The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F

The GP64 and F proteins were previously identified as the sole functional envelope fusion proteins in Baculoviridae. F-like proteins, present only in group I nucleopolyhedroviruses (NPVs), are remnant, nonfunctional F proteins. In this report, we describe the effect of the presence or absence of the F-like protein Ac23 in a gp64-null Autographa californica multinucleocapsid NPV pseudotyped with the F protein from Spodoptera exigua multicapsid NPV (SeF). We found that the presence of Ac23 elevates the infectivity of the pseudotyped virus. This is in contrast to the results of Lung et al. (J. Virol. 76:5729-5736, 2002), who found no such effect. The possible reasons for the differing results are discussed.     

Early synthesis of budded virus envelope fusion protein GP64 enhances Autographa californica multicapsid nucleopolyhedrovirus virulence in orally infected Heliothis virescens

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the type species of the Nucleopolyhedrovirus genus (Baculoviridae family), has two highly unusual traits shared by several baculovirus species. First, the occlusion-derived virus (ODV) that establishes primary infection in the midgut following its ingestion by host larvae contains multiple nucleocapsids, all of which enter the same midgut cell. Second, GP64, the envelope fusion protein of the budded virus (BV) that spreads infection beyond the midgut, is synthesized both early and late during infection. We tested the hypothesis that, together, these two traits enable parental ODV nucleocapsids to bud from infected midgut cells, essentially as BV, to establish secondary infections prior to completion of viral replication within the midgut. This "pass-through" strategy would enable the virus to counter the host's principal defense, sloughing of infected midgut cells, by accelerating the onset of systemic infections. To test this hypothesis, we created an AcMNPV recombinant, AcLate21/20-64HB, that can express gp64 only during the late phase of infection (coincident with the other structural proteins). We then compared the virulence of this virus to that of a control recombinant virus that expresses gp64 in a wild-type manner. We found that when administered orally, the control virus was far more virulent and established secondary infection earlier than AcLate21/20-64HB, but when administered intrahemocoelically, infectivity and virulence of the two recombinants were identical. Our results demonstrate that early gp64 expression is a key component of a unique and highly adaptive baculovirus infection strategy.     

Palmitoylation of the Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64: mapping,functional studies,and lipid rafts

Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.     

The immediate-early protein IE0 of the Autographa californica nucleopolyhedrovirus is not essential for viral replication

The role of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein IE0 in the baculoviral infection is not clear. In this study, we constructed the recombinant virus vAcDeltaie0 null for ie0 expression by targeted mutagenesis replacing exon0 with the cat gene. We found that vAcDeltaie0 replicated efficiently in Spodoptera littoralis SL2 cells, which are poorly permissive for AcMNPV. In contrast, in Spodoptera frugiperda SF9 cells, which are fully permissive for AcMNPV, vAcDeltaie0 DNA replication and budded virus production were delayed. These results and recently published data (X. Dai et al., J. Virol. 78:9633-9644, 2004) indicate that ie0 is not essential for AcMNPV replication but enhances it in permissive SF9 cells.     

Abnormal formation of polyhedra resulting from a single mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus

A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.     

Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.     

Identification of a GP64 subdomain involved in receptor binding by budded virions of the baculovirus Autographica californica multicapsid nucleopolyhedrovirus

Enveloped virus entry into host cells is typically initiated by an interaction between a viral envelope glycoprotein and a host cell receptor. For budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, the envelope glycoprotein GP64 is involved in host cell receptor binding, and GP64 is sufficient to mediate low-pH-triggered membrane fusion. To better define the role of GP64 in receptor binding, we generated and characterized a panel of antisera against subdomains of GP64. Eight subdomain-specific antisera were generated, and their reactivities with GP64 proteins and neutralization of virus infectivity and binding were examined. Antibodies directed against the N-terminal region of GP64 (amino acids 21 to 159) showed strong neutralization of infectivity and effectively inhibited binding of (35)S-labeled budded virions to Sf9 cells. In addition, we generated virions displaying truncated GP64 constructs. A construct displaying the N-terminal 274 amino acids (residues 21 to 294) of the ectodomain was sufficient to mediate virion binding. Additional studies of antisera directed against small subdomains revealed that an antiserum against a 40-amino-acid region (residues 121 to 160) neutralized virus infectivity. Site-directed mutagenesis was subsequently used for functional analysis of that region. Recombinant viruses expressing GP64 proteins with single amino acid substitutions within amino acids 120 to 124 and 142 to 148 replicated to high titers, suggesting that those amino acids were not critical for receptor binding or other important GP64 functions. In contrast, GP64 proteins with single amino acid substitutions of residues 153 and 156 were unable to substitute for wild-type GP64 and did not rescue a gp64 knockout virus. Further analysis showed that these substitutions substantially reduced binding of recombinant virus to Sf9 cells. Thus, the amino acid region from positions 21 to 159 was identified as a putative receptor binding domain, and amino acids 153 and 156 appear to be important for receptor binding.     

