Enhanced virulence of Autographa californica multiple nucleopolyhedrovirus in Spodoptera frugiperda is mediated by an Ac34 mutation that promotes nucleocapsid envelopment within occlusion bodies

Spodoptera frugiperda larvae are highly resistant to oral infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) (LD(50), approximately 9200 occlusions), but extremely susceptible to budded virus within the haemocoel (LD(50), <1 p.f.u.). The inability of AcMNPV occlusion-derived virus (ODV) to establish primary infections readily within midgut cells accounts for a major proportion of oral resistance. To determine whether inappropriate binding of AcMNPV ODV to S. frugiperda midgut cells contributes to lack of oral infectivity, the binding and fusion properties of AcMNPV ODV were compared with those of the ODV of a new isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) obtained from a field-collected larva (oral LD(50), 12 occlusions). By using a fluorescence-dequenching assay conducted in vivo, it was found that AcMNPV ODV bound to the midgut epithelia of S. frugiperda larvae at approximately 15 % of the level of SfMNPV ODV, but that, once bound, the efficiencies of fusion for the two ODVs were similar: 60 % for AcMNPV and 53 % for SfMNPV. Whilst the difference in binding efficiencies was significant, it could not account entirely for the observed differences in infectivity. Competition experiments, however, revealed that, in S. frugiperda larvae, SfMNPV ODV bound to a midgut cell receptor that was not bound by AcMNPV ODV, indicating that ODV interaction with a specific receptor(s) was necessary for productive infection of midgut columnar epithelial cells. Fusion in the absence of this ligand-receptor interaction did not result in productive infections.

Disruption of the baculovirus core gene ac78 results in decreased production of multiple nucleocapsid-enveloped occlusion-derived virions and the failure of primary infection in vivo

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) E18 (AC143, ODV-E18) is an envelope protein common to both occlusion-derived virions (ODVs) and budded virions (BVs). The e18 gene has been demonstrated to be essential for generating infectious BVs. However, its functional role in virion morphogenesis remains unclear. In this study, we constructed an e18 knockout virus and an e18 repair virus to investigate the effects of e18 deletion on virion morphogenesis. Our data indicated that e18 is required for normal intranuclear microvesicle (IMV) formation and accumulation, for intranuclear envelopment and nuclear egress of nucleocapsids, as well as for embedding of ODVs into occlusion bodies (OBs) and BV production. Additionally, we created and characterized a series of recombinant viruses with truncated e18 of varying lengths to identify domains involved in nuclear translocation and virion morphogenesis. We identified two low-complexity domains (LCDs) in E18, in addition to a known transmembrane domain (TM). The AA30-34 sequence within the TM was found to be essential, but not sufficient for nuclear translocation. However, an α-helix structure encompassing the TM domain proved adequate to mediate a fusion protein's trafficking into the nucleus in the context of additional viral factors. Furthermore, we discovered that the TM was required for the accumulation of IMVs, while both the TM and LCD 1 were necessary for intranuclear envelopment, nuclear egress of nucleocapsids, and the embedding of ODVs into OBs; LCD 2 influenced the processing of IMVs and ODV formation. Both the TM and the two LCDs were essential for BV production.IMPORTANCEThe envelope protein E18 is a conserved component common to both ODV and BV virion types of baculoviruses, yet its functional role in virion morphogenesis remains unclear. This study investigated the e18 gene of Autographa californica multiple nucleopolyhedrovirus, determining that it is essential for normal IMV formation and accumulation, intranuclear envelopment and nuclear egress of nucleocapsids, as well as for the embedding of ODVs into occlusion bodies and BV production. The functional roles of the single TM domain and two LCD domains within E18 during virion morphogenesis were identified. Furthermore, it was found that an α-helix structure encompassing the TM domain is sufficient to facilitate the trafficking of a fusion protein into the nucleus in the context of other viral factors, with AA30-34 being critical for the nuclear import of E18.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.

