Developments in the classification and nomenclature of arthropod-infecting large DNA viruses that contain pif genes.

Viruses of four families of arthropod-specific, large dsDNA viruses (the nuclear arthropod large DNA viruses, or NALDVs) possess homologs of genes encoding conserved components involved in the baculovirus primary infection mechanism. The presence of such homologs encoding per os infectivity factors (pif genes), along with their absence from other viruses and the occurrence of other shared characteristics, suggests a common origin for the viruses of these families. Therefore, the class Naldaviricetes was recently established, accommodating these four families. In addition, within this class, the ICTV approved the creation of the order Lefavirales for three of these families, whose members carry homologs of the baculovirus genes that code for components of the viral RNA polymerase, which is responsible for late gene expression. We further established a system for the binomial naming of all virus species in the order Lefavirales, in accordance with a decision by the ICTV in 2019 to move towards a standardized nomenclature for all virus species. The binomial species names for members of the order Lefavirales consist of the name of the genus to which the species belongs (e.g., Alphabaculovirus), followed by a single epithet that refers to the host species from which the virus was originally isolated. The common names of viruses and the abbreviations thereof will not change, as the format of virus names lies outside the remit of the ICTV.

The C-termini of the baculovirus per os infectivity factors 1 and 2 mediate ODV oral infectivity by facilitating the binding of PIF0 and PIF8 to the core of the entry complex.

Oral infection of caterpillars by baculoviruses is initiated by occlusion-derived virus particles (ODVs) that infect midgut epithelium cells. The ODV envelope therefore contains at least ten different proteins, which are called per os infectivity factors (PIFs). Nine of these PIFs form the so-called ODV entry complex that consists of a stable core formed by PIF1, 2, 3 and 4, to which the other PIFs [PIF0, 6, 7, 8 and 9 (ac108)] bind with lower affinity. PIF1 and 2 are not only essential for complex formation, but also mediate ODV-binding to the epithelial brush border, probably via the C-termini. To study the involvement of these PIFs during midgut infection in greater detail, we assessed the oral infectivity and the ability to form the complex of a series of PIF1 and PIF2 C-terminal truncation mutants of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), which were constructed in this study. Limited truncation of either PIF1 or 2 already severely impaired the ODV oral infectivity, but did not affect the formation of the core complex. However, the entry complex as a whole was not assembled in these mutants as PIF0 and 8 failed to bind to the core. This suggests that the interactions between the core and the loosely associated PIFs are important for the ODV infectivity and that complex formation complicates the determination of the exact roles of PIF1 and 2 during midgut infection. We also showed that the presence of PIF0, 6 and the ZF-domain of PIF8 are crucial for complex formation.

ICTV Virus Taxonomy Profile: Nudiviridae.

Members of the family Nudiviridae are large dsDNA viruses with distinctive rod-shaped nucleocapsids and circular genomes of 96-232 kbp. Nudiviruses have been identified from a diverse range of insects and crustaceans and are closely related to baculoviruses. This is a summary of the International Committee on Taxonomy of Viruses Report on the taxonomy of the family Nudiviridae, which is available at ictv.global/report/nudiviridae.

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

During 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 both ac141 and vubi restricted 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.IMPORTANCE Baculoviruses 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.

The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: implications for baculovirus phylogeny and host specificity.