Autographa californica multiple nucleopolyhedrovirus GP64 protein: roles of histidine residues in triggering membrane fusion and fusion pore expansion

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein mediates membrane fusion during entry. Fusion results from a low-pH-triggered conformational change in GP64 and subsequent interactions with the membrane bilayers. The low-pH sensor and trigger of the conformational change are not known, but histidine residues are implicated because the pK(a) of histidine is near the threshold for triggering fusion by GP64. We used alanine substitutions to examine the roles of all individual and selected clusters of GP64 histidine residues in triggering and mediating fusion by GP64. Three histidine residues (H152, H155, and H156), located in fusion loop 2, were identified as important for membrane fusion. These three histidine residues were important for efficient pore expansion but were not required for the pH-triggered conformational change. In contrast, a cluster of three histidine residues (H245, H304, and H430) located near the base of the central coiled coil was identified as a putative sensor for low pH. Three alanine substitutions in cluster H245/H304/H430 resulted in dramatically reduced membrane fusion and the apparent loss of the prefusion conformation at neutral pH. Thus, the H245/H304/H430 cluster of histidines may function or participate as a pH sensor by stabilizing the prefusion structure of GP64.     

Altering the surface properties of baculovirus Autographa californica NPV by insertional mutagenesis of the envelope protein gp64.

The envelope protein gp64 of the baculovirus Autographa californica nuclear polyhedrosis virus is essential for viral entry into insect cells, as the glycoprotein both mediates pH-dependent membrane fusion and binds to host cell receptors. Surface modification of baculovirus particles by genetic engineering of gp64 has been demonstrated by various strategies and thus has become an important and powerful tool in molecular biology. To improve further the presentation of peptides on the surface of baculovirus particles, several insertion sites within the gp64 envelope protein were selected by their theoretical maximum surface probability and investigated for efficient peptide presentation. The ELDKWA peptide of the gp41 of HIV-1, specific for the human mAb 2F5, was inserted into 17 different positions of the glycoprotein gp64. Propagation of viruses was successful in 13 cases, mutagenesis at four positions did not result in production of intact virus particles. Western blotting, FACS analysis and ELISA were used for characterization of the different binding properties of the mutants. Insertion of this peptide into the native envelope protein resulted in high avidity display on the surface of baculovirus particles. This approach offers the possibility of effective modification of surface properties in regard to host range specificity and antigen display.     

Functional analysis of the transmembrane (TM) domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: substitution of heterologous TM domains

GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.     

Mixed-genotype infections of Trichoplusia ni larvae with Autographa californica multicapsid nucleopolyhedrovirus: Speed of action and persistence of a recombinant in serial passage

Fast-acting recombinant baculoviruses have potential for improved insect pest suppression. However, the ecological impact of using such viruses must be given careful consideration. One strategy for mitigating risks might be simultaneous release of a wild-type baculovirus, so as to facilitate rapid displacement of the recombinant baculovirus by a wild-type. However, at what ratio must the two baculoviruses be released? An optimum release ratio must ensure both fast action, and the eventual competitive displacement of the recombinant virus and fixation of the wild-type baculovirus in the insect population. Here we challenged Trichoplusia ni larvae with different ratios of wild-type Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and a derived recombinant, vEGTDEL, which has the endogenous egt gene (coding for ecdysteroid UDP-glucosyltransferase) deleted. Time to death increased with the proportion wild-type virus in the inoculum mixture, although a 1:10 ratio (wild-type: recombinant) resulted in equally rapid insecticidal action as vEGTDEL alone. Five serial passages of three different occlusion body (OB) mixtures of the two viruses were also performed. OBs from 10 larval cadavers were pooled and used to initiate the following passage. Although the wild-type baculovirus was maintained over five passages, it did not go to fixation in most replicates of the serial passage experiment (SPE), and there was no good evidence for selection against the recombinant. Long-term maintenance of a recombinant in serial passage suggests an ecosystem safety risk. We conclude that for assessing ecological impact of recombinant viruses, SPEs in single and multiple larvae are relevant because of potential modulating effects at the between-host level.     

