Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells

Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, then some products will comprise a chimeric molecule with part of the polyhedrin protein. In this study, we showed that if AUU is placed in the frame with a Strep tag and eGFP coding region, we could identify a protein product with both sequences present. Further work examined if alternative codons in lieu of AUG might reduce translation initiation further. We found that AUA was used slightly more efficiently than AUU, whereas AUC was the least efficient at initiating translation. The use of this latter codon suggested that there might also be a slight improvement of protein yield if this is incorporated into expression vectors.

Comparative Transcriptome Analysis of Bombyx mori (Lepidoptera) Larval Hemolymph in Response to Autographa californica Nucleopolyhedrovirus in Differentially Resistant Strains

Bombyx mori nucleopolyhedrovirus (BmNPV) is a kind of pathogen that causes huge economic losses to silkworm production. Although Autographa californica nucleopolyhedrovirus (AcMNPV) and BmNPV are both baculoviruses, the host domains of these two viruses have almost no intersection in nature. Recently, it has been found that some silkworms could be infected by recombinant AcMNPV through a puncture, which provided valuable material for studying the infection mechanism of baculovirus to silkworm. In this study, comparative transcriptomics was used to analyse the hemolymph of two differentially resistant strains following AcMNPV inoculation. There were 678 DEGs in p50 and 515 DEGs in C108 following viral infection. Among them, the upregulation and downregulation of DEGs were similar in p50; however, the upregulated DEGs were nearly twice as numerous as the downregulated DEGs in C108. The DEGs in different resistant strains differed by GO enrichment. Based on KEGG enrichment, DEGs were mainly enriched in metabolic pathways in p50 and the apoptosis pathway in C108. Moreover, 13 genes involved in metabolic pathways and 11 genes involved in the apoptosis pathway were analysed. Among the DEGs involved in apoptosis, the function of BmTex261 in viral infection was analysed. The BmTex261 showed the highest expression in hemolymph and a significant response to viral infection in the hemolymph of C108, indicating that it is involved in anti-AcMNPV infection. This was further validated by the significantly decreased expression of viral gene lef3 after overexpression of BmTex261 in BmN cells. The results provide a theoretical reference for the molecular mechanism of resistance to BmNPV in silkworms.

Comparison of the potential activities of viral and bacterial chitinases

Background Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. Results In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Conclusion Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

Identification of miRNAs encoded by Autographa californica nucleopolyhedrovirus

Two Autographa californica nucleopolyhedrovirus (AcMNPV) encoded miRNAs, AcMNPV-miR-1 and AcMNPV-miR-3, have been reported by us in 2013 and 2019, respectively. Here, we present an integrated investigation of AcMNPV-encoded miRNAs, which include the above two miRNAs and three additional newly identified miRNAs. Six candidate miRNAs were predicted through small RNA deep sequencing and bioinformatics, of which, five were validated. Three miRNAs are located opposite the coding sequences, the other two are located in the coding sequences of viral genes. Targets in both virus and host were predicted and subsequently tested using dual-luciferase reporter assays. The validated targets were found mainly in AcMNPV, except for the targets of AcMNPV-miR-4, which are all host genes. Based on reporter assays, the five miRNAs predominantly function by down-regulating their targets. The transcription start sites of these miRNAs were bioinformatic screened based on known baculovirus promoter motifs. Our study reveals that AcMNPV-encoded miRNAs function as fine modulators of the interactions between host and virus by regulating viral and/or host genes.

Characterization of miRNAs encoded by Autographa californica nucleopolyhedrovirus

Two Autographa californica nucleopolyhedrovirus (AcMNPV) encoded miRNAs, AcMNPV-miR-1 and -miR-3, have been reported in 2013 and 2019. Here, we present an integrated investigation of AcMNPV-encoded miRNAs. Six candidate miRNAs were predicted through small RNA deep sequencing and bioinformatics, of which, five validated by experiments. Three miRNAs perfectly matched the coding sequence of viral genes. The other two are located in coding sequences of viral genes. Targets in both virus and host were predicted and subsequently tested using dual-luciferase reporter assay. The validated targets were found mainly in AcMNPV, except for the targets of AcMNPV-miR-4, which are all host genes. Based on reporter assays, the five miRNAs predominantly function by down-regulating their targets, though individual target is slightly up-regulated. The transcription start sites of these miRNAs were analyzed. Our results suggest that AcMNPV-encoded miRNAs function as fine modulators of the interactions between host and virus by regulating viral and/or host genes.Author summaryVirus-encoded miRNAs have been widely reported as modulators participating in almost all biological processes. However, among Baculoviridae, which consists of a large family of dsDNA viruses that infect numerous beneficial insects and agricultural pests, only several have been reported encoding miRNAs. To clarify the roles of AcMNPV-encoded miRNAs in host–pathogen interactions, we employed RNA deep sequencing and series of experimental approaches identifying AcMNPV-encoded miRNAs, followed by target validation and function deduction. Among them, AcMNPV-miR-1 and AcMNPV–miR-3 have been reported in 2013 and 2019, respectively. This study reveals the sites of these miRNAs in the genome, both in coding sequences and complements, suggesting diverse functions. These miRNAs target genes in the virus itself and in the host, largely by suppressing expression, with some enhancing it. The transcription initiations of the miRNAs were analyzed. Our results provide some insight into the finely regulated process of baculovirus infection.

Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress

ABSTRACTDuring the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of bothac141andvubirestricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV.IMPORTANCEBaculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus, where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV, but not those found in ODV, using a virus-encoded ubiquitin (vUbi). Therefore, ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV, thus ensuring lethal infection of the host.

Phosphorylation Induces Structural Changes in the Autographa californica Nucleopolyhedrovirus P10 Protein

ABSTRACT Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10. IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.