Search

The correct formation of native disulfide bonds is critical for the proper structure and function of many proteins. Cellular disulfide bond formation pathways commonly consist of two parts: sulfhydryl oxidase-mediated oxidation and disulfide isomerase-mediated isomerization. Some large DNA viruses, such as baculoviruses, encode sulfhydryl oxidases, but viral disulfide isomerases have not yet been identified, although G4L in poxvirus has been suggested to serve such a function. Here, we report that the baculovirus core gene ac81 encodes a putative disulfide isomerase. ac81 is conserved in baculoviruses, nudiviruses, and hytrosaviruses. We found that AC81 homologs contain a typical thioredoxin fold conserved in disulfide isomerases. To determine the role of AC81, a series of Autographa californica nucleopolyhedrovirus (AcMNPV) bacmids containing ac81 knockout or point mutations was generated, and the results showed that AC81 is essential for budded virus production, multinucleocapsid occlusion-derived virus (ODV) formation, and ODV embedding in occlusion bodies. Nonreducing Western blot analysis indicated that disulfide bond formation in per os infectivity factor 5 (PIF5), a substrate of the baculoviral sulfhydryl oxidase P33, was abnormal when ac81 was knocked out or mutated. Pulldown assays showed that AC81 interacted with PIF5 and P33 in infected cells. In addition, two critical regions that harbor key amino acids for function were identified in AC81. Taken together, our results suggest that AC81 is a key component involved in the baculovirus disulfide bond formation pathway and likely functions as a disulfide isomerase. IMPORTANCE Many large DNA viruses, such as poxvirus, asfarvirus, and baculovirus, encode their own sulfhydryl oxidase to facilitate the disulfide bond formation of viral proteins. Here, we show that AC81 functions as a putative disulfide isomerase and is involved in multiple functions of the baculovirus life cycle. Interestingly, AC81 and P33 (sulfhydryl oxidase) are conserved in baculoviruses, nudiviruses, and hytrosaviruses, which are all insect-specific large DNA viruses replicating in the nucleus, suggesting that viral disulfide bond formation is an ancient mechanism shared by these viruses.

The engineering of viral genomes facilitates both fundamental and applied research on viruses. However, the multiloci manipulation of DNAs of viruses with large DNA genomes, such as baculoviruses, herpesviruses, and poxviruses, is technically challenging, particularly for highly homologous or repetitive sequences. Homologous regions ( hrs ) have multiple copies in many large DNA viruses and play pivotal roles in the viral life cycle. Here, we used synthetic biology to investigate the fundamental function of baculoviral hrs by conducting multiloci manipulation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA that contains eight hrs scattered in the genome. Using transformation-associated recombination in yeast, we generated recombinant AcMNPV genomes in which we deleted all hrs or retained a single hr ( hr1 , hr2 , or hr3 ). Infectious viruses were rescued after transfecting the synthetic viral genomes into host cells, and their replication features were characterized. The results demonstrated that deletion of all hrs severely compromised viral DNA replication and progeny production, whereas retaining only a single hr was essential for efficient viral DNA replication and progeny production. The synthetic virus with hr2 or hr3 showed a growth curve similar to that of the parental virus. Transcriptomic analysis revealed that hr1 , hr2 , and hr3 could enhance gene transcription within a surrounding region of 14.6 kb, 13.8 kb, and 29.8 kb, respectively. Overall, this study revealed the advantages of synthetic biology in multiloci engineering and functional studies of large DNA viruses. In addition, our findings on hrs will be helpful for the design and improvement of baculovirus-based expression vectors.

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 ( lef-1 and lef-3 , respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9 . No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.

Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome. Recently, we generated a synthetic baculovirus AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration; its DNA copy numbers could be induced to a higher level in E. coli; and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection., (© 2021. Wuhan Institute of Virology, CAS.)

