Identification and Functional Analysis of the Pre-piRNA 3' Trimmer in Silkworms.

PIWI-interacting RNAs (piRNAs) play a crucial role in transposon silencing in animal germ cells. In piRNA biogenesis, single-stranded piRNA intermediates are loaded into PIWI-clade proteins and cleaved by Zucchini/MitoPLD, yielding precursor piRNAs (pre-piRNAs). Pre-piRNAs that are longer than the mature piRNA length are then trimmed at their 3' ends. Although recent studies implicated the Tudor domain protein Papi/Tdrkh in pre-piRNA trimming, the identity of Trimmer and its relationship with Papi/Tdrkh remain unknown. Here, we identified PNLDC1, an uncharacterized 3'-5' exonuclease, as Trimmer in silkworms. Trimmer is enriched in the mitochondrial fraction and binds to Papi/Tdrkh. Depletion of Trimmer and Papi/Tdrkh additively inhibits trimming, causing accumulation of ∼35-40-nt pre-piRNAs that are impaired for target cleavage and prone to degradation. Our results highlight the cooperative action of Trimmer and Papi/Tdrkh in piRNA maturation.

Zucchini consensus motifs determine the mechanism of pre-piRNA production.

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.

The piRNA cluster torimochi is an expanding transposon in cultured silkworm cells.

PIWI proteins and PIWI-interacting RNAs (piRNAs) play a central role in repressing transposable elements in animal germ cells. It is thought that piRNAs are mainly produced from discrete genomic loci named piRNA clusters, which often contain many "dead" transposon remnants from past invasions and have heterochromatic features. In the genome of silkworm ovary-derived cultured cells called BmN4, a well-established model for piRNA research, torimochi was previously annotated as a unique and specialized genomic region that can capture transgenes and produce new piRNAs bearing a trans-silencing activity. However, the sequence identity of torimochi has remained elusive. Here, we carefully characterized torimochi by utilizing the updated silkworm genome sequence and the long-read sequencer MinION. We found that torimochi is in fact a full-length gypsy-like LTR retrotransposon, which is exceptionally active and has massively expanded its copy number in BmN4 cells. Many copies of torimochi in BmN4 cells have features of open chromatin and the ability to produce piRNAs. Therefore, torimochi may represent a young, growing piRNA cluster, which is still "alive" and active in transposition yet capable of trapping other transposable elements to produce de novo piRNAs.

Non-gonadal somatic piRNA pathways ensure sexual differentiation, larval growth, and wing development in silkworms.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans' gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.

The two Gtsf paralogs in silkworms orthogonally activate their partner PIWI proteins for target cleavage.

The PIWI-interacting RNA (piRNA) pathway is a protection mechanism against transposons in animal germ cells. Most PIWI proteins possess piRNA-guided endonuclease activity, which is critical for silencing transposons and producing new piRNAs. Gametocyte-specific factor 1 (Gtsf1), an evolutionarily conserved zinc finger protein, promotes catalysis by PIWI proteins. Many animals have multiple Gtsf1 paralogs; however, their respective roles in the piRNA pathway are not fully understood. Here, we dissected the roles of Gtsf1 and its paralog Gtsf1-like (Gtsf1L) in the silkworm piRNA pathway. We found that Gtsf1 and Gtsf1L preferentially bind the two silkworm PIWI paralogs, Siwi and BmAgo3, respectively, and facilitate the endonuclease activity of each PIWI protein. This orthogonal activation effect was further supported by specific reduction of BmAgo3-bound Masculinizer piRNA and Siwi-bound Feminizer piRNA, the unique piRNA pair required for silkworm feminization, upon depletion of Gtsf1 and Gtsf1L, respectively. Our results indicate that the two Gtsf paralogs in silkworms activate their respective PIWI partners, thereby facilitating the amplification of piRNAs.

A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses.

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.

Two Complete Genomes of Male-Killing Wolbachia Infecting Ostrinia Moth Species Illuminate Their Evolutionary Dynamics and Association with Hosts.

Wolbachia is an extremely widespread intracellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of Ostrinia furnacalis and Ostrinia scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species' infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, two possible scenarios were proposed: (1) Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis, or (2) Wolbachia infection in these species was introgressively transferred from a currently unidentified relative. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint.

Expression and localization of Bombyx mori nucleopolyhedrovirus GP37.

