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Baculoviruses are large DNA viruses of insects that are highly pathogenic in many hosts. In the infection cycle, baculoviruses produce two types of virions. These virion phenotypes are physically and functionally distinct, and each serves a critical role in the biology of the virus. One phenotype, the occlusion-derived virus (ODV), is occluded within a crystallized protein that facilitates oral infection of the host. A large complex of at least nine ODV envelope proteins called per os infectivity factors are critically important for ODV infection of insect midgut epithelial cells. Viral egress from midgut cells is by budding to produce a second virus phenotype, the budded virus (BV). BV binds, enters, and replicates in most other tissues of the host insect. Cell recognition and entry by BV are mediated by a single major envelope glycoprotein: GP64 in some baculoviruses and F in others. Entry and egress by the two virion phenotypes occur by dramatically different mechanisms and reflect a life cycle in which ODV is specifically adapted for oral infection while BV mediates dissemination of the infection within the animal.

Baculoviruses are large double-stranded DNA viruses that are virulent pathogens of certain insect species. In a natural host, Trichoplusia ni, infection by the model baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) begins when the occluded form of the virus disassembles in the midgut and virions infect midgut epithelial cells to establish the primary phase of the infection. To better understand the primary phase of the AcMNPV infection cycle, newly molted 5th-instar T. ni larvae were orally infected with AcMNPV occlusion bodies and the transcriptional responses of the T. ni midgut were analyzed at various times from 0 to 72 h postinfection, using transcriptome sequencing analysis and a T. ni reference genome. The numbers of differentially expressed host genes increased as the infection progressed, and we identified a total of 3,372 differentially expressed T. ni transcripts in the AcMNPV-infected midgut. Genes encoding orthologs of HMG176, atlastin, and CPH43 were among the most dramatically upregulated in response to AcMNPV infection. A number of cytochrome P450 genes were downregulated in response to infection. We also identified the effects of AcMNPV infection on a large variety of genes associated with innate immunity. This analysis provides an abundance of new and detailed information on host responses to baculovirus infection during the primary phase of the infection in the midgut and will be important for understanding how baculoviruses establish productive infections in the organism. IMPORTANCE Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni, infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.

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.

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 β-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.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Alphabaculovirus genus of the family Baculoviridae, is an enveloped double-stranded DNA virus. Budded virions (BVs) of AcMNPV enter host cells via clathrin-mediated endocytosis. However, the route of functional intracellular trafficking of AcMNPV BVs during entry is not well established. In the current study, we found that entering BVs were colocalized mainly with cellular Rab5 and Rab11. Expression of dominant-negative (DN) Rab5 and Rab11 or RNAi-mediated down regulation of these two cellular transcripts significantly reduced BVs entry into but not egress from Spodoptera frugiperda cells (Sf9), whereas similar treatments for Rab4 and Rab7 had no apparent effect on virus infection. Combined with data from RNAi knockdowns of dynamin, and dynasore inhibition assays, our results support a model in which AcMNPV BVs enter permissive host cells by clathrin-mediated endocytosis, followed by de-envelopment of BVs predominantly within early and maturing endosomes rather than within late endosomes. Additionally, Rab11 suppression studies suggest the Rab11-dependent recycling endosomal pathway is involved in virion entry.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model enveloped DNA virus that infects and replicates in lepidopteran insect cells, and can efficiently enter a wide variety of non-host cells. Budded virions of AcMNPV enter cells by endocytosis and traffic to the nucleus where the virus initiates gene expression and genome replication. While trafficking of nucleocapsids by actin propulsion has been studied in detail, other important components of trafficking during entry remain poorly understood. We used a recombinant AcMNPV virus expressing an EGFP reporter in combination with an RNAi screen in Drosophila DL1 cells, to identify host proteins involved in AcMNPV entry. The RNAi screen targeted 86 genes involved in vesicular trafficking, including genes coding for VPS and ESCRT proteins, Rab GTPases, Exocyst proteins, and Clathrin adaptor proteins. We identified 24 genes required for efficient virus entry and reporter expression, and 4 genes that appear to restrict virus entry.

The retroviral Gag protein is frequently used to generate ‘virus-like particles’ (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.

Serine protease (SP) and serine protease homolog (SPH) genes in insects encode a large family of proteins involved in digestion, development, immunity, and other processes. While 68 digestive SPs and their close homologs are reported in a companion paper (Kuwar et al., in preparation), we have identified 125 other SPs/SPHs in Manduca sexta and studied their structure, evolution, and expression. Fifty-two of them contain cystine-stabilized structures for molecular recognition, including clip, LDLa, Sushi, Wonton, TSP, CUB, Frizzle, and SR domains. There are nineteen groups of genes evolved from relatively recent gene duplication and sequence divergence. Thirty-five SPs and seven SPHs contain 1, 2 or 5 clip domains. Multiple sequence alignment and molecular modeling of the 54 clip domains have revealed structural diversity of these regulatory modules. Sequence comparison with their homologs in Drosophila melanogaster, Anopheles gambiae and Tribolium castaneum allows us to classify them into five subfamilies: A are SPHs with 1 or 5 group-3 clip domains, B are SPs with 1 or 2 group-2 clip domains, C, D1 and D2 are SPs with a single clip domain in group-1a, 1b and 1c, respectively. We have classified into six categories the 125 expression profiles of SP-related proteins in fat body, brain, midgut, Malpighian tubule, testis, and ovary at different stages, suggesting that they participate in various physiological processes. Through RNA-Seq-based gene annotation and expression profiling, as well as intragenomic sequence comparisons, we have established a framework of information for future biochemical research of nondigestive SPs and SPHs in this model species.