Autographa californica multiple nucleopolyhedrovirus 38K is a novel nucleocapsid protein that interacts with VP1054, VP39, VP80, and itself

It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusion-derived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.     

ORF98 of Autographa californica nucleopolyhedrosisvirus is an auxiliary factor in late gene expression

Autographa californica nucleopolyhedrosisvirus (AcMNPV) is the type member of the family Baculoviridae. Gene expression of AcMNPV during virus infection is temporally regulated. A series of late expression factors (LEFs) are required for late gene expression to take place. A number of additional factors have also been shown to more modestly influence late gene expression. Using the LEF transient assay, we scanned the AcMNPV genome for such factors by replacing plasmids using the LEF genes with larger clones and then looked for increases in late gene expression using a reporter plasmid under the control of a late promoter. Using this approach, ORF98 was identified as having a stimulatory effect on late gene expression. The ability of ORF98 to influence early, late, and very late gene expression was established. Furthermore, tagged versions of ORF98 were localized to the nuclei of transfected cells and were shown to interact with each other as homo-oligomers. Potential roles of ORF98 in baculovirus infection are discussed.     

Non-polar distribution of green fluorescent protein on the surface of Autographa californica nucleopolyhedrovirus using a heterologous membrane anchor

Heterogeneous proteins can be displayed on the surface of the budded form of Autographa californica nucleopolyhedrovirus (AcMNPV) after fusion of the display protein to the AcMNPV major envelope glycoprotein, gp64. However, display is restricted to the poles of the virion and is relatively low level. To investigate the use of alternative membrane anchor sequences that would be compatible with virus surface display, we have constructed a display vector containing the gp64 signal peptide and a membrane anchor from the vesicular stomatitis virus (VSV) G glycoprotein. Introduction of a gene encoding green fluorescent protein (GFP) between these signals led to abundant display of GFP on the surface of insect cells and on recombinant budded virions. In addition, and in contrast to gp64 based fusion proteins, GFP was localized to the lateral virion surfaces.     

The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection.

To demonstrate the essential nature of the baculovirus GP64 envelope fusion protein (GP64 EFP) and to further examine the role of this protein in infection, we inactivated the gp64 efp gene of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and examined the biological properties of this virus in vivo. To provide GP64 EFP during construction of the recombinant GP64 EFP-null AcMNPV baculovirus, we first generated a stably transfected insect cell line (SfpOP64-6) that constitutively expressed the GP64 EFP of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The AcMNPV gp64 efp gene was inactivated by inserting the bacterial lacZ gene in frame after codon 131 of the gp64 efp gene. The inactivated gp64 gene was cloned into the AcMNPV viral genome by replacement of the wild-type gp64 efp locus. When propagated in the stably transfected insect cells (Sf9OP64-6 cells), budded virions produced by the recombinant AcMNPV GP64 EFP-null virus (vAc64z) contained OpMNPV GP64 EFP supplied by the Sf9OP64-6 cells. Virions propagated in Sf9OP64-6 cells were capable of infecting wild-type Sf9 cells, and cells infected by vAc64z exhibited a blue phenotype in the presence of X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside). Using cytochemical staining to detect vAc64z infected cells, we demonstrated that this GP64 EFP-null virus is defective in cell-to-cell propagation in cell culture. Although defective in cell-to-cell propagation, vAc64z produces occlusion bodies and infectious occlusion-derived virions within the nucleus. Occlusion bodies collected from cells infected by vAc64z were infectious to midgut epithelial cells of Trichoplusia ni larvae. However, in contrast to infection by a control virus, infection by vAc64z did not proceed into the hemocoel. Analysis of vAc64z occlusion bodies in a standard neonate droplet feeding assay showed no virus-induced mortality, indicating that occluded virions produced from vAc64z could not initiate a productive (lethal) infection in neonate larvae. Thus, GP64 EFP is an essential virion structural protein that is required for propagation of the budded virus from cell to cell and for systemic infection of the host insect.