Development of liposomes and pseudovirions with fusion activity for efficient gene delivery

For efficient gene delivery, chimeric vectors combining non-viral vectors with viral components have been developed. In particular, increasing attention has been paid to viral fusion activity. HVJ (hemagglutinating virus of Japan; Sendai virus) fuses with the cell membrane at neutral pH, and HN and F, fusion proteins of the virus, contribute to the cell fusion. For fusion-mediated gene transfer, DNA-loaded liposomes were fused with UV-inactivated HVJ to form the fusion liposome, HVJ-liposome. Fusion-mediated delivery protects the molecules incorporated in the liposome from degradation in endosomes and lysosomes before reaching the cytoplasm. Reconstituted pseudovirions of fusion-competent viruses such as HVJ and influenza virus have been also developed by a detergent-lysis and-removal method. A more direct and practical approach is the conversion of fusion-competent virions to non-viral gene delivery particles. Based on this concept, the HVJ envelope vector was developed using inactivated particles of HVJ and has been utilized for gene therapy experiments and functional screening for therapeutic genes. A tissue-targeting HVJ envelope vector was also constructed.     

Construction and characterization of expression systems for the env gene of ts1, a mutant of Moloney murine leukemia virus-TB

A temperature-sensitive mutant of the Moloney murine leukemia virus-TB, ts1, causes hindlimb paralysis and immunodeficiency in mice. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly, and this is associated with the neurovirulence of ts1. To test the hypothesis that expression of the envelope proteins of ts1 alone without infectious virus production can induce paralysis, it is necessary to use either transmissible retroviral expression vectors or microinjection of eukaryotic gene expression plasmid to introduce the env gene of ts1 into germlines of mice. In this study, we have constructed three retrovirus vectors and three gene expression plasmids, all of which contain the env gene of ts1. By comparing the different expression systems, we found that one construct, pts1-env(F) can express the envelope proteins at a level comparable to the level expressed in ts1-infected cells. Furthermore, the expressed envelope proteins of pts1-env(F)-transfected cells possess the phenotypes of the proteins expressed by the env gene of ts1.     

Toll-like receptor 4 is not involved in host defense against respiratory tract infection with Sendai virus

Toll-like receptors (TLR) induce innate immune responses upon stimulation by a wide variety of pathogens. TLR4 has been implicated in innate immunity against respiratory syncytial virus (RSV) by an interaction with the viral envelope fusion (F) protein. Sendai virus (mouse parainfluenza type 1) shares many features with RSV, including a structurally and functionally similar F protein. To determine the role of TLR4 in host defense against Sendai virus respiratory tract infection, TLR4 mutant and wildtype mice were intranasally infected with Sendai virus. Sendai infection resulted in an increase in viral RNA copies in lung homogenates peaking on day 4. Pulmonary viral loads, histopathology, cytokine levels and leukocyte influx were similar in TLR4 mutant and wildtype mice. In spite of the structural similarities shared by the F proteins of Sendai virus and RSV, TLR4 is not involved in host defense against respiratory tract infection with Sendai virus.     

Improved retroviral packaging lines derived from spleen necrosis virus

Using highly efficient gene expression vectors, we constructed new retroviral packaging lines derived from spleen necrosis virus. Core proteins are expressed from the murine leukemia virus promoter and enhancer followed by the tripartite leader sequence of an adenovirus. Using different plasmids for envelope expression, we found that the efficiency of vector transduction is dependent on the level of gag-pol expression. The level of envelope expression did not have a measurable impact on vector virus titers. The new helper cell lines do not contain any sequences homologous to vector genomes. They transduce standard retrovirus vectors with titers up to 10(6) colony forming units per milliliter of supernatant tissue culture medium. No replication-competent virus was observed.     

Efficient propagation of single gene deleted recombinant Sendai virus vectors

Recombinant Sendai virus vectors (SeVV) have become an attractive tool for basic virological as well as for gene transfer studies. However, to (i) reduce the cellular injury induced by basic recombinant SeV vectors (encoding all six SeV genes as being present in SeV wild-type (wt) genomes) and to (ii) improve SeV vector safety, deletions of viral genes are necessary for the construction of superior SeVV generations. As a strong expression system recombinant replication-incompetent adenoviruses, coding for SeV proteins hemagglutinin-neuraminidase (HN), fusion (F), or matrix (M), were generated and successfully employed for the propagation of single gene deleted (DeltaHN, DeltaF, DeltaM) recombinant SeVV. Further investigations of the propagation procedures required for single gene deleted recombinant SeVV demonstrated (i) modifications of the cell culture medium composition as well as (ii) incubation with vitamin E as crucial steps for the enhancement of SeVV-DeltaHN, -DeltaF, or -DeltaM viral particle yield. Such optimized propagation procedures even led to a successful propagation of HN-deleted viral particles (SeVV-DeltaHN), which has not been reported before.     