Mitochondria play an essential role in intracellular energy metabolism. This study described the involvement of Bombyx mori nucleopolyhedrovirus (BmNPV) GP37 (BmGP37) in host mitochondria. Herein, the proteins associated with host mitochondria isolated from BmNPV-infected or mock-infected cells by two-dimensional gel electrophoresis were compared. One mitochondria-associated protein in virus-infected cells was identified as BmGP37 by liquid chromatography-mass spectrometry analysis. Furthermore, the BmGP37 antibodies were generated, which could react specifically with BmGP37 in the BmNPV-infected BmN cells. Western blot experiments showed that BmGP37 was expressed at 18 h post-infection and was verified as a mitochondria-associated protein. Immunofluorescence analysis demonstrated that BmGP37 localized to the host mitochondria during BmNPV infection. Furthermore, western blot analysis revealed that BmGP37 is a novel component protein of the occlusion-derived virus (ODV) of BmNPV. The present results indicated that BmGP37 is one of the ODV-associated proteins and may have important roles in host mitochondria during BmNPV infection.

Horizontal Gene Transfer and Gene Duplication of ß-Fructofuranosidase Confer Lepidopteran Insects Metabolic Benefits.

Horizontal gene transfer (HGT) is a potentially critical source of material for ecological adaptation and the evolution of novel genetic traits. However, reports on posttransfer duplication in organism genomes are lacking, and the evolutionary advantages conferred on the recipient are generally poorly understood. Sucrase plays an important role in insect physiological growth and development. Here, we performed a comprehensive analysis of the evolution of insect ß-fructofuranosidase transferred from bacteria via HGT. We found that posttransfer duplications of ß-fructofuranosidase were widespread in Lepidoptera and sporadic occurrences of ß-fructofuranosidase were found in Coleoptera and Hymenoptera. ß-fructofuranosidase genes often undergo modifications, such as gene duplication, differential gene loss, and changes in mutation rates. Lepidopteran ß-fructofuranosidase gene (SUC) clusters showed marked divergence in gene expression patterns and enzymatic properties in Bombyx mori (moth) and Papilio xuthus (butterfly). We generated SUC1 mutations in B. mori using CRISPR/Cas9 to thoroughly examine the physiological function of SUC. BmSUC1 mutant larvae were viable but displayed delayed growth and reduced sucrase activities that included susceptibility to the sugar mimic alkaloid found in high concentrations in mulberry. BmSUC1 served as a critical sucrase and supported metabolic homeostasis in the larval midgut and silk gland, suggesting that gene transfer of ß-fructofuranosidase enhanced the digestive and metabolic adaptation of lepidopteran insects. These findings highlight not only the universal function of ß-fructofuranosidase with a link to the maintenance of carbohydrate metabolism but also an underexplored function in the silk gland. This study expands our knowledge of posttransfer duplication and subsequent functional diversification in the adaptive evolution and lineage-specific adaptation of organisms.

Mutations in the polyhedrin NLS affect the assembly and polyhedral shape of alphabaculovirus occlusion bodies.

Alphabaculoviruses produce occlusion bodies (OBs) in the nucleus of the infected cells at the late stage of infection. OBs are mainly composed of a single viral protein called polyhedrin (POLH). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) POLH possesses a monopartite nuclear localization signal sequence (NLS), KRKK, from 32nd to 35th residues. However, the functions of POLH NLS of other alphabaculoviruses remain unknown. Here, POLH NLS mutants of Bombyx mori nucleopolyhedrovirus (BmNPV) were generated and NLS function as well as the relationship between NLS and OB localization or morphology was investigated. Deletion or mutation of BmNPV POLH NLS severely affected POLH and OB intracellular localization. Additionally, viruses in which the arginine residue at the 33rd position of POLH was mutated produced a lower number of OBs, which was presumably due to decreased POLH accumulation in the infected cells. Furthermore, cytoplasmic OBs were morphologically aberrant, even though nuclear OB morphology was normal in the same cell. These results indicate that NLS is required for nuclear localization and efficient accumulation of BmNPV POLH, which heavily affect the number and morphology of OBs.

Transcriptome analysis in the silkworm Bombyx mori overexpressing piRNA-resistant Masculinizer gene.