The Trichoplusia ni single nucleopolyhedrovirus tn79 gene encodes a functional sulfhydryl oxidase enzyme that is able to support the replication of Autographa californica multiple nucleopolyhedrovirus lacking the sulfhydryl oxidase ac92 gene

Baculovirus dual-phase infection strategy and biotechnological applications: A structural and regulatory review.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of virus host range. To assess the role of ODV-E56 in oral infectivity and host range, we constructed recombinant AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lacZ) in which ODV-E56 protein synthesis was eliminated by inserting a beta-galactosidase (lacZ) expression cassette into the odv-e56 open reading frame. We also constructed a recombinant virus, Ac69GFP-Roe56, in which the native AcMNPV odv-e56 coding sequence was replaced with that of Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The odv-e56 recombinant viruses exhibited no alterations in polyhedron production and morphogenesis or in the production of infectious budded virus in cell culture. In bioassays using three lepidopteran host species, the oral infectivities of the odv-e56 mutant viruses Ac69GFP-e56lacZ and AcIEGFP-e56lacZ were profoundly impaired compared with those of wild-type and control recombinant viruses. Oral infectivity was restored fully by marker rescue of the odv-e56 mutant viruses with either the AcMNPV or the RoMNPV odv-e56 gene. In bioassays using two host species that are more susceptible to RoMNPV than to AcMNPV, Ac69GFP-Roe56 killed larvae with LC50 values similar to those of recombinant viruses expressing AcMNPV ODV-E56. This result indicated that replacement of the AcMNPV odv-e56 gene with the RoMNPV orthologue did not increase virulence against these two species.

The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host-range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with EGFP fused to the nucleocapsid to monitor virus entry into primary midgut epithelium cells ex vivo using confocal fluorescence microscopy. The EGFP-labelled virus showed similar BV virulence and ODV infectivity as WT virus. The ability to bind and enter host cells was then visualized for WT AcMNPV and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, whilst PIF1 and PIF2 play important roles in the entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.

Functional Characterization of the Group I Alphabaculovirus Specific Gene ac73.

Cysteines 128 and 250 are essential for the functions of the baculovirus core gene ac109

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.

Deletion of AcMNPV ac146 eliminates the production of budded virus

This research investigated the function of envelope protein P74 of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) in primary infection to host. A p74-inactivation recombinant baculovirus, rAc-gfp(Delta) p74, was constructed by inserting gfp driven by AcMNPV polyhedrin promoter into the p74 locus of AcMNPV genome. Bioassays showed that the P74-null occlusion bodies (OBs) failed to infect its natural host larvae, Spodoptera exigua, per os, while the p74-null budded virus (BVs) could infect host larvae by injection. However, its inability for oral infectivity was rescued by a mixed infection with wild-type OBs or with the purified P74 protein expressed in Spodoptera frugiperda Sf-9 cells, and the P74 protein rescue was in a dosage-dependent manner. The 50% lethal dosage (LD50) value of a P74 overexpression recombinant virus, rAc-p74(++)-polh+, which contained two copies of p74 gene, was not significantly different from that of wild-type virus. One-step growth curve assays of viruses suggested that BV production from cells infected with p74-null virus was similar to that from cells infected with wild-type virus or the P74 overexpression virus. ELISA analysis indicated that P74 protein could bind its host brush border membrane vesicles (BBMV) efficiently with saturation, but it could only bind its sensitive midgut BBMV specifically. In vitro pull-down assay showed that a protein of approximately 35 kDa in the BBMV was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of occlusion-derived virus (ODV) and plays a role in midgut attachment and fusion.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf114 (ac114) is one of the highly conserved unique genes in the lepidopteran group I nucleopolyhedrovirus. So far, the biological function of ac114 is unknown. To study the function of ac114 in the virus life cycle, an ac114 knockout baculovirus shuttle vector (bacmid) was generated. Fluorescence and light microscopy showed that the ac114 knockout mutant was able to produce infectious budded viruses (BVs) and occlusion bodies (OBs). Titration assays demonstrated that the ac114 knockout virus had similar growth kinetics to the control virus during the infection phase. Electron microscopy indicated that ac114 did not affect the morphogenesis of BVs and occlusion-derived viruses (ODVs); however, the numbers of ODVs per OB of the ac114 knockout virus were significantly lower than those of the control virus. RT-PCR demonstrated that ac114 was a late stage expression gene and that its transcription initiated at an A residue, 16 nucleotides upstream of the ATG start codon. Intracellular localization analysis revealed that the Ac114-GFP fusion protein localized predominantly as punctate patches in the cytoplasm of infected Sf9 cells. Bioassays showed that the ac114 knockout did not change the killing speed of AcMNPV in Spodoptera exigua larvae, but reduced its viral infectivity significantly. Taken together, these data indicate that ac114 is an auxiliary gene that facilitates embedding of ODVs into OBs, thus affecting the per os infectivity of the virus.