The Mythimna unipuncta nucleopolyhedrovirus isolate KY310 (MyunNPV-KY310) is an alphabaculovirus isolated from a true armyworm (Mythimna unipuncta) population in Kentucky, USA. Occlusion bodies of this virus were examined by electron microscopy and the genome sequence was determined by 454 pyrosequencing. MyunNPV-KY310 occlusion bodies consisted of irregular polyhedra measuring 0.8-1.8 µm in diameter and containing multiple virions, with one to six nucleocapsids per virion. The genome sequence was determined to be 156,647 bp with a nucleotide distribution of 43.9% G+C. 152 ORFs and six homologous repeat (hr) regions were annotated for the sequence, including the 38 core genes of family Baculoviridae and an additional group of 26 conserved alphabaculovirus genes. BLAST queries and phylogenetic inference confirmed that MyunNPV-KY310 is most closely related to the alphabaculovirus Leucania separata nucleopolyhedrovirus isolate AH1, which infects Mythimna separata. In contrast, MyunNPV-KY310 did not exhibit a close relationship with Mythimna unipuncta nucleopolyhedrovirus isolate #7, an alphabaculovirus from the same host species. MyunNPV-KY310 lacks the gp64 envelope glycoprotein, which is a characteristic of group II alphabaculoviruses. However, this virus and five other alphabaculoviruses lacking gp64 are placed outside the group I and group II clades in core gene phylogenies, further demonstrating that viruses of genus Alphabaculovirus do not occur in two monophyletic clades. Potential instances of MyunNPV-KY310 ORFs arising by horizontal transfer were detected. Although there are now genome sequences of four different baculoviruses from M. unipuncta, comparison of their genome sequences provides little insight into the genetic basis for their host specificity.

ICTV Virus Taxonomy Profile: Baculoviridae.

The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped, rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15-5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera, Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae, which is available at www.ictv.global/report/baculoviridae.

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin ß superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.

Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes.

Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene.IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

Baculoviruses require an intact ODV entry-complex to resist proteolytic degradation of per os infectivity factors by co-occluded proteases from the larval host.

Baculoviruses orally infect caterpillars in the form of occlusion-derived viruses (ODVs). The ODV-envelope contains a number of proteins which are essential for oral infectivity, called per os infectivity factors (PIFs). Most of these PIFs are involved in the formation of an ODV-entry complex that consists of a stable core, formed by PIF1, PIF2, PIF3 and PIF4, and the more loosely associated PIFs P74 (PIF0) and P95 (PIF8). PIF1, PIF2 and PIF3 are essential for formation of the stable core, whereas deletion of the pif4 gene results in the formation of a smaller complex. P74 is not needed for formation of the stable core. We show here in larva-derived ODVs of the Autographa californica multicapsid nucleopolyhedrovirus that PIF-proteins are degraded by host-derived proteases after deletion of a single pif-gene. Constituents of the stable core-complex appeared to be more resistant to proteases as part of the complex than as monomer, as in ODVs of a p74 deletion mutant only the stable core was found but no PIF monomers. When the stable core lacks PIF4, it lost its proteolytic resistance as the resulting smaller core complex was degraded in a pif4 deletion mutant. We also identified PIF6 as a loosely associated component of the entry complex that appeared nevertheless important for the proteolytic resistance of the stable core, which was degraded after deletion of pif6. We conclude from these results that an intact entry-complex in the ODV-envelope is prerequisite for proteolytic resistance of PIF-proteins under the alkaline conditions of the larval midgut.

Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus.

UNLABELLED: The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with ß-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE: In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

Nuclear Translocation Sequence and Region in Autographa californica Multiple Nucleopolyhedrovirus ME53 That Are Important for Optimal Baculovirus Production.