Dosage compensation is a process that produces a similar expression of sex-linked and autosomal genes. In the silkworm Bombyx mori with a WZ sex-determination system, the expression from the single Z in WZ females matches that of ZZ males due to the suppression of Z-linked genes in males. A primary maleness determinant gene, Masculinizer (Masc), is also required for dosage compensation. In females, silkworm Piwi is complexed with the W chromosome-derived female-specific Feminizer (Fem) PIWI-interacting RNA (piRNA) and cleaves Masc mRNA. When Fem piRNA-resistant Masc cDNA (Masc-R) is overexpressed in both sexes, only female larvae are dead during the larval stage. In this study, transcriptome analysis was performed in neonate larvae to examine the effects of Masc-R overexpression on a global gene expression profile. Z-linked genes were globally repressed in Masc-R-overexpressing females due to force-driven dosage compensation. In contrast, Masc-R overexpression had little effect on the expression of Z-linked genes and the male-specific isoform of B. mori insulin-like growth factor II mRNA-binding protein in males, indicating that excessive Masc expression strengthens neither dosage compensation nor maleness in males. Fourteen genes were differentially expressed between Masc-R-overexpressing and control neonate larvae in both sexes, suggesting Masc functions other than dosage compensation and masculinization.

Masculinizer-induced dosage compensation is achieved by transcriptional downregulation of both copies of Z-linked genes in the silkworm, Bombyx mori.

The evolution of dosage compensation produces similar expression of sex-linked and autosomal genes in the heterogametic sex. The silkworm (Bombyx mori), a lepidopteran insect, has a female heterogametic WZ sex determination system. A Z-linked gene, Masculinizer (Masc), is the primary determinant of maleness and dosage compensation in B. mori. However, it remains unknown whether one of the two Z chromosomes is inactivated or both Z chromosomes are suppressed in B. mori males. Hence, we performed transcriptome analysis using hybrids between two B. mori strains and analysed allele-specific expression to distinguish these alternatives. Our analysis revealed that genes on both the maternal and paternal Z chromosomes are transcriptionally upregulated in Masc knocked down males. We therefore conclude that both Z chromosomes are transcriptionally downregulated in B. mori males, similar to the system in Caenorhabditis elegans.

H3K4me3 histone modification in baculovirus-infected silkworm cells.

Baculovirus infection modulates the chromatin states and gene expression of host insect cells. Here we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of H3 trimethylated at Lys4 (H3K4me3) histone modification in Bombyx mori nucleopolyhedrovirus-infected Bombyx mori cells. The ChIP-seq data revealed the changes of the genome-wide distribution and accumulation of euchromatic histone marks in host insect cells during the progression of baculovirus infection.

High-resolution analysis of baculovirus-induced host manipulation in the domestic silkworm, Bombyx mori.

Many parasites manipulate host behaviour to enhance their transmission. Baculoviruses induce enhanced locomotory activity (ELA) combined with subsequent climbing behaviour in lepidopteran larvae, which facilitates viral dispersal. However, the mechanisms underlying host manipulation system are largely unknown. Previously, larval locomotion during ELA was summarized as the distance travelled for a few minutes at several time points, which are unlikely to characterize ELA precisely, as ELA typically persists for several hours. In this study, we modified a recently developed method using time-lapse recording to characterize locomotion of Bombyx mori larvae infected with Bombyx mori nucleopolyhedrovirus (BmNPV) for 24 h at 3 s resolution. Our data showed that the locomotion of the mock-infected larvae was restricted to a small area, whereas the BmNPV-infected larvae exhibited a large locomotory area. These results indicate that BmNPV dysregulates the locomotory pattern of host larvae. Furthermore, both the mock- and BmNPV-infected larvae showed periodic cycles of movement and stationary behavior with a similar frequency, suggesting the physiological mechanisms that induce locomotion are unaffected by BmNPV infection. In contrast, the BmNPV-infected larvae exhibited fast and long-lasting locomotion compared with mock-infected larvae, which indicates that locomotory speed and duration are manipulated by BmNPV.

Potential for small RNA production against Bombyx mori latent virus in Bombyx mori ovaries.

Bombyx mori latent virus (BmLV) is a positive, single-stranded insect RNA virus closely related to plant maculaviruses. BmLV was first isolated from Bombyx mori ovary-derived cell line BmN-4, and this virus has already infected most B. mori-derived cultured cell lines. We previously reported that small interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) pathways function cooperatively to maintain the amount of BmLV RNA for normal BmN-4 cell growth. On the other hand, BmLV does not propagate in B. mori larvae. Here we conducted BmLV injection into the larval body cavities of B. mori, and examined BmLV accumulation in larval ovaries where siRNA and piRNA pathways are both active, to investigate whether this in vivo resistance is governed by small RNA pathways. Expression levels of RNA-dependent RNA polymerase, coat protein, and p15 genes in BmLV-injected larval ovaries were extremely low compared with those in B. mori cultured cells, indicating that B. mori larval ovaries are more resistant to BmLV than B. mori cultured cells. We also sequenced small RNAs prepared from BmLV-injected larval ovaries and mapped them onto the BmLV genome. Although their amounts were very small, we were able to detect BmLV-derived small RNAs in the ovaries. According to their length distribution and nucleotide bias, they were likely to be siRNAs and piRNAs. These results suggest that B. mori ovaries can potentially produce small RNAs against BmLV, but the resistance of larval ovaries against BmLV is not dependent on RNA silencing pathways.