Baculoviridae constitutes a family of insect-specific, large DNA viruses with a unique life cycle characterized by the production of two morphologically distinct virions, the budded virus (BV) and the occlusion-derived virus (ODV). ODV and BV, with different tissue tropisms, have been widely applied in the areas of biological control and biotechnology, respectively. In nature, baculovirus infection of susceptible host larvae is initiated by ODV-mediated primary infection, followed by the production of BV for spreading infection within larval body. Across millions of years of co-evolution with their hosts, baculoviruses have developed dedicated mechanisms for efficient entry/egress, genome replication/transcription, and virion assembly by employing either their own proteins or host machineries. They have also adopted versatile strategies to precisely regulate the immunity, behaviors and physiology of hosts to facilitate their own replication and dispersal. In this chapter, research advances relating to key aspects of the baculovirus life cycle are reviewed, and the application of a newly-developed baculovirus synthetic biology technology is introduced. Finally, future avenues for baculovirus research are discussed.

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However, the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins (GPs), such as vesicular stomatitis virus G protein (VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64. Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus (AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4-12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher pH conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0 , - 4 , - 6 , - 7 , or - 9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1 , - 2 , or - 3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection. IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex., (Copyright © 2019 American Society for Microbiology.)

Baculoviridae is a family of large DNA viruses that infect insects. They have been extensively used as safe and efficient biological agents for the control of insect pests. As a result of coevolution with their hosts, baculoviruses developed unique life cycles characterized by the production of two distinctive virion phenotypes, occlusion-derived virus and budded virus, which are responsible for mediating primary infection in insect midgut epithelia and spreading systemic infection within infected insects, respectively. In this article, advances associated with virus-host interactions during the baculovirus life cycle are reviewed. We mainly focus on how baculoviruses exploit versatile strategies to overcome diverse host barriers and establish successful infections. For example, in the midgut, baculoviruses encode enzymes to degrade peritrophic membranes and use a series of per os infectivity factors to initiate primary infection. A viral fibroblast growth factor is expressed to attract tracheoblasts that spread the virus for systemic infection. Baculoviruses use different strategies to suppress host defence systems, including apoptosis, melanization and RNA interference. Additionally, baculoviruses can manipulate host physiology and induce 'tree-top disease' for optimal virus replication and dispersal. These advances in our understanding of baculoviruses will greatly inform the development of more effective baculoviral pesticides. This article is part of the theme issue 'Biotic signalling sheds light on smart pest management'.

Oxyplax ochracea (Moore) is a pest that causes severe damage to a wide range of crops, forests and fruit trees. The complete genome sequence of Oxyplax ochracea nucleopolyhedrovirus (OxocNPV) was determined using a Roche 454 pyrosequencing system. OxocNPV has a double-stranded DNA (dsDNA) genome of 113,971 bp with a G+C content of 31.1%. One hundred and twenty-four putative open reading frames (ORFs) encoding proteins of >50 amino acids in length and with minimal overlapping were predicted, which covered 92% of the whole genome. Six baculoviral typical homologous regions (hrs) were identified. Phylogenetic analysis and gene parity plot analysis showed that OxocNPV belongs to clade "a" of Group I alphabaculoviruses, and it seems to be close to the most recent common ancestor of Group I alphabaculoviruses. Three unique ORFs (with no homologs in the National Center for Biotechnology Information database) were identified. Interestingly, OxocNPV lacks three auxiliary genes (lef7, ie-2 and pcna) related to viral DNA replication and RNA transcription. In addition, OxocNPV has significantly different sequences for several genes (including ie1 and odv-e66) in comparison with those of other baculoviruses. However, three dimensional structure prediction showed that OxocNPV ODV-E66 contain the conserved catalytic residues, implying that it might possess polysaccharide lyase activity as AcMNPV ODV-E66. All these unique features suggest that OxocNPV represents a novel species of the Group I alphabaculovirus lineage.

