Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids

Autographa californica nucleopolyhedrovirus (AcMNPV) orf93 (ac93) is a highly conserved uncharacterized gene that is found in all of the sequenced baculovirus genomes except for Culex nigripalpus NPV. In this report, using bioinformatics analyses, ac93 and odv-e25 (ac94) were identified as baculovirus core genes and thus p33-ac93-odv-e25 represent a cluster of core genes. To investigate the role of ac93 in the baculovirus life cycle, an ac93 knockout AcMNPV bacmid was constructed via homologous recombination in Escherichia coli. Fluorescence and light microscopy showed that the AcMNPV ac93 knockout did not spread by infection, and titration assays confirmed a defect in budded virus (BV) production. However, deletion of ac93 did not affect viral DNA replication. Electron microscopy indicated that ac93 was required for the egress of nucleocapsids from the nucleus and the formation of intranuclear microvesicles, which are precursor structures of occlusion-derived virus (ODV) envelopes. Immunofluorescence analyses showed that Ac93 was concentrated toward the cytoplasmic membrane in the cytoplasm and in the nuclear ring zone in the nucleus. Western blot analyses showed that Ac93 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV. Our results suggest that ac93, although not previously recognized as a core gene, is one that plays an essential role in the formation of the ODV envelope and the egress of nucleocapsids from the nucleus.     

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.     

Baculovirus VP80 protein and the F-actin cytoskeleton interact and connect the viral replication factory with the nuclear periphery

Recently, we showed that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) VP80 protein is essential for the formation of both virion types, budded virus (BV) and occlusion-derived virus (ODV). Deletion of the vp80 gene did not affect assembly of nucleocapsids. However, these nucleocapsids were not able to migrate from the virogenic stroma to the nuclear periphery. In the current paper, we constructed a baculovirus recombinant with enhanced-green fluorescent protein (EGFP)-tagged VP80, allowing visualization of the VP80 distribution pattern during infection. In baculovirus-infected cells, the EGFP-VP80 protein is entirely localized in nuclei, adjacent to the virus-triggered F-actin scaffold that forms a highly organized three-dimensional network connecting the virogenic stroma physically with the nuclear envelope. Interaction between VP80 and host actin was confirmed by coimmunoprecipitation. We further showed that VP80 is associated with the nucleocapsid fraction of both BVs and ODVs, typically at one end of the nucleocapsids. In addition, the presence of sequence motifs with homology to invertebrate paramyosin proteins strongly supports a role for VP80 in the polar transport of nucleocapsids to the periphery of the nucleus on their way to the plasma membrane to form BVs and for assembly in the nuclear periphery to form ODVs for embedding in viral occlusion bodies.     

核型多角体病毒侵染及其与宿主免疫系统互作的研究进展

核型多角体病毒(Nucleopolyhedrovirus,NPV)应用广泛,已被开发成微生物杀虫剂和用于重组蛋白表达等.NPV具有两种病毒颗粒:包埋型病毒粒子(occlusion-derived virus,ODV)和芽生型病毒粒子(budded virus,BV),两者的构成和组装存在差异.病毒包涵体在肠道中溶解后释...     

Concomitant primary infection of the midgut epithelial cells and the hemocytes of Trichoplusia ni by Autographa californica nucleopolyhedrovirus

We have constructed a modified Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) to express the green fluorescent protein (GFP) under the polyhedrin promoter and used it to study the infection process of AcMNPV in Trichoplusia ni larvae. T. ni larvae that ingested the virus showed localized expression of GFP in the midgut epithelial cells and the hemocytes at 12 h post infection (hpi). The presence of GFP-related fluorescence in the midgut columnar cells indicated that the virus was not only replicating, but also synthesizing the late viral proteins. Studies using the transmission electron microscope showed that the virus infected the midgut columnar cells. At the same time a proportion of the parental virus travelled through the midgut epithelial layer, possibly utilizing the plasma membrane reticular system, entered the hemocoel and infected the hemocytes. This resulted in the simultaneous infection of the midgut epithelial cells and the hemocytes. Subsequently, the budded virus (BV) released from the infected hemocytes into the hemolymph caused secondary infection within the tracheal epithelial cells. The virus then rapidly spread through the tracheal system allowing the infection of a variety of other tissues such as the epidermis and the fat body.     

Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) exon0 (orf141) has been shown to be required for the efficient production of budded virus (BV). The deletion of exon0 reduces the level of BV production by up to 99% (X. Dai, T. M. Stewart, J. A. Pathakamuri, Q. Li, and D. A. Theilmann, J. Virol. 78:9633-9644, 2004); however, the function or mechanism by which EXON0 affects BV production is unknown. In this study, we further elucidated the function of EXON0 by investigating the localization of EXON0 in infected Sf9 cells and in virions and by identifying interactions between EXON0 and other viral proteins. In addition, electron microscopy was used to study the cellular localization of nucleocapsids in cells transfected with an exon0 knockout (KO) virus. The results showed that EXON0 was localized to both the cytoplasm and the nuclei of infected Sf9 cells throughout the infection. Western blotting results also showed that EXON0 was purified along with BV and occlusion-derived virus (ODV). The fractionation of BV into the nucleocapsid and envelope components showed that EXON0 localized to the BV nucleocapsid. Yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy revealed that it interacted with nucleocapsid proteins FP25 and BV/ODV-C42. Cells transfected with the exon0 KO virus exhibited normally appearing nucleocapsids in the nuclei in numbers equal to those in the nuclei of cells transfected with the EXON0 repaired virus. In contrast, the numbers of nucleocapsids in the cytoplasm of cells transfected with the exon0 KO virus were significantly lower than those in the cytoplasm of cells transfected with the repaired virus. These results support the conclusion that EXON0 is required in the BV pathway for the efficient egress of nucleocapsids from the nucleus to the cytoplasm.     

P74 mediates specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to primary cellular targets in the midgut epithelia of Heliothis virescens Larvae

P74, an envelope protein of the occlusion-derived virus (ODV) of Autographa californica M nucleopolyhedrovirus (AcMNPV), is critical for oral infection of Trichoplusia ni larvae. The role of P74 during primary infection, however, is unknown. Here we provide evidence that P74 facilitates binding of AcMNPV ODV to a specific receptor within the larval midgut epithelia of another host species, Heliothis virescens. We adapted a fluorescence dequenching assay to compare binding, fusion, and competition of wild-type AcMNPV ODV in vivo with itself and with the ODV of a p74-deficient AcMNPV mutant. We found that relative to wild-type ODV, binding and fusion of ODV deficient in P74 were both qualitatively and quantitatively different. Unlike wild-type ODV, an excess of P74-deficient ODV failed to compete effectively with wild-type ODV binding, and the overall binding level of the mutant ODV was one-third that of the wild type. These results implicated P74 as an ODV attachment protein that binds to a specific receptor on primary target cells within the midgut.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus (HearNPV) ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-1, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.     

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.     

Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD), ranging from 48 (Spodoptera litura multicapsid NPV [MNPV]) to 78 (Adoxophyes honmai NPV) amino acid (aa) residues, with a nonassigned function. This CTD is much longer than the CTD of GP64-like envelope fusion proteins (7 aa), which appear to be nonessential for BV infectivity. Here we have investigated the functional role of the CTD of Helicoverpa armigera single-capsid NPV (HearNPV), a group II NPV. We combined a newly constructed HearNPV f-null bacmid knockout-repair system and an Autographa californica MNPV (AcMNPV) gp64-null bacmid knockout-pseudotype system with mutation and rescue experiments to study the functional role of the baculovirus F protein CTD. We show that except for the 16 C-terminal aa, the HearNPV F CTD is essential for virus spread from cell to cell. In addition, the CTD of HearNPV F is involved in BV production in a length-dependent manner and is essential for BV infectivity. The tyrosine residue Y658, located 16 aa from the C terminus, seems to be critical. However, HearNPV F without a CTD still rescues the infectivity of gp64-null AcMNPV BV, indicating that the CTD is not involved in processing and fusogenicity. Altogether, our results indicate that the F protein is essential for baculovirus BV infectivity and that the CTD is important for F protein incorporation into BV.     

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.     

杆状病毒与昆虫宿主相互作用的研究进展

杆状病毒与昆虫宿主相互作用是一种基本的分子和生态问题, 不仅在农业上, 而且在真核表达系统、 基因治疗、 蛋白表面展示 系统以及基因工程疫苗等方面都有重要的实际应用。杆状病毒还是一种很有潜力的病毒杀虫剂, 而且对环境来说是安全的。研究这些相互 作用也产生了许多重要和有价值的发现。杆状病毒生命循环中存在两种不同形式的病毒, 即包埋型病毒粒子(occlusion derived virus, ODV) 和出芽型病毒粒子(budded virus, BV)。ODV包裹于多角体中, 主要负责宿主的原发感染; 而BV由感染的宿主细胞释放后引发继发 感染。病毒侵染起始于敏感的昆虫宿主食用了污染包涵体病毒的植物。在宿主中肠的碱性环境中, 多角体溶解释放ODV, ODV与宿主肠道 柱状上皮细胞细胞膜融合, 通过内吞体进入细胞。之后核衣壳从内吞体中逃脱并被转运到细胞核。病毒转录和复制在细胞核进行, 新生 的BV粒子从基底膜出芽引起全身感染。杆状病毒与宿主细胞相互作用包括从病毒结合和进入时的相互作用, 到宿主基因表达调节, 以及 修饰与调节细胞和机体所发生的生理和防御的相互作用的复杂和微妙的机制。本文主要以杆状病毒侵染昆虫宿主的过程为线索, 总结和评 述了杆状病毒与昆虫宿主相互作用方面研究的最新进展, 特别是杆状病毒基因在病毒入侵过程中所起的作用。     

Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.     

Multiple Nucleocapsid Packaging of Autographa californica Nucleopolyhedrovirus Accelerates the Onset of Systemic Infection in Trichoplusia ni

Among the nucleopolyhedroviruses (Baculoviridae), the occlusion-derived virus (ODV), which initiates infection in host insects, may contain only a single nucleocapsid per virion (the SNPVs) or one to many nucleocapsids per virion (the MNPVs), but the significance of this difference is unclear. To gain insight into the biological relevance of these different packaging strategies, we compared pathogenesis induced by ODV fractions enriched for multiple nucleocapsids (ODV-M) or single nucleocapsids (ODV-S) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) containing a β-galactosidase reporter gene. In time course experiments wherein newly molted fourth-instar Trichoplusia ni were challenged with doses of ODV-S or ODV-M that yielded the same final mortality (~70%), we characterized viral foci as either being restricted to the midgut or involving tracheal cells (the secondary target tissue, indicative of systemic infection). We found that while the timing of primary infection by ODV-S and ODV-M was similar, ODV-S established significantly more primary midgut cell foci than ODV-M, but ODV-M infected tracheal cells at twice the rate of ODV-S. The more efficient establishment of tracheal infections by ODV-M decreased the probability that infections were lost by midgut cell sloughing, explaining why higher numbers of primary infections established by ODV-S within larvae were needed to achieve the same final mortality. These results showed that the multiple nucleocapsid packaging strategy of AcMNPV accelerates the onset of irreversible systemic infections and may indicate why MNPVs have wider individual host ranges than SNPVs.     

A GP64-null baculovirus pseudotyped with vesicular stomatitis virus G protein

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein is an essential virion protein that is involved in both receptor binding and membrane fusion during viral entry. Genetic studies have shown that GP64-null viruses are unable to move from cell to cell and this results from a defect in the assembly and production of budded virions (BV). To further examine requirements for virion budding, we asked whether a GP64-null baculovirus, vAc(64-), could be pseudotyped by introducing a heterologous viral envelope protein (vesicular stomatitis virus G protein [VSV-G]) into its membrane and whether the resulting virus was infectious. To address this question, we generated a stably transfected insect Sf9 cell line (Sf9(VSV-G)) that inducibly expresses the VSV-G protein upon infection with AcMNPV Sf9(VSV-G) and Sf9 cells were infected with vAc(64-), and cells were monitored for infection and for movement of infection from cell to cell. vAc(64-) formed plaques on Sf9(VSV-G) cells but not on Sf9 cells, and plaques formed on Sf9(VSV-G) cells were observed only after prolonged intervals. Passage and amplification of vAc(64-) on Sf9(VSV-G) cells resulted in pseudotyped virus particles that contained the VSV-G protein. Cell-to-cell propagation of vAc(64-) in the G-expressing cells was delayed in comparison to wild-type (wt) AcMNPV, and growth curves showed that pseudotyped vAc(64-) was generated at titers of approximately 10(6) to 10(7) infectious units (IU)/ml, compared with titers of approximately 10(8) IU/ml for wt AcMNPV. Propagation and amplification of pseudotyped vAc(64-) virions in Sf9(VSV-G) cells suggests that the VSV-G protein may either possess the signals necessary for baculovirus BV assembly and budding at the cell surface or may otherwise facilitate production of infectious baculovirus virions. The functional complementation of GP64-null viruses by VSV-G protein was further demonstrated by identification of a vAc(64-)-derived virus that had acquired the G gene through recombination with Sf9(VSV-G) cellular DNA. GP64-null viruses expressing the VSV-G gene were capable of productive infection, replication, and propagation in Sf9 cells.     

Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115

Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.     

