Plant and insect virus-like particles: emerging nanoparticles for agricultural pest management.

Virus-like particles (VLPs) represent a biodegradable, biocompatible nanomaterial made from viral coat proteins that can improve the delivery of antigens, drugs, nucleic acids, and other substances, with most applications in human and veterinary medicine. Regarding agricultural viruses, many insect and plant virus coat proteins have been shown to assemble into VLPs accurately. In addition, some plant virus-based VLPs have been used in medical studies. However, to our knowledge, the potential application of plant/insect virus-based VLPs in agriculture remains largely underexplored. This review focuses on why and how to engineer coat proteins of plant/insect viruses as functionalized VLPs, and how to exploit VLPs in agricultural pest control. The first part of the review describes four different engineering strategies for loading cargo at the inner or the outer surface of VLPs depending on the type of cargo and purpose. Second, the literature on plant and insect viruses the coat proteins of which have been confirmed to self-assemble into VLPs is reviewed. These VLPs are good candidates for developing VLP-based agricultural pest control strategies. Lastly, the concepts of plant/insect virus-based VLPs for delivering insecticidal and antiviral components (e.g., double-stranded RNA, peptides, and chemicals) are discussed, which provides future prospects of VLP application in agricultural pest control. In addition, some concerns are raised about VLP production on a large scale and the short-term resistance of hosts to VLP uptake. Overall, this review is expected to stimulate interest and research exploring plant/insect virus-based VLP applications in agricultural pest management. © 2023 Society of Chemical Industry.

Structural diversity and phylogenetic distribution of valyl tRNA-like structures in viruses.

Viruses commonly use specifically folded RNA elements that interact with both host and viral proteins to perform functions important for diverse viral processes. Examples are found at the 3' termini of certain positive-sense ssRNA virus genomes where they partially mimic tRNAs, including being aminoacylated by host cell enzymes. Valine-accepting tRNA-like structures (TLSVal) are an example that share some clear homology with canonical tRNAs but have several important structural differences. Although many examples of TLSVal have been identified, we lacked a full understanding of their structural diversity and phylogenetic distribution. To address this, we undertook an in-depth bioinformatic and biochemical investigation of these RNAs, guided by recent high-resolution structures of a TLSVal We cataloged many new examples in plant-infecting viruses but also in unrelated insect-specific viruses. Using biochemical and structural approaches, we verified the secondary structure of representative TLSVal substrates and tested their ability to be valylated, confirming previous observations of structural heterogeneity within this class. In a few cases, large stem-loop structures are inserted within variable regions located in an area of the TLS distal to known host cell factor binding sites. In addition, we identified one virus whose TLS has switched its anticodon away from valine, causing a loss of valylation activity; the implications of this remain unclear. These results refine our understanding of the structural and functional mechanistic details of tRNA mimicry and how this may be used in viral infection.

Phylogenetic analysis of sacbrood virus structural polyprotein and non-structural RNA dependent RNA polymerase gene: Differences in Turkish strains.

Sacbrood virus (SBV) is one of the most damaging viruses in honey bee colonies. Genetic differences among sacbrood viruses detected in honey bees in different locales have been reported in previous studies. The aim of this study was to construct phylogenetic trees based on the structural polyprotein and non-structural RNA dependent RNA polymerase gene regions and to make a molecular characterization of the Tur/Bur/Sac01 and Tur/Bur/Sac02 strains identified in Apis mellifera in Turkey. As a result of the study, the tree based on the structural polyprotein region separated into four lineages: Tur/Bur/Sac01 and Tur/Bur/Sac02 were in the same branch as the Turkish sacbrood virus strains identified in previous studies and formed the Turkish clade. Strains isolated from adjacent geographical areas were in the same clade in this tree. The phylogenetic tree based on the non-structural RNA dependent RNA polymerase gene region divides into two main branches, reflecting host affiliation: Apis cerana and A. mellifera. Strains formed clusters based on their geographic distribution and host affiliation. The Tur/Bur/Sac01 and Tur/Bur/Sac02 strains formed a separate cluster among the European strains. Sacbrood viruses from Turkey were genetically different from SBV strains detected in other countries and in A. cerana.

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV).

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2.

Drosophila use small-interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila. Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence.

Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein.

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.

Direct interactions between bidensovirus BmDNV-Z proteins and midgut proteins from the virus target Bombyx mori.