Characterization of nuclear localization signal in Ostrinia furnacalis Masculinizer protein.

Bombyx mori Masculinizer protein (BmMasc) is essential for both masculinization and dosage compensation in B. mori. We previously identified a bipartite nuclear localization signal (NLS) of BmMasc and two essential residues (lysine at 274 [K274] and arginine at 275 [R275]) implicated in its function. Sequence comparison showed the presence of putative NLSs in lepidopteran Masc proteins, but their functional properties and critical residues are unknown. Here we characterized a putative NLS of Ostrinia furnacalis Masc (OfMasc) using B. mori ovary-derived BmN-4 cell line. Deletion and alanine scanning mutagenesis revealed that a putative NLS is required for nuclear localization of OfMasc. However, mutations at both K227 and R228, which correspond to K274 and R275 of BmMasc, respectively, do not greatly abolish the NLS activity. Additional mutagenesis analysis revealed that triple mutations at K227, R228, and K240 almost completely inhibited OfMasc nuclear localization. These results suggest that lepidopteran Masc proteins possess a common functional NLS, but the critical residues for its activity are different. Moreover, we examined the masculinizing activity of OfMasc derivatives and found that nuclear localization is not required for the masculinizing activity of OfMasc. The results from our studies indicate that lepidopteran Masc proteins function in the cytoplasm to drive masculinizing cascade.

Bombyx mori nucleopolyhedrovirus ptp and egt genes are dispensable for triggering enhanced locomotory activity and climbing behavior in Bombyx mandarina larvae.

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors.

Whole-genome sequencing and comparative transcriptome analysis of Bombyx mori nucleopolyhedrovirus La strain.

The Bombyx mori nucleopolyhedrovirus (BmNPV) La is a variant BmNPV strain isolated in Laos. La has different features from BmNPV type strain T3 in virulence, production of the polyhedrin protein, and the formation of multicapsid occlusion-derived viruses. Here, the whole-genome sequence of La was compared to the sequences of nine BmNPV and two Bombyx mandarina nucleopolyhedrovirus strains. The complete La genome consisted of 127,618 base pairs with a G + C content of 40.3% and contained putative 136 open reading frames encoding more than 60 amino acids. The La genome lacked the bro-b gene and had the highest identity with that of the T3 strain. A comparison of the transcriptomes of La- and T3-infected cells showed that the expression levels of the polyhedrin and cathepsin genes were greater in cells infected with La as compared to those infected with T3. Interestingly, the virus genes with different RNA levels between the two BmNPV strains were assembled into five clusters in the genome of La. Also, the RNA levels of host ribosomal protein genes were significantly decreased in cells infected with La as compared to those infected with T3.

A single female-specific piRNA is the primary determiner of sex in the silkworm.

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Characterization of Bombyx mori nucleopolyhedrovirus infection in fat body-derived Bombyx mori cultured cells.

Bombyx mori nucleopolyhedrovirus (BmNPV) is known to replicate in many tissues of Bombyx mori larvae. However, the cell lines used for BmNPV research are predominantly derived from B. mori ovaries or early embryos. In the present study, we examined the properties of NIAS-Bm-aff3 (aff3), a cell line that was established from B. mori larval fat body, which is one of the major tissues for BmNPV propagation. aff3 is a floating cell line, and cell adhesion was enhanced following the coating of the culture dish with poly-d-lysine. RT-qPCR assays demonstrated that the expression of germ cell markers, Vasa, Siwi, and BmAgo3, was much lower in aff3 cells as compared to the B. mori ovary-derived cell line BmN-4. Conversely, aff3 cells express an adipocyte marker, Fabp1, at higher levels, indicating that this cell line retains the characteristics of fat body cells. BmNPV infection induces unique cell fusion in aff3 cells, which was also observed following infection with Autographa californica multiple nucleopolyhedrovirus, a virus that does not cause productive infection in B. mori cells. Occlusion bodies (OBs) produced in BmNPV-infected aff3 cells exhibit large cuboidal shapes as compared to those produced in BmN-4 cells. Furthermore, extremely large OBs (~25 µm in side length) were produced in aff3 cells when infected with a cuboidal polyhedrin mutant. Taking into account these unusual properties, we conclude that aff3 could prove to be a useful resource for conducting baculovirus research.