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.

The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Spodoptera frugiperda . Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an “egress complex” that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane. IMPORTANCE The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an “egress complex” involved in the nuclear release or transport of viral nucleocapsids.

In eukaryotic cells, the s oluble N -ethylmaleimide- s ensitive f actor (NSF) a ttachment protein re ceptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.

IE0 and IE1 of the baculovirus Autographa californica multiple nucleopolyhedrovirus are essential transregulatory proteins required for both viral DNA replication and transcriptional transactivation. IE0 is identical to IE1 except for 54 amino acids at the N-terminus but the functional differences between these two proteins remain unclear. The purpose of this study was to determine the separate roles of these critical proteins in the virus life cycle. Unlike prior studies, IE0 and IE1 were analyzed using viruses that expressed ie0 and ie1 from an identical promoter so that the timing and levels of expression were comparable. IE0 and IE1 were found to equally support viral DNA replication and budded virus (BV) production. However, specific viral promoters were selectively transactivated by IE0 relative to IE1 but only when expressed at low levels. These results indicate that IE0 preferentially transactivates specific viral genes at very early times post-infection enabling accelerated replication and BV production.

The GP64 envelope glycoprotein of the Autographa californica nucleopolyhedrovirus (AcMNPV) is a class III viral membrane fusion protein that is triggered by low pH during entry. Unlike most other viral fusion protein trimers, the monomers of GP64 are covalently linked to each other within the trimer by a single intermolecular disulfide bond (Cys24—Cys372). Single or paired alanine substitutions for Cys24 and Cys372 resulted in lower-efficiency transport of GP64 to the cell surface. Surprisingly, these mutated GP64s induced syncytium formation, and normalized fusion activities were approximately 30% of that from wild-type (WT) GP64. Heat treatment (37°C) did not further reduce fusion activity of GP64 constructs with a disrupted intermolecular disulfide bond, suggesting that the GP64 trimers were relatively thermostable in the absence of the intermolecular disulfide bond. In addition, analysis of binding by a conformation-specific monoclonal antibody (MAb) suggested that the low-pH-induced refolding of those GP64 constructs was generally similar to that of WT GP64. In addition to its critical role in membrane fusion, GP64 is also necessary for efficient budding. When GP64 constructs containing a disrupted intermolecular disulfide bond (Cys24—Cys372) were displayed at the cell surface at levels comparable to those of WT GP64, virion budding efficiency ranged from approximately 39 to 88%, indicating that the intermolecular disulfide bond is not required for virion budding. However, GP64 proteins with a disrupted intermolecular disulfide could not rescue a GP64-null bacmid. We also examined the 6 conserved intramolecular disulfide bonds using single and paired alanine substitution mutations. None of the GP64 constructs with disrupted intramolecular disulfide bonds were capable of mediating pH-triggered membrane fusion, indicating that the intramolecular disulfide bonds are all necessary for membrane fusion. Thus, while the intramolecular disulfide bonds of GP64 appear to serve critical roles in membrane fusion, the unusual intermolecular disulfide bond was not critical for membrane fusion or virion budding yet appears to play an unknown role in viral infectivity.

The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64 null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.

GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. Previous transmembrane (TM) domain replacement studies showed that the TM domain serves a critical role in GP64 function. To extend the prior studies and examine specific sequence requirements of the TM domain, we generated a variety of GP64 TM domain mutations. The mutations included 4- to 8-amino-acid deletions, as well as single and multiple point mutations. While most TM domain deletion constructs remained fusion competent, those containing deletions of eight amino acids from the C terminus did not mediate detectable fusion. The addition of a hydrophobic amino acid (A, L, or V) to the C terminus of construct C8 (a construct that contains a TM domain deletion of eight amino acids from the C terminus) restored fusion activity. These data suggest that the membrane fusion function of GP64 is dependent on a critical length of the hydrophobic TM domain. All GP64 proteins with a truncated TM domain mediated detectable virion budding with dramatically lower levels of efficiency than wild-type GP64. The effects of deletions of various lengths and positions in the TM domain were also examined for their effects on viral infectivity. Further analysis of the TM domain by single amino acid substitutions and 3-alanine scanning mutations identified important but not essential amino acid positions. These studies showed that amino acids at positions 485 to 487 and 503 to 505 are important for cell surface expression of GP64, while amino acids at positions 483 to 484 and 494 to 496 are important for virus budding. Overall, our results show that specific features and amino acid sequences, particularly the length of the hydrophobic TM domain, play critical roles in membrane anchoring, membrane fusion, virus budding, and infectivity.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 envelope glycoprotein is essential for virus entry and plays an important role in virion budding. An AcMNPV construct that contains a deletion of the gp64 gene is unable to propagate infection from cell to cell, and this defect results from both a severe reduction in the production of budded virions and the absence of GP64 on virions. In the current study, we examined GP64 proteins containing N- and C-terminal truncations of the ectodomain and identified a minimal construct capable of targeting the truncated GP64 to budded virions. The minimal budding and targeting construct of GP64 contained 38 amino acids from the mature N terminus of the GP64 ectodomain and 52 amino acids from the C terminus of GP64. Because the vesicular stomatitis virus (VSV) G protein was previously found to rescue infectivity of a gp64 null AcMNPV, we also examined a small C-terminal construct of the VSV G protein. We found that a construct containing 91 amino acids from the C terminus of VSV G (termed G-stem) was capable of rescuing AcMNPV gp64 null virion budding to wild-type (wt) or nearly wt levels. We also examined the display of chimeric proteins on the gp64 null AcMNPV virion. By generating viruses that expressed chimeric influenza virus hemagglutinin (HA) proteins containing the GP64 targeting domain and coinfecting those viruses with a virus expressing the G-stem construct, we demonstrated enhanced display of the HA protein on gp64 null AcMNPV budded virions. The combined use of gp64 null virions, VSV G-stem-enhanced budding, and GP64 domains for targeting heterologous proteins to virions should be valuable for biotechnological applications ranging from targeted transduction of mammalian cells to vaccine production.