In this study we aimed to identify the protein-protein interactions between Bombyx mori midgut and the bidensovirus BmDNV-Z via a yeast two-hybrid (Y2H) system. To achieve this we constructed a Gal4 activation domain fusion library that expresses the host genes and Gal4 DNA binding domain fusion bait vectors that express BmDNV-Z genes. Y2H assay revealed 15 potential interactions between host and viral proteins. To verify the interactions, we modified and reconstructed a pair of bimolecular fluorescence complementation (BiFC) vectors and achieved the co-expressions of the candidate host genes and viral genes in insect culture cells. The BiFC assay confirmed the specificity of the interactions including B. mori 35 kDa protease and two BmDNV-Z proteins encoded by VD1-ORF2 and VD2-ORF1; B. mori transgelin and BmDNV-Z protein encoded by VD2-ORF3; and B. mori serine protease precursor and BmDNV-Z encoded by VD2-ORF3 in vitro. Our findings revealed that the specific host midgut proteins are involved in the interactions between B. mori and BmDNV-Z, which will facilitate our understanding of the molecular mechanisms of BmDNV-Z infection.

Proteomic footprints of a member of Glossinavirus (Hytrosaviridae): an expeditious approach to virus control strategies in tsetse factories.

The Glossinavirus (Glossina pallidipes salivary gland hypertrophy virus (GpSGHV)) is a rod-shaped enveloped insect virus containing a 190,032 bp-long, circular dsDNA genome. The virus is pathogenic for the tsetse fly Glossina pallidipes and has been associated with the collapse of selected mass-reared colonies. Maintenance of productive fly colonies is critical to tsetse and trypanosomiasis eradication in sub-Saharan Africa using the Sterile Insect Technique. Proteomics, an approach to define the expressed protein complement of a genome, was used to further our understanding of the protein composition, morphology, morphogenesis and pathology of GpSGHV. Additionally, this approach provides potential targets for novel and sustainable molecular-based antiviral strategies to control viral infections in tsetse colonies. To achieve this goal, identification of key protein partners involved in virus transmission is required. In this review, we integrate the available data on GpSGHV proteomics to assess the impact of viral infections on host metabolism and to understand the contributions of such perturbations to viral pathogenesis. The relevance of the proteome findings to tsetse and trypanosomiasis management in sub-Sahara Africa is also considered.

Convergent evolution of argonaute-2 slicer antagonism in two distinct insect RNA viruses.

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.

No evidence for translation of pog, a predicted overlapping gene of Solenopsis invicta virus 1.

An overlapping open reading frame (ORF) with a potential to encode a functional protein has been identified within the 3'-proximal ORF of Solenopsis invicta virus 1 (SINV-1) and three bee viruses. This ORF has been referred to as predicted overlapping gene (pog). Protein motif searches of POG revealed weak relationships precluding assignment of a potential function. Neither a transcript nor a protein encoded by the pog ORF has been detected. However, recently, a protein encoded by the corresponding +1 overlapping ORF (termed ORFx) in the Israeli acute paralysis virus (IAPV) was demonstrated by recombinant means as well as in IAPV-infected honey bees. The objective of our study was to attempt to provide empirical evidence for the presence of a pog-derived protein from SINV-1-infected fire ants. A number of different laboratory and field SINV-1-infected Solenopsis invicta preparations were examined by western blotting for the presence of a POG protein sequence. In every case, these preparations failed to yield any detectable bands when probed with a polyclonal antibody preparation raised to a portion of the pog predicted protein sequence. Although impossible to prove a negative result, proper controls used in these studies suggested that the pog ORF is not translated into a functional protein in SINV-1.

Membrane targeting of an alpha-like tetravirus replicase is directed by a region within the RNA-dependent RNA polymerase domain.

The members of the family Tetraviridae are small positive-sense insect RNA viruses that exhibit stringent host specificity and a high degree of tissue tropism, suggesting that complex virus-host interactions are likely to occur during infection and viral replication. The alpha-like replicase of Helicoverpa armigera stunt virus (HaSV) (genus Omegatetravirus) has been proposed to associate with membranes of the endocytic pathway, which is similar to Semliki Forest virus, Sindbis virus and rubella virus. Here, we have used replicase-EGFP fusion proteins and recombinant baculovirus expression to demonstrate that the HaSV replicase associates strongly with cellular membranes, including detergent-resistant membranes, and that this association is maintained through a novel membrane targeting domain within the C-terminal region of the RNA-dependent RNA polymerase domain. We show a similar subcellular localization and strong association with detergent-resistant membranes for the carmo-like replicase of another tetravirus, Providence virus, in replicating cells, suggesting a common site of replication for these two tetraviruses.

[The structural protein Gag of the gypsy retrovirus forms virus-like particles in the bacterial cell].

The amino acid sequence of the drosophila retrovirus MDG4 (gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, the protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated an ability of the each product to form virus-like particles in the bacterial cell, i.e. in the absence of eukaryotic cell factors. The results obtained showed that the both variants of the gypsy protein Gag form globular particles in the bacterial cell.

RNA viruses in hymenopteran pollinators: evidence of inter-Taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species.