Melanization, an important insect defense mechanism, is mediated by clip-domain serine protease (cSP) cascades and is regulated by serpins. Here we show that proteolytic activation of prophenoloxidase (PPO) and PO-catalyzed melanization kill the baculovirus in vitro. Our quantitative proteomics and biochemical experiments revealed that baculovirus infection of the cotton bollworm, Helicoverpa armigera, reduced levels of most cascade members in the host hemolymph and PO activity. By contrast, serpin-9 and serpin-5 were sequentially upregulated after the viral infection. The H. armigera serpin-5 and serpin-9 regulate melanization by directly inhibiting their target proteases cSP4 and cSP6, respectively and cSP6 activates PPO purified from hemolymph. Furthermore, serpin-5/9-depleted insects exhibited high PO activities and showed resistance to baculovirus infection. Together, our results characterize a part of the melanization cascade in H. armigera, and suggest that natural insect virus baculovirus has evolved a distinct strategy to suppress the host immune system.

Baculoviruses are a family of arthropod-specific large DNA viruses that infect insect species belonging to the orders Lepidoptera, Hymenoptera and Diptera. In nature, occlusion-derived viruses (ODVs) initiate baculovirus primary infection in the midgut epithelium of insect hosts, and this process is largely dependent on a number of ODV envelope proteins designated as per os infectivity factors (PIFs). Interestingly, PIF homologs are also present in other invertebrate large DNA viruses, which is indicative that per os infection is an ancient and phylogenetically conserved entry mechanism shared by these viruses. Here, we review the advances in the knowledge of the functions of individual PIFs and recent discoveries about the PIF complex, and discuss the evolutionary implications of PIF homologs in invertebrate DNA viruses. Furthermore, future research highlights on the per os infection mechanism are also prospected.

Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cell culture. An f homologue gene is absent in gammabaculoviruses. Here we characterized the putative F-homologue (Cuni-F), encoded by (ORF) 104 of Culex nigripalpus nucleopolyhedrovirus (CuniNPV), the only deltabaculovirus member. When expressed alone, this protein seems to locate on the cell surface and is able to induce cell-cell fusion. When expressed by an alphabaculovirus (Autographa california nucleopolyhedrovirus), it was found to be incorporated into BVs. Western blot analyses detected the uncleaved Cuni-F0 and the furin-cleaved F1 forms. Treatment of infected cells with tunicamycin showed that Cuni-F contains N-glycans. Mutagenesis analysis identified the canonical furin cleavage site 126RARR129 as being responsible for the cleavage of Cuni-F in insect cells. The collective evidence suggests that CuniNPV encodes a functional F protein.

Baculoviruses are insect-specific pathogens with a generally narrow host ranges. Successful primary infection is initiated by the proper interaction of at least 8 conserved per os infectivity factors (PIFs) with the host's midgut cells, a process that remains largely a mystery. In this study, we investigated the host specificities of the four core components of the PIF complex, P74, PIF1, PIF2 and PIF3 by using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) backbone. The four pifs of HearNPV were replaced by their counterparts from a group I Autographa californica multiple nucleopolyhedrovirus (AcMNPV) or a group II Spodoptera litura nucleopolyhedrovirus (SpltNPV). Transfection and infection assays showed that all the recombinant viruses were able to produce infectious budded viruses (BVs) and were lethal to H. armigera larvae via intrahaemocoelic injection. However, feeding experiments using very high concentration of occlusion bodies demonstrated that all the recombinant viruses completely lost oral infectivity except SpltNPV pif3 substituted pif3-null HearNPV (vHaBacΔpif3-Sppif3-ph). Furthermore, bioassay result showed that the median lethal concentration (LC50) value of vHaBacΔpif3-Sppif3-ph was 23-fold higher than that of the control virus vHaBacΔpif3-Hapif3-ph, indicating that SpltNPV pif3 can only partially substitute the function of HearNPV pif3. These results suggested that most of PIFs tested have strict host specificities, which may account, at least in part, for the limited host ranges of baculoviruses.