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.

The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.

Pattern recognition receptors (PRRs) detect microbial pathogens and trigger innate immune responses. Previous biochemical studies have elucidated the physiological functions of eleven PRRs in Manduca sexta but our understanding of the recognition process is still limited, lacking genomic perspectives. While 34 C-type lectin-domain proteins and 16 Toll-like receptors are reported in the companion papers, we present here 120 other putative PRRs identified through the genome annotation. These include 76 leucine-rich repeat (LRR) proteins, 14 peptidoglycan recognition proteins, 6 EGF/Nim-domain proteins, 5 β-1,3-glucanase-related proteins, 4 galectins, 4 fibrinogen-related proteins, 3 thioester proteins, 5 immunoglobulin-domain proteins, 2 hemocytins, and 1 Reeler. Sequence alignment and phylogenetic analysis reveal the evolution history of a diverse repertoire of proteins for pathogen recognition. While functions of insect LRR proteins are mostly unknown, their structure diversification is phenomenal: In addition to the Toll homologs, 22 LRR proteins with a signal peptide are expected to be secreted; 18 LRR proteins lacking signal peptides may be cytoplasmic; 36 LRRs with a signal peptide and a transmembrane segment may be non-Toll receptors on the surface of cells. Expression profiles of the 120 genes in 52 tissue samples reflect complex regulation in various developmental stages and physiological states, including some likely by Rel family transcription factors via κB motifs in the promoter regions. This collection of information is expected to facilitate future biochemical studies detailing their respective roles in this model insect.

Signal transduction pathways and their coordination are critically important for proper functioning of animal immune systems. Our knowledge of the constituents of the intracellular signaling network in insects mainly comes from genetic analyses in Drosophila melanogaster. To facilitate future studies of similar systems in the tobacco hornworm and other lepidopteran insects, we have identified and examined the homologous genes in the genome of Manduca sexta. Based on 1:1 orthologous relationships in most cases, we hypothesize that the Toll, Imd, MAPK-JNK-p38 and JAK-STAT pathways are intact and operative in this species, as are most of the regulatory mechanisms. Similarly, cellular processes such as autophagy, apoptosis and RNA interference probably function in similar ways, because their mediators and modulators are mostly conserved in this lepidopteran species. We have annotated a total of 186 genes encoding 199 proteins, studied their domain structures and evolution, and examined their mRNA levels in tissues at different life stages. Such information provides a genomic perspective of the intricate signaling system in a non-drosophiline insect.

In insects, chitin is a major structural component of the cuticle and the peritrophic membrane (PM). In nature, chitin is always associated with proteins among which chitin-binding proteins (CBPs) are the most important for forming, maintaining and regulating the functions of these extracellular structures. In this study, a genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin-binding domains (CBDs) was conducted. A total of 53 genes encoding 56 CBPs were identified, including 15 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 11 CPAP3s (CPAPs with 3 CBDs) and 17 PMPs (PM proteins) with a variable number of CBDs, which are structural components of cuticle or of the PM. CBDs were also identified in enzymes of chitin metabolism including 6 chitinases and 7 chitin deacetylases encoded by 6 and 5 genes, respectively. RNA-seq analysis confirmed that PMP and CPAP genes have differential spatial expression patterns. The expression of PMP genes is midgut-specific, while CPAP genes are widely expressed in different cuticle forming tissues. Phylogenetic analysis of CBDs of proteins in insects belonging to different orders revealed that CPAP1s from different species constitute a separate family with 16 different groups, including 6 new groups identified in this study. The CPAP3s are clustered into a separate family of 7 groups present in all insect orders. Altogether, they reveal that duplication events of CBDs in CPAP1s and CPAP3s occurred prior to the evolutionary radiation of insect species. In contrast to the CPAPs, all CBDs from individual PMPs are generally clustered and distinct from other PMPs in the same species in phylogenetic analyses, indicating that the duplication of CBDs in each of these PMPs occurred after divergence of insect species. Phylogenetic analysis of these three CBP families showed that the CBDs in CPAP1s form a clearly separate family, while those found in PMPs and CPAP3s were clustered together in the phylogenetic tree. For chitinases and chitin deacetylases, most of phylogenetic analysis performed with the CBD sequences resulted in similar clustering to the one obtained by using catalytic domain sequences alone, suggesting that CBDs were incorporated into these enzymes and evolved in tandem with the catalytic domains before the diversification of different insect orders. Based on these results, the evolution of CBDs in insect CBPs is discussed to provide a new insight into the CBD sequence structure and diversity, and their evolution and expression in insects.

