苜蓿丫纹夜蛾核型多角体病毒fp25k基因的改造及用于稳定转化Sf9昆虫细胞系

【目的】苜蓿丫纹夜蛾核型多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)在昆虫细胞中连续传代以后,会出现从多多角体表型到少多角体表型的转变,这种转变与一个编码25 kDa蛋白的基因（few polyhedra, fp25k）突变失活有关。杆状病毒的fp25k基因突变后产生的包涵体（多角体）衍生病毒粒子变少而出芽型病毒粒子增加,会降低外源基因在杆状病毒表达系统中的表达。本研究拟改造fp25k并构建能持续表达FP25K蛋白的转基因昆虫细胞,以克服杆状病毒, fp25k基因易突变导致的表达系统缺陷。【方法】本实验通过改造杆状病毒, fp25k基因在细胞传代过程中容易产生突变的位点,得到 mfp25k,并将mfp25k构建到pIZT/V5-His载体上,重组载体转染Sf9细胞,通过Zeocin抗性筛选逐步淘汰未成功转化的Sf9细胞。【结果】成功改造AcMNPV的, fp25k基因的TTAA位点,得到pIZT-mfp25k重组载体。重组载体成功转染Sf9细胞,通过Zeocin抗性筛选后获得基因组中带有mfp25k的Sf9-mfp25k稳定的转基因细胞系。用AcMNPV的fp25k突变型病毒AcP2感染转基因Sf9-mfp25k昆虫细胞系与正常Sf9细胞,发现转基因Sf9-mfp25k昆虫细胞系表达的FP25K蛋白可弥补病毒, fp25k基因突变的缺陷。【结论】建立的Sf9-mfp25k转基因昆虫细胞系通过细胞表达FP25K蛋白,可以弥补因杆状病毒fp25k基因突变产生的缺陷。研究结果为构建稳定的杆状病毒 昆虫细胞表达系统提供了新途径。     

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

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

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californica, Autographa gamma, Trichoplusia ni, Rachiplusia ou, Anagrapha falcifera, Galleria mellonella, and Heliothis virescens. Alignment and phylogenetic inference from partial nucleotide sequences of three highly conserved genes (lef-8, lef-9, and polh) indicated that 45 of 74 samples contained isolates of AcMNPV, while six samples contained isolates of Rachiplusia ou multiple nucleopolyhedrovirus strain R1 (RoMNPV-R1) and 25 samples contained isolates of the species Trichoplusia ni single nucleopolyhedrovirus (TnSNPV; Alphabaculovirus). One sample from A. californica contained a previously undescribed NPV related to alphabaculoviruses of the armyworm genus Spodoptera. Data from PCR and sequence analysis of the ie-2 gene and a region containing ORF ac86 in samples from the AcMNPV and RoMNPV clades indicated a distinct group of viruses, mostly from G. mellonella, that are characterized by an unusual ie-2 gene previously found in the strain Plutella xylostella multiple nucleopolyhedrovirus CL3 (PlxyMNPV-CL3) and a large deletion within ac86 previously described in the AcMNPV isolate 1.2 and PlxyMNPV-CL3. PCR and sequence analysis of baculovirus repeated ORF (bro) genes revealed that the bro gene ac2 was split into two separate bro genes in some samples from the AcMNPV clade. Comparison of sequences in this region suggests that ac2 was formed by a deletion that fused the two novel bro genes together. In bioassays of a selection of isolates against T. ni, significant differences were observed in the insecticidal properties of individual isolates, but no trends were observed among the AcMNPV, TnSNPV, or RoMNPV groups of isolates. This study expands on what we know about the variation of AcMNPV, AcMNPV-like and TnSNPV viruses, provides novel information on the distinct groups in which AcMNPV isolates occur, and contributes to data useful for the registration, evaluation, and improvement of AcMNPV, AcMNPV-like, and TnSNPV isolates as biological control agents.     

Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus,is a viral pathogenicity factor

Viral envelope fusion proteins are important structural proteins that mediate viral entry and may affect or determine the host range of a virus. The acquisition, exchange, and evolution of such envelope proteins may dramatically affect the success and evolutionary divergence of viruses. In the family Baculoviridae, two very different envelope fusion proteins have been identified. Budded virions of group I nucleopolyhedroviruses (NPVs) such as the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), contain the essential GP64 envelope fusion protein. In contrast group II NPVs and granuloviruses have no gp64 gene but instead encode a different envelope protein called F. F proteins from group II NPVs can functionally substitute for GP64 in gp64null AcMNPV viruses, indicating that GP64 and these F proteins serve a similar functional role. Interestingly, AcMNPV (and other gp64-containing group I NPVs) also contain an F gene homolog (Ac23) but the AcMNPV F homolog cannot compensate for the loss of gp64. In the present study, we show that Ac23 is expressed and is found in budded virions. To examine the function of F protein homologs from the gp64-containing baculoviruses, we generated an Ac23null AcMNPV genome by homologous recombination in E. coli. We found that Ac23 was not required for viral replication or pathogenesis in cell culture or infected animals. However, Ac23 accelerated the mortality of infected insect hosts by approximately 28% or 26 h. Thus, Ac23 represents an important viral pathogenicity factor in larvae infected with AcMNPV.     

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

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

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

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

Pseudotyping Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV): F proteins from group II NPVs are functionally analogous to AcMNPV GP64

GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 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 MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) 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 AcMNPV 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 LdMNPV or SeMNPV 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 AcMNPV 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.     

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

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

Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K gene

38K (ac98) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of 38K in the baculovirus life cycle, a 38K knockout bacmid containing the AcMNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a 38K repair bacmid was constructed by transposing the 38K open reading frame with its native promoter region into the polyhedrin locus of the 38K knockout bacmid. After transfection of these viruses into Spodoptera frugiperda cells, the 38K knockout bacmid led to a defect in production of infectious budded virus, while the 38K repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Slot blot analysis indicated that 38K deletion did not affect the levels of viral DNA replication. Subsequent immunoelectron-microscopic analysis revealed that masses of electron-lucent tubular structures containing the capsid protein VP39 were present in cells transfected with 38K knockout bacmids, suggesting that nucleocapsid assembly was interrupted. In contrast, the production of normal nucleocapsids was restored when the 38K knockout bacmid was rescued with a copy of 38K. Recombinant virus that expresses 38K fused to green fluorescent protein as a visual marker was constructed to monitor protein transport and localization within the nucleus during infection. Fluorescence was first detected along the cytoplasmic periphery of the nucleus and subsequently localized to the center of the nucleus. These results demonstrate that 38K plays a role in nucleocapsid assembly and is essential for viral replication in the AcMNPV life cycle.     

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin

Antimicrobial peptides are generated in insects exposed to pathogens for combating infection. Gloverin is a small cationic antibacterial protein whose expression is induced in the hemocytes and fat body cells of Trichoplusia ni larvae exposed to bacteria. The purpose of this study was to determine the role of gloverin during baculovirus infection. We found that gloverin expression is induced in T. ni systemically infected with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV). Two gloverin genes were cloned using RNA isolated from the hemocytes of T. ni larvae that were systemically infected with AcMNPV budded virus (BV) and C-terminal 6x-His and V5 epitope tags were incorporated to facilitate gloverin isolation, detection and functional studies. The supernatants of Sf9 cells stably transfected with the two gloverin expression plasmids and affinity purified gloverin proteins reduced the quantity of infectious AcMNPV BV as measured in vitro by plaque assay with untransfected Sf9 cells. Nanomolar concentrations of affinity column purified gloverin protein caused calcein to be rapidly released from unilamellar vesicles comprised of phosphatidylglycerol, but not from vesicles made up of phosphatidylcholine, suggesting that gloverin interaction with membranes is rapid and affected by membrane charge. Both the BV inactivation and calcein release activities of gloverin increased with higher concentrations of gloverin. These results demonstrate that gloverin is an antiviral protein that interacts with vesicle membranes to cause the contents to be released.     

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

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