Although overall pollinator populations have declined over the last couple of decades, the honey bee (Apis mellifera) malady, colony collapse disorder (CCD), has caused major concern in the agricultural community. Among honey bee pathogens, RNA viruses are emerging as a serious threat and are suspected as major contributors to CCD. Recent detection of these viral species in bumble bees suggests a possible wider environmental spread of these viruses with potential broader impact. It is therefore vital to study the ecology and epidemiology of these viruses in the hymenopteran pollinator community as a whole. We studied the viral distribution in honey bees, in their pollen loads, and in other non-Apis hymenopteran pollinators collected from flowering plants in Pennsylvania, New York, and Illinois in the United States. Viruses in the samples were detected using reverse transcriptase-PCR and confirmed by sequencing. For the first time, we report the molecular detection of picorna-like RNA viruses (deformed wing virus, sacbrood virus and black queen cell virus) in pollen pellets collected directly from forager bees. Pollen pellets from several uninfected forager bees were detected with virus, indicating that pollen itself may harbor viruses. The viruses in the pollen and honey stored in the hive were demonstrated to be infective, with the queen becoming infected and laying infected eggs after these virus-contaminated foods were given to virus-free colonies. These viruses were detected in eleven other non-Apis hymenopteran species, ranging from many solitary bees to bumble bees and wasps. This finding further expands the viral host range and implies a possible deeper impact on the health of our ecosystem. Phylogenetic analyses support that these viruses are disseminating freely among the pollinators via the flower pollen itself. Notably, in cases where honey bee apiaries affected by CCD harbored honey bees with Israeli Acute Paralysis virus (IAPV), nearby non-Apis hymenopteran pollinators also had IAPV, while those near apiaries without IAPV did not. In containment greenhouse experiments, IAPV moved from infected honey bees to bumble bees and from infected bumble bees to honey bees within a week, demonstrating that the viruses could be transmitted from one species to another. This study adds to our present understanding of virus epidemiology and may help explain bee disease patterns and pollinator population decline in general.

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella.

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.

Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus.

The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3'-ends (3'-RACE) and RT-PCR. Direct sequencing of 3'-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3'-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3'-RACE or RT-PCR. A series of 5'-RACE reactions were conducted on selected ORFs that were identified by 3'-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3' untranslated region (3'-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3'-RACE products without cloning served as an alternative approach to detect both 3'-UTRs and transcript variants of this large DNA virus.

Examining the relative activity of several dicistrovirus intergenic internal ribosome entry site elements in uninfected insect and mammalian cell lines.

Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 5' IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.

An insect virus-encoded microRNA regulates viral replication.

MicroRNAs (miRNAs) are small ( approximately 22 nucleotides) noncoding RNAs which play an essential role in gene regulation and affect a wide range of processes, including development, differentiation, and oncogenesis. Here we report the identification of the first miRNA from an insect virus, derived from the major capsid protein (MCP) gene in Heliothis virescens ascovirus (HvAV) (HvAV-miR-1). Although MCP was abundantly expressed at all time points 24 h after infection, HvAV-miR-1 expression was strictly regulated and specifically detected from 96 h postinfection. HvAV-miR-1 expression coincided with a marked reduction of the expression of HvAV DNA polymerase I, which is a predicted target. Ectopic expression of full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs transcriptional degradation of DNA polymerase I and regulates HvAV replication. These findings are congruent with recent reports that miR-BART-2 regulates Epstein-Barr virus DNA polymerase expression and suggest that virus-encoded miRNA regulation of virus replication may be a general phenomenon.

Sequence analysis of a reovirus isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae) and its parasitoid wasp Phobocampe tempestiva (Hymenoptera: Ichneumonidae).

A reovirus was isolated from Operophtera brumata (ObRV) and its parasitoid wasp Phobocampe tempestiva. Each of the 10 dsRNA genome segments of ObRV was sequenced and shown to contain a single open reading frame (ORF). Conserved motifs ([+ve] 5'-AAATAAA ...(G)/(T)AGGTT-3') were found at the termini of each segment, with the exception of Seg-6 and Seg-8, where the 5' termini were 5'-AACAAA...-3'. The putative proteins encoded by each segment were compared with those of other members of the family Reoviridae. Phylogenetic comparisons to published sequences for the RNA-dependent RNA polymerase genes from other reoviruses indicated that ObRV is most closely related to members of the genus Cypovirus. However, unlike the cypoviruses, ObRV has a double-layered capsid structure. When the protein encoded by ObRV Seg-10 was expressed (by inserting the open reading frame into a baculovirus expression vector) no 'occlusion bodies' were observed in the recombinant baculovirus infected insect cell cultures. This suggests that unlike the cypoviruses, Seg-10 of ObRV does not contain a polyhedrin gene. Further phylogenetic comparisons also identified relationships between Seg-2 and Seg-10 of ObRV, and genes of Diadromus pulchellus Idnoreovirus 1 (DpIRV1), suggesting that ObRV represents a new species from the genus Idnoreovirus.