious BV production are listed in Table 2, and they appear to play important roles in viral replication, transcription, structure, or regulation of host activities.
Gene Protein Function Impact on BV production (references) ac1 Proteins tyrosine phosphatase (ptp) Regulation of host activities Important Takagi et al. (1998) ac6* Lef-2 Replication Essential Wu et al. (2010) ac9 pp78/83 Structure Essential Vialard and Richardson (1993) ac10 Protein kinase-1 (PK-1) Other Essential Liang et al. (2013) ac11 Ac11 Other Essential Tao et al. (2015) ac13 Ac13 Other Important This study ac14* Lef-1, DNA primase Replication Essential Mikhailov and Rohrmann, (2002) ac24 Protein kinase interacting protein (PKIP) Nuleocapsid assembly Essential Fan et al. (1998) ac25 Single-stranded DNA binding protein (DBP) Replication Essential Vanarsdall et al. (2007a) ac34 Ac34 Other Important Cai et al. (2012) ac37 Lef-11 Replication Essential Lin and Blissard (2002) ac40* P47, RNA polymerase subunit Transcription Essential Carstens et al. (1993) ac50* Lef-8, RNA polymerase subunit Transcription Important Gauthier et al. (2012) ac51 DnaJ domain protein Other Important This study; Qiu et al. (2019) ac53* Ac53 Nucleocapsid assembly Essential Liu et al. (2008) ac53a Lef-10 Replication Essential Xu et al. (2016) ac54* VP1054 Nucleocapsid assembly Essential Marek et al. (2013) ac62* Lef-9, RNA polymerase subunit Transcription Essential This study ac65* DNA polymerase Replication Essential Vanarsdall et al. (2005) ac66 Ac66 Other Important Ke et al. (2008) ac67 Lef-3 Replication Important Nie et al. (2012) ac73 Ac73 Other Important Shao et al. (2019) ac75 Ac75 Other Essential Shi et al. (2018) ac76 Ac76 Other Essential Hu et al. (2010) ac77* Very late factor-1 (Vlf-1) Replication Essential Li et al. (2005) ac78* Ac78 Structure Essential Tao et al. (2013) ac80* GP41, tegument protein Other Essential Li et al. (2018) ac81* Ac81 Other Essential Dong et al. (2016) ac82 Telokin-like protein (TLP) Other Essential This study ac83* VP91, PIF8 Nuleocapsid assembly Essential Zhu et al. (2013) ac89* VP39 Structure Essential Thiem and Miller (1989) ac90* Lef-4 Transcription Essential Knebel-Mörsdorf et al. (2006) ac92 P33, sulfhydryl oxidase Other Essential Wu and Passarelli (2010) ac93* Ac93 Transcription Essential Yuan et al. (2011) ac94* ODV-E25 Structure Essential Chen et al. (2012) ac95* DNA helicase, P143 Replication Essential Gordon and Carstens (1984) ac98* 38K Nucleocapsid assembly Essential Wu et al. (2006) ac99* Lef-5 Transcription Essential Su et al. (2011) ac100* P6.9 Nucleocapsid assembly Essential Wang et al. (2010) ac101* BV/ODV-C42 Nucleocapsid assembly Essential Wang et al. (2008) ac102 P12 Replication Essential Gandhi et al. (2012) ac103* P45 Other Essential Yuan et al. (2008) ac104 VP80 Other Essential Marek et al. (2011) ac106/ 107 Ac106/107 Other Essential This study ac109* Ac109 Nuleocapsid assembly Essential Lin et al. (2009) ac120 Ac120 Other Important This study ac128 GP64 Structure, entry Essential Monsma et al. (1996) ac132 Ac132 Other Essential Yang et al. (2014); Fang et al. (2016) ac133* Alkaline nuclease (AN) Transcription Essential Okano et al. (2004) ac139 ME53 Replication Essential Xi et al. (2007) ac141 exon0 Other Essential Dai et al. (2004) ac142* Ac142 Other Essential McCarthy et al. (2008) ac143* ODV-E18 Structure Essential McCarthy and Theilmann (2008) ac144* Ac144 Other Essential Vanarsdall et al. (2007b) ac146 Ac146 Other Essential Dickison et al. (2012) ac153 PE38 Replication Important Milks et al. (2003) ac147-0 IE-0 Transcription ie0-ie1 is essential Stewart et al. (2005) ac147-1 IE-1 Transcription *Core gene.
Table 2. AcMNPV genes essential or important for infectious BV production