The genome sequence of Catopsilia pomona nucleopolyhedrovirus (CapoNPV) was determined by the Roche 454 sequencing system. The genome consisted of 128,058 bp and had an overall G+C content of 40%. There were 130 hypothetical open reading frames (ORFs) potentially encoding proteins of more than 50 amino acids and covering 92% of the genome. Among all the hypothetical ORFs, 37 baculovirus core genes, 23 lepidopteran baculovirus conserved genes and 10 genes conserved in Group I alphabaculoviruses were identified. In addition, the genome included regions of 8 typical baculoviral homologous repeat sequences (hrs). Phylogenic analysis showed that CapoNPV was in a distinct branch of clade "a" in Group I alphabaculoviruses. Gene parity plot analysis and overall similarity of ORFs indicated that CapoNPV is more closely related to the Group I alphabaculoviruses than to other baculoviruses. Interesting, CapoNPV lacks the genes encoding the fibroblast growth factor (fgf) and ac30, which are conserved in most lepidopteran and Group I baculoviruses, respectively. Sequence analysis of the F-like protein of CapoNPV showed that some amino acids were inserted into the fusion peptide region and the pre-transmembrane region of the protein. All these unique features imply that CapoNPV represents a member of a new baculovirus species.

Clostera anastomosis (Lepidoptera: Notodontidae) is a defoliating forest insect pest. Clostera anastomosis granulovirus-B (ClasGV-B) belonging to the genus Betabaculovirus of family Baculoviridae has been used for biological control of the pest. Here we reported the full genome sequence of ClasGV-B and compared it to other previously sequenced baculoviruses. The circular double-stranded DNA genome is 107,439 bp in length, with a G+C content of 37.8% and contains 123 open reading frames (ORFs) representing 93% of the genome. ClasGV-B contains 37 baculovirus core genes, 25 lepidopteran baculovirus specific genes, 19 betabaculovirus specific genes, 39 other genes with homologues to baculoviruses and 3 ORFs unique to ClasGV-B. Hrs appear to be absent from the ClasGV-B genome, however, two non-hr repeats were found. Phylogenetic tree based on 37 core genes from 73 baculovirus genomes placed ClasGV-B in the clade b of betabaculoviruses and was most closely related to Erinnyis ello GV (ErelGV). The gene arrangement of ClasGV-B also shared the strongest collinearity with ErelGV but differed from Clostera anachoreta GV (ClanGV), Clostera anastomosis GV-A (ClasGV-A, previously also called CaLGV) and Epinotia aporema GV (EpapGV) with a 20 kb inversion. ClasGV-B genome contains three copies of polyhedron envelope protein gene (pep) and phylogenetic tree divides the PEPs of betabaculoviruses into three major clades: PEP-1, PEP-2 and PEP/P10. ClasGV-B also contains three homologues of P10 which all harbor an N-terminal coiled-coil domain and a C-terminal basic sequence. ClasGV-B encodes three fibroblast growth factor (FGF) homologues which are conserved in all sequenced betabaculoviruses. Phylogenetic analysis placed these three FGFs into different groups and suggested that the FGFs were evolved at the early stage of the betabaculovirus expansion. ClasGV-B is different from previously reported ClasGV-A and ClanGV isolated from Notodontidae in sequence and gene arrangement, indicating the virus is a new notodontid betabaculovirus.

ha72 of Helicoverpa armigera nucleopolyhedrovirus (a homologue of ac78) was identified as a conserved late baculovirus gene and characterized. HA72 localizes in the intranuclear ring zone. By generating mutants, we showed that HA72 is essential for budded virus (BD) production and occlusion-derived virus (ODV) embedding. HA72 also interacted with P33, a baculoviral sulfhydryl oxidase. A point mutation of amino acid 22 from lysine to glutamic acid curtailed BV production and precluded ODV occlusion as well as interaction with P33.

The GP64 and F proteins were previously identified as the sole functional envelope fusion proteins in Baculoviridae. F-like proteins, present only in group I nucleopolyhedroviruses (NPVs), are remnant, nonfunctional F proteins. In this report, we describe the effect of the presence or absence of the F-like protein Ac23 in a gp64-null Autographa californica multinucleocapsid NPV pseudotyped with the F protein from Spodoptera exigua multicapsid NPV (SeF). We found that the presence of Ac23 elevates the infectivity of the pseudotyped virus. This is in contrast to the results of Lung et al. (J. Virol. 76:5729-5736, 2002), who found no such effect. The possible reasons for the differing results are discussed.