UNLABELLED: Autographa californicamultiple nucleopolyhedrovirus (AcMNPV) is in the familyBaculoviridae, genusAlphabaculovirus AcMNPVme53is a highly conserved immediate early gene in all lepidopteran baculoviruses that have been sequenced and is transcribed up to late times postinfection. Althoughme53is not essential for viral DNA synthesis, infectious budded virus (BV) production is greatly attenuated when it is deleted. ME53 associates with the nucleocapsid on both budded virus and occlusion-derived virus, but not with the virus envelope. ME53 colocalizes in plasma membrane foci with the envelope glycoprotein GP64 in a GP64-dependent manner. ME53 localizes in the cytoplasm early postinfection, and despite the lack of a reported nuclear localization signal (NLS), ME53 translocates to the nucleus at late times postinfection. To map determinants of ME53 that facilitate its nuclear translocation, recombinant AcMNPV bacmids containing a series of ME53 truncations, internal deletions, and peptides fused with hemagglutinin (HA) or green fluorescent protein (GFP) tags were constructed. Intracellular-localization studies identified residues within amino acids 109 to 137 at the N terminus of ME53 that acted as the nuclear translocation sequence (NTS), facilitating its nuclear transport at late times postinfection. The first 100 N-terminal amino acids and the last 50 C-terminal amino acids of ME53 are dispensable for high levels of budded virus production. The region within amino acids 101 to 398, which also contains the NTS, is critical for optimal levels of budded virus production. IMPORTANCE: Baculovirusme53is a conserved immediate early gene found in all sequenced lepidopteran alpha- and betabaculoviruses. We first identified residues within amino acids 109 to 137 at the N terminus that act as the ME53 nuclear translocation sequence (NTS) to facilitate its nuclear translocation and defined an internal region within amino acids 101 to 398, which includes the NTS, as being necessary for optimal budded virus production. Altogether, these results indicate a previously unidentified nuclear role that ME53 plays in virus replication.

Microscopic investigation of AcMNPV infection in the Trichoplusia ni midgut.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the type species for the genus Alphabaculovirus in the family Baculoviridae. In nature, AcMNPV infection begins with ingestion of viral occlusion bodies (OBs) from which occlusion-derived viruses (ODV) are released to infect midgut cells. This study explored the early stages of Trichoplusia ni midgut infection using recombinant viruses expressing green fluorescent protein (GFP) and/or a VP39-mCherry fusion protein under the control of early and late promoters, respectively. Using a recombinant ie1:GFP virus, the anterior midgut region was identified as the predominant site for primary infection. Infection of midguts using the GFP-VP39mCherry-dual labelled recombinant virus revealed that active viral replication and cell-to-cell spread was required for the formation of infection foci and the subsequent spread to uninfected midgut cells and tracheoblasts. The spread of the infection from primary infected cells to secondary cells within the midgut was shown to be dependent upon the membrane fusion protein GP64.

Mamestra configurata nucleopolyhedrovirus-A transcriptome from infected host midgut.

Infection of an insect by a baculovirus occurs in two distinct phases, an initial infection of host midgut by occlusion-derived virions (ODVs) and subsequent systemic infection of other tissues by budded virions (BV). A vast majority of investigations of the infection process have been restricted to cell culture studies using BV that emulate the systemic phase of infection. This is one of the first studies to investigate baculovirus gene expression in ODV infected midgut cells. We have focused on the critical first phase of in vivo infection by Mamestra configurata nucleopolyhedrovirus-A in M. configurata larvae, using qPCR and RNAseq mass sequencing to measure virus gene expression in midgut cells. The earliest genes detected by each method had significant overlap, including known early genes as well as genes unique to MacoNPV-A and genes of unknown function. The RNAseq data also revealed a large range of expression levels across all ORFs, which could not be measured using qPCR. This dataset provides a first whole genome transcriptomic analysis of viral genes required for virus infection in vivo and will provide the basis for functionally analyzing specific genes that may be critical elements in baculovirus midgut infectivity and host range.

Analysis of the autographa californica multiple nucleopolyhedrovirus overlapping gene pair lef3 and ac68 reveals that AC68 is a per os infectivity factor and that LEF3 is critical, but not essential, for virus replication.

