利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一,产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒,建立双病毒感染昆虫细胞以生产AAV的技术体系。首先,进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增,并检测杆状病毒滴度及其感染细胞的效果;然后,使用双杆状病毒共感染昆虫细胞,并优化感染条件;最后,基于优化条件进行AAV生产,并检测评估产量、质量等相关指标。结果表明,AAV生产用多基因缺失型杆状病毒滴度较野生型无差异,感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产,Bac4.0-1的基因组滴度为1.63×10¹¹ VG/mL,Bac5.0-2的基因组滴度为1.02×10¹¹ VG/mL,较野生型产量分别提升了240%和110%。电镜下,3组均具有正常的AAV病毒形态,且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系,显著提高了AAV产量,具有一定的应用价值。     

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.     

RGD motifs on the surface of baculovirus enhance transduction of human lung carcinoma cells

Baculovirus vectors have been shown to enter a variety of mammalian cell lines and gene transfer with wild-type baculovirus (WT) has been demonstrated both in vitro and in vivo. Different protein motifs have been displayed on the viral surface to serve as ligands for cell-specific receptor molecules. We have generated recombinant baculovirus vectors displaying an RGD-motif, recognized by alphaV integrin, on the viral surface. The RGD motifs within the C-terminus of coxsackie virus A9 and human parechovirus 1 VP1 proteins were fused to the N-terminus of the major envelope glycoprotein, gp64, of Autographa californica multiple nucleopolyhedrovirus. The recombinant RGD-presenting viruses bound more efficiently to the surface of human lung carcinoma cells (A549), known to contain alphaV integrins, as compared to WT baculovirus. In addition, the binding pattern of the RGD-displaying baculovirus showed extensive clustering. This most likely represents clustering of the integrin molecules on the cell surface, induced by binding of the RGD-displaying baculovirus. Finally, the transduction efficiency of an RGD-representing virus increased by almost three-fold as monitored by light emission measurements. In conclusion, these results suggest that the RGD-motif is functional on the surface of baculovirus and thereby these tropism-modified viruses bind more efficiently as well as enhance the transduction efficiency of human cancer cells expressing alphaV integrins.     

Stable expression of mammalian beta 1,4-galactosyltransferase extends the N-glycosylation pathway in insect cells

An established lepidopteran insect cell line (Sf9) was cotransfected with expression plasmids encoding neomycin phosphotransferase and bovine beta 1,4-galactosyltransferase. Neomycin-resistant transformants were selected, assayed for beta 1,4-galactosyltransferase activity, and the transformant with the highest level of enzymatic activity was characterized. Southern blots indicated that this transformed Sf9 cell derivative contained multiple copies of the galactosyltransferase- encoding expression plasmid integrated at a single site in its genome. One-step growth curves showed that these cells supported normal levels of baculovirus replication. Baculovirus infection of the transformed cells stimulated beta 1,4-galactosyltransferase activity almost 5-fold by 12 h postinfection. This was followed by a gradual decline in activity, but the infected cells still had about as much activity as uninfected controls as late as 48 h after infection and they were able to produce a beta 1,4-galactosylated virion glycoprotein during infection. Infection of the transformed cells with a conventional recombinant baculovirus expression vector encoding human tissue plasminogen activator also resulted in the production of a galactosylated end-product. These results demonstrate that stable transformation can be used to add a functional mammalian glycosyltransferase to lepidopteran insect cells and extend their N- glycosylation pathway. Furthermore, stably-transformed insect cells can be used as modified hosts for conventional baculovirus expression vectors to produce foreign glycoproteins with "mammalianized" glycans which more closely resemble those produced by higher eucaryotes.      

Baculovirus GP64-mediated entry into mammalian cells

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.     

Increase of host cell longevity by the expression of 30K protein originating from silkworm hemolymph in an insect cell–baculovirus system

A recombinant baculovirus was constructed by the homologous recombination between wild-type AcMNPV DNA and a baculovirus transfer vector containing a gene coding for the 30K protein originating from silkworm hemolymph. The 30K protein was successfully expressed in Sf9 cells infected with the recombinant baculovirus (AcMNPV/30K). To investigate the effect produced by the expression of the 30K protein, host cell viability after infection was compared with that of Sf9 cells infected with AcMNPV/β-gal. The viability of the cells infected with AcMNPV/β-gal began to decrease exponentially 3 days after infection, whereas that of the cells infected with AcMNPV/30K remained at a high level until 5 days after infection. This indicates that the 30K protein increases cell longevity after viral infection. This increased cell longevity is considered to be due to the inhibition of host cell apoptosis induced by a baculovirus, and the extent of apoptosis was measured by the flow cytometric method. The percentage of the sub-G1 fraction, which represents the extent of apoptosis, was decreased by the expression of the 30K protein. This indicates that the expression of the 30K protein in insect cells increases host cell longevity by inhibiting apoptosis.     

pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN

The severe acute respiratory syndrome coronavirus (SARS-CoV) synthesizes several putative viral envelope proteins, including the spike (S), membrane (M), and small envelope (E) glycoproteins. Although these proteins likely are essential for viral replication, their specific roles in SARS-CoV entry have not been defined. In this report, we show that the SARS-CoV S glycoprotein mediates viral entry through pH-dependent endocytosis. Further, we define its cellular tropism and demonstrate that virus transmission occurs through cell-mediated transfer by dendritic cells. The S glycoprotein was used successfully to pseudotype replication-defective retroviral and lentiviral vectors that readily infected Vero cells as well as primary pulmonary and renal epithelial cells from human, nonhuman primate, and, to a lesser extent, feline species. The tropism of this reporter virus was similar to that of wild-type, replication-competent SARS-CoV, and binding of purified S to susceptible target cells was demonstrated by flow cytometry. Although myeloid dendritic cells were able to interact with S and to bind virus, these cells could not be infected by SARS-CoV. However, these cells were able to transfer the virus to susceptible target cells through a synapse-like structure. Both cell-mediated infection and direct infection were inhibited by anti-S antisera, indicating that strategies directed toward this gene product are likely to confer a therapeutic benefit for antiviral drugs or the development of a SARS vaccine.     