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.     

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA. Starting with genomic negative sense (?) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low. Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (?)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (?)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5′ ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein. Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (?)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.     

Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes

The Sendai virus ribonucleoprotein (RNP) showed only very low plaque-forming titers upon transfection and the virus yields after one-step growth were quite limited. We tried to enhance the Sendai virus yield by supplying the viral L and P/C gene products through vaccinia vectors. A combination of the recombinant vaccinia viruses carrying the L gene (Vac-HL) and the P/C gene (Vac-HPC), both of which were driven by the promoter of the vaccinia virus 7.5K protein gene, enhanced the yield only a little whereas another combination of Vac-HLd7.5, the L gene insert of which was driven by the promoter of the vaccinia virus thymidine kinase gene in place of the 7.5K promoter, and Vac-HPC greatly enhanced the Sendai virus yield. This seemed to correlate with the fact that the Vac-HL interfered with Sendai virus growth markedly while the Vac-HLd7.5 did not. These results strongly suggest that the L and P/C gene products act in cooperation as the RNA polymerase, and overproduction of the L protein is inhibitory for Sendai virus growth. This system seems to be of value as a tool for analyzing the functions of L and P/C genes of Sendai virus.     

The fate of protein subunits of parainfluenza (Sendai) virus after adsorption to NIL8 hamster embryo cells.

Adsorption of u.v.-inactivated Sendai virus on to NIL8 hamster cells causes fusion of the cells into polykaryocytes within 2 h. "Infected" cells were incubated at 37 degrees C for periods of 10 min to 8 h and their surface proteins iodinated with [125I] catalysed by peroxidase. Structural components of the viral envelope, such as haemagglutin-neuraminidase (HN) and probably also the fusion protein (F) were detected in the cell membrane for periods up to 4 h post infection.     

Generation of replication-defective helper-free vectors based on simian immunodeficiency virus

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial beta-galactosidase gene (beta-gal). These vectors had a technical difficulty: "pseudotransduction" of beta-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10(6)/ml.     

麻疹病毒血凝素和融合蛋白基因的克隆与表达及其重组蛋白的免疫学评价

采用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）的方法从麻疹病毒Ｅｄｍｏｎｓｔｏｎ株基因组中扩增出血凝素Ｈ基因和融合蛋白Ｆ基因，并利用转移质粒将这两个基因分别重组到杆状病毒多角体蛋白启动子（ＰＨ）控制之下，获得重组病毒ｖＢＭＶＨ和ｖＢＭＶＦ。重组杆状病毒感染Ｓｆ９昆虫细胞，表达的重组蛋白分别具有血凝、血溶活性。红细胞吸附抑制试验、免疫印迹、酶联免疫试验结果显示重组蛋白在生物学、生化学特性与天然蛋白类似，并能被天然麻疹免疫血清（人、鼠、兔）所识别。动物实验表明，重组蛋白具有诱生中和抗体的能力。重组血凝素免疫血清还具有血凝抑制抗体活性。这些结果说明杆状病毒－昆虫细胞体系表达的麻疹病毒血凝素和融合蛋白具有较好的生物学活性及免疫原性和抗原性。     

In vivo induction of cellular and humoral immune responses by hybrid DNA vectors encoding simian/human immunodeficiency virus/hepatitis B surface antigen virus particles in BALB/c and HLA-A2-transgenic mice

To improve the immunogenicity of epitopes derived from Gag proteins of simian immunodeficiency virus (SIV) and from the envelope (Env) protein of human immunodeficiency virus type 1 (HIV-1), we have designed hybrid DNA vaccines by inserting sequences encoding antigenic domains of SIV and HIV-1 into the hepatitis B virus envelope gene. This gene encodes the hepatitis B surface antigen (HBsAg) capable of spontaneous assembly into virus-like particles that were used here as carrier. Injections of hybrid vectors encoding B-cell epitopes from the gp41 and the gp120 envelope proteins of HIV-1 induced specific humoral responses in BALB/c mice. Furthermore, high frequencies of IFN-gamma-secreting CD8+ T cells specific for various antigenic determinants of SIV-Gag were observed after intramuscular injections of hybrid DNA vectors in BALB/c mice. Genetic immunization of HLA-A2.1-transgenic mice with HIV-Env/HBsAg-encoding DNA generated a strong CTL response and IFN-gamma-secreting CD8+ T lymphocytes specific for HIV-1 envelope-derived peptide. H-2d-restricted HBs-specific T-cell responses dominated over SIV-Gag responses in BALB/c mice whereas HLA-A2-restricted HIV-Env response was enhanced after fusion with HBsAg. These data demonstrate that different B and T-cell epitopes of vaccine-relevant viral antigens can be expressed in vivo as fusion proteins with HBsAg but that the optimal immunogenicity may differ strikingly between individual epitopes.     

Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer

A reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamin e (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published.     

登革病毒4型E蛋白的表达与多克隆抗体的制备

 目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白III区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体。方法 将登革病毒4型E蛋白与E蛋白III区编码序列克隆到pET-32a（+）质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测。分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白III区的多克隆抗体,并对其进行Western blot鉴定。结果 登革病毒4型E蛋白与E蛋白III区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带。结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础。     

Chemiluminescence: an early event in the interaction of Sendai and influenza viruses with mouse spleen cells. I. The role of the envelope glycoproteins in the stimulation of chemiluminescence

On infection with Sendai virus, non-adherent mouse spleen cells emit a burst of chemiluminescence (CL) starting within a few seconds and peaking at 6–8 min postinfection. The biological reactions leading to CL are not known in mouse spleen cells, but in phagocytic cells are believed to be correlated with the generation of unstable oxygen species by the cells (e.g., H₂0₂, 0₂^?, OH·, singlet oxygen). In this paper, we have investigated the mechanism of CL induction by the virus. Envelope particles, possessing the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins stimulated CL, suggesting that the biochemical reactions leading to light emission are triggered by the interaction of the envelope “spike” glycoproteins with the cell surface. The individual contributions of HN and F to CL stimulation were investigated by removing F from egg-grown virus and by using Sendai virus (grown in MDBK cells) which possesses F₀, the biologically inactive precursor of F. Both viral preparations still induced CL. However, CL was reduced and the peak of light emission shifted from 6–8 to 2.5 min postinfection. In MDBK cell-grown Sendai virus, cleavage of F₀ into F resulted in the increase of CL and shift back of the peak CL to 6–8 min postinfection. These results suggest that the bulk of light emission by the spleen cells is correlated with the action of the F protein. In addition, HN correlates with CL in the initial period following the addition of the virus to the spleen cell suspension, while F is important for the subsequent further increase in light emission. The mechanism of CL induction by the F glycoprotein was investigated using egg-grown nonhemolytic “early harvest” and hemolytic “late harvest” Sendai virus, respectively. “Early harvest” virus, known to possess F and to have fusion activity, was less active in CL induction than “late harvest” Sendai virus which expresses both the fusion and hemolytic activities. This suggests that F stimulates CL by a mechanism related to hemolysis, rather than by envelope-cell membrane fusion. In addition, we show that influenza virus (strain A/RI/5-) also induces CL. The kinetics and extent of CL induction by this virus were similar to those induced by Sendai virus lacking the fusion protein and by nonhemolytic Sendai virus possessing F. As with Sendai virus, Pronase treatment resulted in the loss of CL stimulation while uv-inactivation of the virus did not affect its CL-inducing activity, suggesting that influenza virus also triggers CL by a mechanism involving the envelope glycoproteins.     

Peptide mapping of neutralizing and nonneutralizing epitopes of duck hepatitis B virus pre-S polypeptide.

Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro. Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins. In this study we report the localization of all but one of these epitopes at the amino acid level. All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli. All Mabs in this study reacted with the expressed gene products in Western blots. Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs. Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene. One of the two non-neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes. Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses.     

Baculovirus display of fusion protein of Peste des petits ruminants virus and hemagglutination protein of Rinderpest virus and immunogenicity of the displayed proteins in mouse model

Recombinant Bombyx mori nucleopolyhedroviruses (BmNPV) displaying the immunodominant ectodomains of fusion glycoprotein (F) of Peste des petitis ruminants virus (PPRV) and the hemagglutinin protein (H) of Rinderpest virus (RPV), on the budded virions as well as the surface of the infected host cells have been constructed. The F and H protein sequences were inserted in-frame within the amino-terminal region of BmNPV envelope glycoprotein GP64 expressing under the strong viral polyhedrin (polh) promoter. We improved the recombinant virus selection in BmNPV by incorporating the green fluorescent protein gene (gfp) as selection marker under a separate promoter within the transfer cassette harboring the desired genes. Following infection of the insect larvae or the host-derived BmN cells with these recombinant BmNPVs, the expressed GP64 fusion proteins were displayed on the host cell surface and the budded virions. The antigenic epitopes of the recombinant proteins were properly displayed and the recombinant virus particles induced immune response in mice against PPRV or RPV.