Recent evidence from genome sequence analyses demands a substantial revision of the taxonomy and classification of the family Baculoviridae. Comparisons of 29 baculovirus genomes indicated that baculovirus phylogeny followed the classification of the hosts more closely than morphological traits that have previously been used for classification of this virus family. On this basis, dipteran- and hymenopteran-specific nucleopolyhedroviruses (NPV) should be separated from lepidopteran-specific NPVs and accommodated into different genera. We propose a new classification and nomenclature for the genera within the baculovirus family. According to this proposal the updated classification should include four genera: Alphabaculovirus (lepidopteran-specific NPV), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific NPV) and Deltabaculovirus (dipteran-specific NPV).

Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ∼156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni . We used an ∼60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure. IMPORTANCE Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

The family Baculoviridae represents a large group of enveloped double-stranded DNA viruses that are restricted to invertebrate hosts, mainly insects of the order Lepidoptera (2). Baculoviruses are currently subdivided into two genera, the nucleopolyhedroviruses (NPVs) and the granuloviruses (GVs). Phylogenetic studies also indicate that NPVs can be further subdivided into two subgroups: groups I and II (9, 12, 33). During a single infection cycle, baculoviruses produce two virion phenotypes that play distinctly different roles. Virions of the occlusion-derived virus (ODV) mediate the transmission of infection from animal to animal, while virions of the budded virus (BV) mediate the cell-to-cell spread of infection within the infected animal and in cell culture. Virions of the ODV phenotype are formed very late in the infection cycle when nucleocapsids become enveloped within the nucleus. ODV are later encased in the crystallized occlusion body protein (polyhedrin in NPVs and granulin in GVs) to form occlusion bodies, which are released when the host cells lyse. When occlusion bodies of lepidopteran baculoviruses are consumed by susceptible larvae, the highly alkaline environment of the host's midgut triggers disassembly of the occlusion body and subsequent release of the ODV. ODV are highly infectious to midgut epithelial cells and initiate the infection cycle in the animal. In contrast to the ODV, virions of the BV phenotype are generated earlier in the infection cycle, when nucleocapsids bud through the host cell plasma membrane into the hemocoel. In the hemocoel, BV initially infects the tracheal epithelium and hemocytes and then infects the fat body, muscle, malpighian tubules, and other tissues, leading to the eventual death of the host (5-7). BV enter cells via the endocytic pathway and appear to contain a very limited number of virus-encoded envelope proteins. GP64 is the major glycoprotein found in the BV envelope of Autographa californica multicapsid NPV (AcMNPV) and other members of the group I NPVs. Previous studies showed that GP64 was (i) involved in BV binding to host cells (8), (ii) necessary and sufficient for the low-pH-triggered membrane fusion that occurs during BV entry by endocytosis (3, 11, 20, 31), and (iii) essential for efficient virion budding from the cell surface (21, 23). Recent analyses of genomic sequences from group II NPVs and GVs indicate that they do not possess a gp64 gene. However, BV envelope proteins unrelated to GP64 have been identified from two group II NPVs; these envelope proteins include Ld130 from Lymantria dispar MNPV and Se8 from Spodoptera exigua MNPV (14, 16). Like GP64, both Ld130 and Se8 possess pH-triggered membrane fusion activity (15, 25) and were subsequently named F proteins (32). A recent study demonstrated that Ld130 (or LdF) and Se8 (or SeF) are capable of rescuing virion production and infectivity in a gp64-null AcMNPV (18). Thus, these F proteins are functionally analogous to GP64. While proteolytic cleavage does not appear to be required for activation of GP64, both LdF and SeF contain a conserved proprotein convertase (furin-like) cleavage site, and it was recently demonstrated that cleavage at that site is necessary for low-pH-triggered fusion activity of the SeF protein (18, 32). In addition, SeF cleavage is necessary for rescue of the gp64-null AcMNPV by this F protein (18). F genes have been identified in all NPVs and GVs for which genomic sequences are available. Therefore, group I NPVs such as AcMNPV and the Orgyia pseudotsugata MNPV (OpMNPV) contain both a gp64 gene and a gene encoding an F protein homolog. Comparisons of available F protein sequences show that the conserved region containing the furin-like cleavage site is absent from the AcMNPV and OpMNPV F protein homologs Ac23 and Op21 (28). In addition, pH-triggered membrane fusion activity has not been detected in syncytium formation assays of transiently expressed Ac23 or Op21 (reference 27 and unpublished observations). These observations suggested that the F protein homologs in group I NPVs may play a role distinctly different from that of GP64. However, the presence of F homolog genes in baculovirus genomes suggests that they have a positive impact on viral success. We therefore examined the role of the F protein homolog (Ac23) in the context of the AcMNPV infection cycle. To accomplish this, we used a new PCR and bacmid-based recombination system to delete the Ac23 gene from the AcMNPV genome. Our results indicate that Ac23 is not essential for virus replication and that deletion of Ac23 has no detectable effect on yields of infectious budded virus from cultured cells, or from orally infected animals. However, Ac23 substantially accelerates the death of cultured insect cells and insect larvae.