Statistical modeling of cell-to-cell variability in viral infection during passaging in suspension cell culture: Application in Monte-Carlo simulation

Packaging during the passaging of viruses in cell cultures yields various phenotypes and is regulated by viral protein expression in infected cells. Although such a packaging mechanism has a profound effect in controlling the virus yield, little is known about the underlying statistical models followed by virus packaging and protein expression among cells infected with the virus. A predictive framework combining identification of the probability density function (PDF) based on log-likelihood and using the PDF for Monte-Carlo simulations is developed. The Birnbaum–Saunders distribution was found to be consistent with all three-virus packaging levels, including nucleocapsids/occlusion-derived virus (ODV), ODVs/polyhedra, and polyhedra/cell for both wild-type and genetically modified AcMNPV. Next, it was demonstrated that PDF fitting could be used to compare two viruses having distinctly different genetic configurations. Finally, the identified PDF can be incorporated in RNA synthesis parameters for baculovirus infection to predict the cell-to-cell variability in protein expression using Monte-Carlo simulations. The proposed tool can be used for the estimation of uncertainty in the kinetic parameter and prediction of cell-to-cell variability for other biological systems.     

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc^96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc^96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc^96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).The Baculoviridae comprise a large and diverse group of viruses that are pathogens of insects, mainly from the Lepidoptera, Hymenoptera, and Diptera. During the typical biphasic infection cycle, two structurally and functionally distinct enveloped virion phenotypes are produced: occlusion-derived virus (ODV) and budded virus (BV) (35). The primary infection cycle in animals begins in the midgut cell after occlusion bodies (OBs) are ingested. Upon ingestion, the OBs dissolve in the alkaline environment of the midgut, and the ODVs are released into the lumen of midgut (15, 16, 20). Virions pass through a disrupted peritrophic membrane, a process often facilitated by enhancins, a group of virus-encoded metalloproteases (38). Subsequently, ODVs bind to and fuse directly with the microvilli of midgut columnar epithelial cells. A protein receptor is proposed to mediate the process, since binding is proteinase sensitive and saturable (15, 16, 20). After the nucleocapsids are transported to the nuclei of the midgut cells, viral DNA is released, followed by gene expression, DNA replication, and assembly of progeny nucleocapsids. In the late phase of infection, newly formed nucleocapsids are transported to the cell membrane, bud from the cell, and acquire a new envelope from the basal membrane. The BVs spread via the hemolymph (16) and the tracheal system (8) into the other tissues of the insect, causing the secondary infection.Baculoviruses encode per os infectivity factors (PIFs) on the envelope surface of ODV to initiate the efficient primary infection in midgut. So far, four highly conserved core genes, p74 (pif-0), pif-1, pif-2, and pif-3, have been identified. The deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p74 gene results in the complete elimination of the per os infectivity of OBs, while virions purified from mutant OBs were infectious when injected into the hemocoels of Trichoplusia ni or Heliothis virescens larvae (13, 17, 22). P74 is proposed to function as an ODV attachment protein that binds to a specific 30-kDa receptor protein on the primary target cells within the midgut (17, 39). PIF-1 was originally identified in Spodoptera littoralis NPV, where the deletion of pif-1 (spli7) resulted in viruses that were unable to infect S. littoralis larvae per os (21). PIF-2 was first identified in Spodoptera exigua MNPV, and the disruption of pif-2 resulted in the complete loss of per os infectivity for the host (11, 31). PIF-1 and PIF-2 have also been shown to participate in the binding of ODV to target cells in the midgut (28). PIF-3 (ac115) is also an essential factor for oral infection of AcMNPV. Although PIF-3 is not required for ODV attachment and fusion, it may mediate a critical downstream event, such as the translocation of ODV along microvilli during primary infection (28).AcMNPV, the archetype Alphabaculovirus of the Baculoviridae, has a double-stranded DNA genome of approximately 134 kbp that contains 154 predicted open reading frames (ORFs) (1). Comparative analysis of the 49 completely sequenced baculovirus genomes reveals 31 core genes that are conserved in all baculovirus genomes and are therefore likely to serve important roles in baculovirus life cycles (14, 26, 32, 37). Most core genes are related either to DNA replication, gene expression, packaging and assembly, or per os infection (37). Four core genes, ac68, p33 (ac92), ac96, and ac109, still have no known function or sequence similarities to proteins of known functions.In this study, an ac96-null mutant was constructed utilizing an AcMNPV bacmid, and the results showed that in tissue culture, ac96 was nonessential and was not required for viral DNA replication, ODV production, or BV production. However, in vivo assays demonstrated that the ac96-null virus was unable to infect midgut tissue when T. ni larvae were inoculated per os. The core gene ac96 therefore encodes a new per os infectivity factor, PIF-4.