An immunological assay for determination of baculovirus titers in 48 hours

Th baculovirus expression system is a system of choice for expressing eukaryotic proteins. Large amounts of biologically active material can be generated using this system by infecting insect cells with a baculovirus expressing the target protein. At several stages during the production of a baculovirus stock, it is necessary to titer the virus. Current methods have long time lines and are either technically difficult or are limited to viruses expressing a reporter gene. The new assay described here yields titers in 48 h, is easy to perform using 96-well plates, and is applicable to any Autographa californica nucleopolyhedrovirus-based recombinant baculovirus. This assay uses an antibody to a viral envelope glycoprotein to detect infected cells via immunostaining. The titer is determined by counting foci of infection under a light microscope. The required incubation period is shortened considerably because infected cells express viral antigens long before the macroscopic signs of infection scored in other assays become apparent. Titers determined using this immunological assay are comparable, both in value and variability, to those obtained using a traditional method, provided that the stocks have titers above 10(4) pfu/ml.     

Expression of genes coding for animal virus glycoproteins in heterologous systems.

The outermost layers of animal viruses are usually composed of glycoproteins. They are responsible not only for the entrance of viruses into, and release from host cells but also for the initial interaction of a viral particle with immunological defense of the host. It is therefore not surprising that many laboratories devote a lot of effort to study viral glycoproteins at the molecular level. Very often such studies are possible only after the introduction of a glycoprotein gene into a heterologous system. Expression of glycoprotein genes is usually obtained in mammalian or insect cells. Expression in mammalian cells yields viral glycoproteins with glycan chains indistinguishable from the original counterparts in virion particles but the level of synthesis of glycoproteins is very low. Vaccinia virus is the most common vector for expression in mammalian cells. It is easy to grow, the introduction of foreign genes is relatively simple and, due to the size of the vaccinia genome, it can accept large pieces of foreign DNA. Glycosylation in insect cells is not as complex as in mammalian cells and usually glycoproteins produced in insect cells are of slightly lower molecular mass than those produced in mammalian cells. The most common vector for expression of glycoproteins in insect cells is a baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The great advantage of this system is a very high level of expression of foreign genes.     

Improved efficient gene transfer into insect and mammalian cells by baculovirus vectors

The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.     

Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.     

Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses

We previously reported that the baculovirus induced a strong host immune response against infections and malignancies. Among the immune cells, the dendritic cells were most strongly infected and activated by the baculovirus, although the exact mechanism remained unclear. Here, we evaluated the non-specific immune responses of bone marrow-derived dendritic cells (BMDCs) after infection by a wild-type baculovirus. MHC class I and II molecules and co-stimulation molecules (CD40, CD80, and CD86) on BMDCs were up-regulated by baculovirus infection. At the same time, the BMDCs produced pre-inflammatory cytokines (IL-6, IL12p70, and TNF-α) and IFN-α. NK cells showed IFN-γ production, CD69 up-regulation, and enhanced cytotoxicity when they were co-cultured with baculovirus-infected BMDCs. T cells showed IFN-γ production, CD69 up-regulation, and cell proliferation. Ex vivo analysis performed in vitro produced similar results. These findings suggested that baculovirus-infected dendritic cells induce non-specific immune responses and can be used as an immunotherapeutic agent against viral infections and malignancies, together with present therapeutic drug regimens.     

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells

Background  

Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells.     

Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids

We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.     

In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.     

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.     

Development of viral vectors and the application for viral entry mechanisms

Virus is identified as one of the obligate intracellular parasites, which only amplify in cells of specific living things. Viral vectors, which are developed by utilizing these properties, are available in the various fields such as basic research of medical biology or application of gene therapy. Our research group has studied development of viral vectors using properties of baculovirus or vesicular stomatitis virus (VSV). Due to the development of new baculoviral vectors for mammalian cells, it is possible to be more efficient transduction of foreign gene in mammalian cells and animals. Furthermore, pseudotype or recombinant VSV possessing the envelope proteins of hepatitis C virus, Japanese encephalitis virus or baculovirus were constructed, and characteristics of the envelope proteins or entry mechanisms of these viruses were analyzed.     

Engineering the protein N-glycosylation pathway in insect cells for production of biantennary,complex N-glycans

Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors.     

Infectious Epstein-Barr virus lacking major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands

The binding of the viral major glycoprotein BLLF1 (gp350/220) to the CD21 cellular receptor is thought to play an essential role during infection of B lymphocytes by the Epstein-Barr virus (EBV). However, since CD21-negative cells have been reported to be infectible with EBV, additional interactions between viral and cellular molecules seem to be probable. Based on a recombinant genomic EBV plasmid, we deleted the gene that encodes the viral glycoprotein BLLF1. We tested the ability of the viral mutant to infect different lymphoid and epithelial cell lines. Primary human B cells, lymphoid cell lines, and nearly all of the epithelial cell lines that are susceptible to wild-type EBV infection could also be successfully infected with the viral mutant in vitro, although the efficiency of infection with BLLF1-negative virus was clearly lower than the one observed with wild-type EBV. Our studies show that the interaction between BLLF1 and CD21 is not absolutely required for the infection of lymphocytes and epithelial cells, indicating that viral molecules other than BLLF1 can mediate the binding of EBV to its target cells. In this context, our results further suggest the hypothesis that additional cellular molecules, apart from CD21, allow virus entry into these cells.