Baculoviridae is a family of large double-stranded DNA viruses of invertebrates. Baculovirus genomes are circular and range in size from approximately 80 to 180 kbp. Members of the family infect a large number of insect species, with most hosts found in the insect order Lepidoptera (5). The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the most intensively studied baculovirus. The AcMNPV genome is approximately 134 kbp in size and encodes 154 predicted genes (2). After entry into the host cell, AcMNPV nucleocapsids are transported to the nuclei of infected cells, where viral transcription and DNA replication occur. The infection cycle can be subdivided into three major phases of gene expression: early, late, and very late. Genes expressed in the early phase are transcribed by the host RNA polymerase II (10). Some of the early genes encode products necessary for DNA replication and late gene expression. The late phase of gene expression begins concomitant with or shortly after replication of the viral genome, and viral DNA replication appears to be a necessary prerequisite to late gene transcription. Inhibitors of viral DNA replication (such as aphidicolin) also block late gene expression in infected cells (19). Late genes are transcribed by a viral RNA polymerase (13, 14) that recognizes distinct late promoters. Most, if not all, late gene promoters contain the conserved sequence 5′-TAAG-3′ (33) at the late transcription start site. The conserved core TAAG sequence plus nonconserved flanking sequences of at least 8 to 12 nucleotides (nt) appear to comprise the late promoter sequences that are necessary for wild-type levels of late transcription (11, 24, 31). Except for the conserved core TAAG motif, little is known regarding the sequence specificity requirements for late promoter recognition and activation. The very late phase of gene expression follows late gene expression and is characterized by the hyperexpression of two genes, polyhedrin and p10. Very late promoters appear to be similar to late promoters in that they also include the conserved core TAAG motif and flanking sequences, but differ in that they also require an additional sequence called a “burst” sequence. The exceptionally high levels of transcription from the polyhedrin and p10 genes appear to be regulated or mediated by binding of viral protein VLF-1 to the “burst” DNA sequence, downstream of the transcription start site. The burst sequence is so called because it appears to regulate the burst of very late transcription (22, 26, 34, 36-38). The transcription of late genes requires a number of early gene products. In previous studies using a transient late expression assay (29) to identify genes necessary for transient transcription from an AcMNPV late promoter, 19 late expression factor (lef) genes were identified (16, 20, 32). Because DNA replication is a necessary prerequisite for late gene transcription, a transient origin-dependent DNA replication assay was used to identify a subset of lef genes necessary for DNA replication, and these genes were proposed to constitute the subset of lef genes associated with viral DNA replication (15, 20). Transient assays for late gene expression and DNA replication have proved to be extremely important tools for the identification of genes associated with DNA replication and late gene expression. However, because viral proteins are expressed transiently from plasmid constructs in these assays, it is likely that the regulation of expression of each protein differs substantially from its expression in the context of a viral infection. Thus, it is possible that artifacts may arise from over- or underexpression of various LEF proteins. It is therefore critical to examine the effects of LEF proteins in the context of the AcMNPV infection cycle. Because many lef genes are likely essential for viral replication, only a few viruses have been generated with knockout or null mutations in lef genes (12). Recently, the p143 gene of AcMNPV was deleted and replaced with the p137 gene from the Trichoplusia ni granulovirus (TnGV), by recombination in Escherichia coli with an AcMNPV genome propagated as a bacterial artificial chromosome (BACmid) (4). That study demonstrated the utility and convenience of manipulating essential AcMNPV genes in an E. coli-based system. The lef-11 gene is located immediately upstream of and overlapping the pp31 open reading frame (ORF). lef-11 is expressed as an early gene, and the LEF-11 protein is localized to the nuclei of infected cells (18). lef-11 was initially identified as a gene necessary for efficient transcription from a late promoter in a transient late expression assay (35). Several studies showed that omission of lef-11 in transient late expression assays resulted in only approximately 1 to 10% of the reporter expression that was observed when plasmids containing all 19 lef genes were present (20, 32, 35). In addition, lef-11 was not identified as necessary for transient origin-dependent plasmid DNA replication in two studies using that technique (15, 20). We initially attempted to delete the lef-11 gene in the AcMNPV genome by recombination in an insect cell line that was stably transfected with the lef-11 gene, but repeated attempts were unsuccessful. Therefore, for the present study, we used a commercially available AcMNPV BACmid to delete the lef-11 gene by recombination in E. coli. After transfection into Sf9 cells, a lef-11-null AcMNPV BACmid (vAclef11KO) was unable to propagate in cell culture. However, a “repair” BACmid (vAclef11KO-REP) generated by transposition of the lef-11 gene into the polyhedrin locus of the vAclef11KO BACmid was able to replicate in a manner similar to wild-type and control AcMNPV. Thus, we found that the lef-11 gene was essential for viral replication in Sf9 cells. The lef-11-null BACmid was subsequently examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in transcription. In transfected Sf9 cells, the viral DNA genome of the lef-11-null BACmid (vAclef11KO) was not amplified, whereas the repair BACmid's genome was amplified in a manner similar to that of either the wild-type or control virus. Repair of the vAclef11KO BACmid confirmed that the defect in DNA replication in vAclef11KO was due to the loss of lef-11. Thus, the lef-11-null virus was deficient in viral DNA replication. Northern blot analysis of early, late, and very late genes showed that late transcription and very late transcription were absent in cells transfected with the lef-11-null BACmid. In contrast to prior studies suggesting a role for lef-11 in late transcriptional regulation, our data obtained with a lef-11-knockout virus indicate that lef-11 is necessary for viral DNA replication in Sf9 cells and that effects on late transcription may represent only secondary effects of the lef-11 knockout.