Autographa californica multiple nucleopolyhedrovirus ac68 is a core gene that overlaps lef3 which encodes the single-stranded DNA binding protein. A knockout (KO) virus lacking both lef3 and ac68 was generated (lef3-ac68 2×KO) to enable the functional study of ac68. To produce an ac68KO virus that did not impact lef3 expression, the lef3-ac68 2×KO virus was repaired with a DNA fragment containing lef3 and ac68, in which ac68 contained point mutations so that only LEF3 was expressed. Repair of lef3-ac68 2×KO with just ac68 generated an lef3KO virus. Analysis of the ac68KO virus showed that viral DNA replication and budded virus (BV) levels were unaffected compared to levels in the double-repair or wild-type (WT) control virus. Bioassay analyses of Trichoplusia ni larvae injected with BV directly into the hemolymph, bypassing the gut, showed no difference in mortality rates between the ac68KO and the WT viruses. However, in oral bioassays the ac68KO occlusion bodies failed to kill larvae. These results show that the core gene ac68 encodes a per os infectivity factor (pif6). The lef3KO virus was also analyzed, and virus replication was drastically reduced compared to WT virus, but very low levels of lef3KO virus DNA replication and BV production could be detected. In addition, in transfected cells P143 was transported to the nucleus in the absence of LEF3. This study therefore shows for the first time that even though the loss of LEF3 severely impairs virus replication, it is not absolutely essential for P143 nuclear import or viral replication.

Characterization of novel components of the baculovirus per os infectivity factor complex.

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.

The genome of a baculovirus isolated from Hemileuca sp. encodes a serpin ortholog.

The genome sequence of a baculovirus from Hemileuca sp. was determined. The genome is 140,633 kb, has a G+C content of 38.1 %, and encodes 137 putative open-reading frames over 50 amino acids. 126 of these ORFs showed similarity to other baculovirus genes in the database including all 37 core genes. Of the remaining 11 predicted genes, one is related to a lepidopteran serpin gene. This is the first report of a baculovirus encoding a member of this family of serine protease inhibitors, and to our knowledge the first report of a viral serpin outside the Poxviridae. The genome also contained three homologous repeat sequences. Phylogenetic analysis indicated that the virus is a group II Alphabaculovirus and belongs to a lineage that includes Orgyia leucostigma, Ectropis obliqua, Apocheima cinerarium, and Euproctis pseudoconspersa nucleopolyhedroviruses.

Immediate-early protein ME53 forms foci and colocalizes with GP64 and the major capsid protein VP39 at the cell membranes of Autographa californica multiple nucleopolyhedrovirus-infected cells.

me53 is an immediate-early/late gene found in all lepidopteran baculoviruses sequenced to date. Deletion of me53 results in a greater-than-1,000-fold reduction in budded-virus production in tissue culture (J. de Jong, B. M. Arif, D. A. Theilmann, and P. J. Krell, J. Virol. 83:7440-7448, 2009). We investigated the localization of ME53 using an ME53 construct fused to green fluorescent protein (GFP). ME53:GFP adopted a primarily cytoplasmic distribution at early times postinfection and a primarily nuclear distribution at late times postinfection. Additionally, at late times ME53:GFP formed distinct foci at the cell periphery. These foci colocalized with the major envelope fusion protein GP64 and frequently with VP39 capsid protein, suggesting that these cell membrane regions may represent viral budding sites. Deletion of vp39 did not influence the distribution of ME53:GFP; however, deletion of gp64 abolished ME53:GFP foci at the cell periphery, implying an association between ME53 and GP64. Despite the association of ME53 and GP64, ME53 fractionated with the nucleocapsid only after budded-virus fractionation. Together these findings suggest that ME53 may be providing a scaffold that bridges the viral envelope and nucleocapsid.

Horizontally transmitted parasitoid killing factor shapes insect defense to parasitoids.

Interkingdom competition occurs between hymenopteran parasitoids and insect viruses sharing the same insect hosts. It has been assumed that parasitoid larvae die with the death of the infected host or as result of competition for host resources. Here we describe a gene family, parasitoid killing factor (pkf), that encodes proteins toxic to parasitoids of the Microgastrinae group and determines parasitism success. Pkfs are found in several entomopathogenic DNA virus families and in some lepidopteran genomes. We provide evidence of equivalent and specific toxicity against endoparasites for PKFs found in entomopoxvirus, ascovirus, baculovirus, and Lepidoptera through a mechanism that elicits apoptosis in the cells of susceptible parasitoids. This highlights the evolutionary arms race between parasitoids, viruses, and their insect hosts.