C-type lectins (CTLs) are a large family of Ca(2+)-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca(2+)-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.

In eukaryotic cells, regulated vesicular trafficking is critical for directing protein transport and for recycling and degradation of membrane lipids and proteins. Through carefully regulated transport vesicles, the endomembrane system performs a large and important array of dynamic cellular functions while maintaining the integrity of the cellular membrane system. Genetic studies in yeast Saccharomyces cerevisiae have identified approximately 50 vacuolar protein sorting (VPS) genes involved in vesicle trafficking, and most of these genes are also characterized in mammals. The VPS proteins form distinct functional complexes, which include complexes known as ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III. Little is known about the orthologs of VPS proteins in insects. Here, with the newly annotated Manduca sexta genome, we carried out genomic comparative analysis of VPS proteins in yeast, humans, and 13 sequenced insect genomes representing the Orders Hymenoptera, Diptera, Hemiptera, Phthiraptera, Lepidoptera, and Coleoptera. Amino acid sequence alignments and domain/motif structure analyses reveal that most of the components of ESCRT, retromer, CORVET, HOPS, GARP, and PI3K-III are evolutionarily conserved across yeast, insects, and humans. However, in contrast to the VPS gene expansions observed in the human genome, only four VPS genes (VPS13, VPS16, VPS33, and VPS37) were expanded in the six insect Orders. Additionally, VPS2 was expanded only in species from Phthiraptera, Lepidoptera, and Coleoptera. These studies provide a baseline for understanding the evolution of vesicular trafficking across yeast, insect, and human genomes, and also provide a basis for further addressing specific functional roles of VPS proteins in insects.

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion.Spodoptera frugiperda(Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused toSf9 cells containing anEGFPreporter construct under the control of a responsivelacoperator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity.Sf9 cells expressing theOrgyia pseudotsugatamulticapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.

Self-incompatibility is a phenomenon that involves recognition of self versus non-self pollen, leading to the rejection of self-related pollen and preventing self-fertilization. In this study, we used a baculovirus-infected insect cell culture system to express two Brassica oleracea stigma-specific proteins required for self-incompatibility: the S-locus glycoprotein, a soluble cell wall-localized glycosylated protein, and the S-locus receptor kinase, a receptor-like integral plasma membrane glycoprotein with serine/threonine kinase activity. Insect cells expressing the S-locus receptor kinase were used in conjunction with immunofluorescence and a whole cell enzyme-linked immunosorbant assay to demonstrate that the receptor is targeted to the cell surface and is oriented with its N-terminal S domain towards the outside of the cell.

GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). To examine the potential role of GP64 as a viral attachment protein in host cell receptor binding, we generated, overexpressed, and characterized a soluble form of the OpMNPV GP64 protein, GP64solOp. Assays for trimerization, sensitivity to proteinase K, and reduction by dithiothreitol suggested that GP64solOp was indistinguishable from the ectodomain of the wild-type OpMNPV GP64 protein. Virion binding to host cells was analyzed by incubating virions with cells at 4°C in the presence or absence of competitors, using a single-cell infectivity assay to measure virion binding. Purified soluble GP64 (GP64solOp) competed with a recombinant AcMNPV marker virus for binding to host cells, similar to control competition with psoralen-inactivated wild-type AcMNPV and OpMNPV virions. A nonspecific competitor protein did not similarly inhibit virion binding. Thus specific competition by GP64solOp for virion binding suggests that the GP64 protein is a host cell receptor-binding protein. We also examined the kinetics of virion internalization into endosomes and virion release from endosomes by acid-triggered membrane fusion. Using a protease sensitivity assay to measure internalization of bound virions, we found that virions entered Spodoptera frugiperda Sf9 cells between 10 and 20 min after binding, with a half-time of approximately 12.5 min. We used the lysosomotropic reagent ammonium chloride to examine the kinetics of membrane fusion and nucleocapsid release from endosomes after membrane fusion. Ammonium chloride inhibition assays indicated that AcMNPV nucleocapsids were released from endosomes between 15 and 30 min after binding, with a half-time of approximately 25 min.

Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5′ capped transcription start sites and 3′ polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (T A AG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3′ ends showed that 92 (77%) of the 3′ PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

Baculovirus interactions with host cells range from the physical interactions that occur during viral binding and entry, to the complex and subtle mechanisms that regulate host gene expression and modify and regulate cellular and organismal physiology and defenses. Fundamental studies of baculovirus biochemistry and molecular biology have yielded many interesting and important discoveries on the mechanisms of these virus-host interactions. Information from such studies has also resulted in exciting new strategies for environmentally sound insect pest control, and in the development and improvement of a valuable eukaryotic expression vector system. In addition a number of important and valuable model biological systems have emerged from studies of baculoviruses. These include robust systems for studies of eukaryotic transcription, viral DNA replication, membrane fusion, and apoptosis. Because functions have been identified for only a small number of baculovirus genes, we can expect many exciting new discoveries in the future and an unfolding of the complex and intricate relationship between baculoviruses and insect cells.

The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein that enters the secretory pathway and is oligomerized and extensively processed during transport to the plasma membrane. The kinetics of GP64 EFP biosynthesis, oligomerization, and processing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of GP64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i., and the maximal rate of synthesis was observed in the period of 24-26 hr pi., a time coincident with the onset of high level production of budded virus in OpMNPV-infected L. dispar cells. To determine the oligomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatography, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP was identified as a trimeric molecule, that migrates as two discrete bands on nonreducing SDS-PAGE. Pulse-chase studies, performed at both early (12 hr pi.) and late (36 hr p.i.) stages of the infection cycle, showed that GP64 EFP oligomerization is complete within 15 min after synthesis. Efficiency of oligomerization however was relatively low, with less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be degraded within 30 to 45 min after synthesis. Analysis of the kinetics of carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for beth early and late phases of infection, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during the late phase. Thus, carbohydrate processing appeared to become less efficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results in a homotrimeric structure for GP64 EFP. While carbohydrate addition is rapid, carbohydrate processing requires prolonged periods of time (with half-times of 45 to 75 min) and appears to become less efficient during the late phase of the infection.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the interaction of GP64 with host cellular membranes and direct GP64-membrane interactions may represent a receptor-binding step prior to a low-pH-triggered conformational change.

The early promoter of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 gene is active when transfected into several insect cell lines and does not require viral gene products for transcription in uninfected cells. Because previous studies have shown that the gp64 early promoter is activated above basal levels in uninfected cells, host transcription factors are likely to play a role in gp64 activation at early times postinfection. By using nuclear extracts from uninfected Sf9 cells for electrophoretic mobility shift analysis of gp64 regulatory regions, host nuclear proteins were shown to bind specifically to the upstream regulatory region of the gp64 early promoter. Host factor binding was mapped to a 24-bp sequence centered approximately 35 bp upstream of the TATA box. Two consensus eukaryotic transcription factor-binding site motifs, GATA and CACGTG, were identified within the 24-bp sequence. Competition assays using oligonucleotides containing either a GATA or a CACGTG motif and similar oligonucleotides with point mutations in these sites showed that each site is required for binding host transcription factors. To investigate the functional significance of host factor binding to GATA and CACGTG motifs, constructs containing point mutations in these motifs were examined in transient expression assays. Mutations in either or both GATA and CACGTG sites decreased reporter activity in transient expression assays, suggesting that binding of host transcription factors to these motifs is important in transcriptional regulation of the gp64 early promoter.

Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells.

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.

Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Conclusions Ao38 cells represent a highly productive new insect cell line that will be useful for heterologous protein expression and other applications in biotechnology.

The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded virus (BV) and is involved in BV entry into the host cell by endocytosis. To determine whether gp64 alone was sufficient to mediate membrane fusion, the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 protein was transiently expressed in uninfected insect cells. Cells expressing the baculovirus gp64 protein were examined for membrane fusion activity by using a syncytium formation assay under various conditions of exposure to low pH. Cells expressing the gp64 protein mediated membrane fusion and syncytium formation in a pH-dependent manner. A pH of 5.5 or lower was required to induce membrane fusion. In addition, exposure of gp64-expressing cells to low pH for as little as 5 s was sufficient to induce gp64-mediated syncytium formation. These studies provide direct evidence that gp64 is a pH-dependent membrane fusion protein and suggest that gp64 is the protein responsible for fusion of the virion envelope with the endosome membrane during BV entry into the host cell by endocytosis.

Expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. To characterize the early promoter and identify sequences involved in the regulation of gp64 early transcription, promoter-reporter gene fusions were generated from the Orygia pseudotsugata nuclear polyhedrosis virus gp64 promoter and were analyzed by transient expression in uninfected insect cells. For these analyses, 5' deletion mutations were constructed in the gp64 upstream regulatory region. Larger promoter constructs were functional in uninfected Lymantria dispar cells, indicating that transcription from the gp64 early promoter required no additional viral gene products. Deletion analysis of the gp64 upstream region revealed several regulatory regions. These included a putative negative regulatory element between -319 and -166 nucleotides (nt) and multiple positive regulatory elements between -166 and -77 nt. Deletion of the TATA box located between -77 and -62 nt resulted in the loss of transcriptional activity. Cotransfections of reporter constructs and a plasmid containing a baculovirus transcriptional transactivator gene (Autographa californica nuclear polyhedrosis virus IE1) resulted in transcriptional transactivation of all constructs containing an intact TATA box. These data demonstrate that sequences upstream of the gp64 TATA box are not essential for IE1 transactivation and that only 34 nt upstream of the early transcription start site were necessary for basal levels of transcription and for transactivation by IE1. Function of the gp64 early promoter was also examined in cell lines from Spodoptera frugiperda and Drosophila melanogaster.

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.

With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)

The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp31 may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968–974] although genetic studies of the closely related BmNPV pp31 gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35–47]. In the current study, we examined the role of the pp31 gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp31 knockout in the AcMNPV genome. The pp31 knockout was subsequently rescued by reinserting the pp31 gene into the polyhedrin locus of the same virus genome. We found that pp31 was not essential for viral replication although the absence of pp31 resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp31 showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp31 gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes.

Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10 6 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10 7 to 10 9 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼10 9 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

Baculovirus F (fusion) proteins are found in the envelopes of budded virions. Recently a Drosophila melanogaster gene (CG4715) that encodes a protein with sequence similarity to baculovirus F proteins was discovered. To examine similarities and differences with baculovirus F proteins, we cloned the D. melanogaster cellular F ( Dm-cF ) protein gene and analyzed Dm-cF expression and localization. The predicted Dm-cF protein sequence lacks a furin cleavage site, and transiently expressed Dm-cF showed no protein cleavage and no detectable membrane fusion activity. In cell localization studies, transiently expressed Dm-cF was localized to intracellular organelles in D. melanogaster S2 cells, unlike baculovirus F proteins, which localize to cellular plasma membranes. Using reverse transcriptase PCR and Western blot analysis to examine Dm-cF expression in animals, we detected Dm-cF expression in both larval and adult D. melanogaster cells. However, Dm-cF expression was detected only in third instar larvae and adults, suggesting that Dm-cF expression may be developmentally regulated. We also identified genes related to Dm-cF in the genomes of two other Drosophila species, Drosophila yakuba and Drosophila pseudoobscura , and the mosquito Anopheles gambiae . These observations suggest that f genes may be present in the genomes of many insects. Conservation within and between 22 baculovirus and 4 insect F proteins was examined in detail. These studies demonstrate that Dm-cF is expressed in D. melanogaster and suggest that if baculovirus f genes are derived from a host cellular f gene, the function appears to have changed substantially upon adaptation to the baculovirus infection cycle.

The Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV) lef-6 gene was previously shown to be necessary for optimal transcription from an Ac M NPV late promoter in transient late expression assays. In the present study, we examined the expression and cellular localization of lef-6 during the Ac M NPV infection cycle and generated a lef-6 -null virus for studies of the role of lef-6 in the infection cycle. Transcription of lef-6 was detected from 4 to 48 h postinfection, and the LEF-6 protein was identified in dense regions of infected cell nuclei, a finding consistent with its potential role as a late transcription factor. To examine lef-6 in the context of the Ac M NPV infection cycle, we deleted the lef-6 gene from an Ac M NPV genome propagated as an infectious BACmid in Escherichia coli . Unexpectedly, the resulting Ac M NPV lef-6 -null BACmid (vAc lef6KO ) was able to propagate in cell culture, although virus yields were substantially reduced. Thus, the lef-6 gene is not essential for viral replication in Sf9 cells. Two “repair” Ac M NPV BACmids (vAc lef6KO-REP-P and vAc lef6KO-REP-ie1P ) were generated by transposition of the lef-6 gene into the polyhedrin locus of the vAc lef6KO BACmid. Virus yields from the two repair viruses were similar to those from wild-type Ac M NPV or a control (BACmid-derived) virus. The lef-6 -null BACmid (vAc lef6KO ) was further examined to determine whether the deletion of lef-6 affected DNA replication or late gene transcription in the context of an infection. The lef-6 deletion did not appear to affect viral DNA replication. Using Northern blot analysis, we found that although early transcription was apparently unaffected, both late and very late transcription were delayed in cells infected with the lef-6 -null BACmid. This phenotype was rescued in viruses containing the lef-6 gene reinserted into the polyhedrin locus. Thus, the lef-6 gene was not essential for either viral DNA replication or late gene transcription, but the absence of lef-6 resulted in a substantial delay in the onset of late transcription. Therefore, lef-6 appears to accelerate the infection cycle of Ac M NPV.

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (Ac M NPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar M NPV (Ld M NPV) and Spodoptera exigua M NPV (Se M NPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an Ac M NPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64 -null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from Ld M NPV or Se M NPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The Ac M NPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

The Autographa californica multiple nucleopolyhedrovirus (Ac M NPV) 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 Ac M NPV 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) Ac M NPV, 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 Ac M NPV. 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.