人免疫缺陷病毒假病毒药物筛选模型的构建及其应用

目的:构建人免疫缺陷病毒(HIV)假病毒模型,用多种HIV逆转录酶和蛋白酶抑制剂作用于该模型,以检测其是否能有效用于HIV抑制药物的筛选。方法:通过载体改造获得最终慢病毒载体puc18-NL4-3-LUC-stop,其中含有萤光素酶基因,将该载体与包膜质粒VSV-G共转染293FT细胞,包装产生HIV假病毒,在假病毒包装和病毒感染293FT细胞的过程中加入蛋白酶和逆转录酶抑制剂,通过检测感染细胞中萤光素酶的表达来检测该模型的有效性,并利用此模型检测药物的抗病毒效果。结果:将HIV逆转录酶和蛋白酶抑制剂作用于该假病毒模型时发现萤光素酶的表达得到很大程度的抑制。结论:建立了HIV假病毒药物筛选模型,该模型以萤光素酶基因作为报告基因,快速灵敏,在抗HIV药物筛选中有一定的应用价值。     

Vesicular Stomatitis Virus G-Pseudotyped Lentivirus Vectors Mediate Efficient Apical Transduction of Polarized Quiescent Primary Alveolar Epithelial Cells

We investigated the use of lentivirus vectors for gene transfer to quiescent alveolar epithelial cells. Primary rat alveolar epithelial cells (AEC) grown on plastic or as polarized monolayers on tissue culture-treated polycarbonate semipermeable supports were transduced with a replication-defective human immunodeficiency virus-based lentivirus vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein and encoding an enhanced green fluorescent protein reporter gene. Transduction efficiency, evaluated by confocal microscopy and quantified by fluorescence-activated cell sorting, was dependent on the dose of vector, ranging from 4% at a multiplicity of infection (MOI) of 0.1 to 99% at an MOI of 50 for AEC grown on plastic. At a comparable titer and MOI, transduction of these cells by a similarly pseudotyped murine leukemia virus vector was approximately 30-fold less than by the lentivirus vector. Importantly, comparison of lentivirus-mediated gene transfer from the apical or basolateral surface of confluent AEC monolayers (R(t) > 2 kOmega. cm(2); MOI = 10) revealed efficient transduction only when VSV-G-pseudotyped lentivirus was applied apically. Furthermore, treatment with EGTA to increase access to the basolateral surface did not increase transduction of apically applied virus, indicating that transduction was primarily via the apical membrane domain. In contrast, differentiated tracheal epithelial cells were transduced by apically applied lentivirus only in the presence of EGTA and at a much lower overall efficiency (approximately 15-fold) than was observed for AEC. Efficient transduction of AEC from the apical cell surface supports the feasibility of using VSV-G-pseudotyped lentivirus vectors for gene transfer to the alveolar epithelium and suggests that differences exist between upper and lower airways in the polarity of available receptors for the VSV-G protein.     

Characterization of Ebola Virus Entry by Using Pseudotyped Viruses: Identification of Receptor-Deficient Cell Lines

Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 × 10⁶ infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.     

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

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

Exploiting heparin-binding properties of MoMLV-based retroviral vectors for affinity chromatography

Chromatography is deemed the most promising technology for large-scale purification of viral vectors. The authors have previously shown that heparin affinity chromatography could be successfully employed for the purification of VSV-G pseudotyped Moloney murine leukemia virus (MoMLV)-derived vectors giving excellent results in terms of recovery of active particles, reproducibility and selectivity. In this study, the authors examined whether the ability of retrovirus particles to specifically bind to heparin ligands is restricted to VSV-G pseudotypes produced by 293-based packaging cells. It is shown that VSV-G deficient retrovirus particles are captured by a heparin chromatography column as efficiently as VSV-G containing particles. Most strikingly, RD114 pseudotyped retrovirus particles derived from a HT1080-based cell line were found to bind heparin with the same affinity as 293-derived VSV-G pseudotypes. RD114 pseudotyped retrovirus particles were successfully isolated using heparin affinity chromatography obtaining good recoveries of functional particles (43%). These results indicate that heparin affinity chromatography can be extended to the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping.     

Oncoretrovirus and Lentivirus Vectors Pseudotyped with Lymphocytic Choriomeningitis Virus Glycoprotein: Generation, Concentration, and Broad Host Range

Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.     

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.     

Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer

Recombinant lentiviral vectors stably transduce both dividing and nondividing cells. Virus pseudotyping with vesicular stomatitis virus envelope G (VSV-G) protein broadens the host range of lentiviral vector and enables vector concentration by ultra-centrifugation. However, as a result of virus vector concentration, contaminating protein debris derived from vector-producing cell culture media is toxic to target cells and reduces the transduction efficiency. Here we report a new and rapid technique for purifying lentivirus vector using the strong anion exchange column that significantly improves gene transfer rates. We purified VSV-G pseudotyped self-inactivating lentivirus vector and obtained two protein elution peaks (Peak 1 and Peak 2) corresponding to transducing activity. Peak 1 viral particles were 4-8 times more effective in transducing target cells than Peak 2 or non-purified (pre-HPLC) viral particles. We used purified lentivirus vector expressing the human Fanconi anemia group A (FANCA) gene to transduce murine hematopoietic stem/progenitor cells. We observed a consistent 2- to 3-fold increase in gene transfer rates using Peak 1 purified virus compared with non-purified virus. We conclude that the purification method using the HPLC system provides the highly purified virus vector that reduces cell toxicity and significantly improves gene transfer in primary cells.     

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.     

Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Tunicamycin treatment of CHO cells abrogates multiple blocks to retrovirus infection, one of which is due to a secreted inhibitor.

Chinese hamster ovary (CHO) cells are resistant to infection by all of the major classes of murine retroviruses and are partially resistant to infection by gibbon ape leukemia virus. Treatment of CHO cells with the glycosylation inhibitor tunicamycin rendered these cells susceptible to infection by retroviral vectors with ecotropic, xenotropic, and amphotropic host ranges and increased the titer of gibbon ape leukemia virus pseudotyped vectors 10-fold. Vectors having a polytropic host range did not infect CHO cells in the presence or absence of tunicamycin, showing that the effect of tunicamycin was specific and related to the pseudotype of the vector. We present evidence for three mechanisms of resistance to infection: lack of viral receptors on CHO cells, the presence of nonfunctional receptors which can be made functional by treatment with tunicamycin, and the secretion of a protein factor that blocks retroviral infection of CHO cells. Several criteria indicate that the secreted inhibitor is not an interferon, and secretion of this factor was not detected in several other cell lines that were examined.     

Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins

Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.     

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.     

Packaging cell lines for simian foamy virus type 1 vectors

Foamy viruses are nonpathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. We have previously demonstrated the utility of simian foamy virus type 1 (SFV-1) as a vector system by transient expression assay (M. Wu et al., J. Virol. 72:3451-3454, 1998). In this report, we describe the first stable packaging cell lines for foamy virus vectors based on SFV-1. We developed two packaging cell lines in which the helper DNA is placed under the control of either a constitutive cytomegalovirus (CMV) immediate-early gene or inducible tetracycline promoter for expression. Although the constitutive packaging expressing cell line had a higher copy number of packaging DNA, the inducible packaging cell line produced four times more vector particles. This result suggested that the structural gene products in the constitutively expressing packaging cell line were expressed at a level that is not toxic to the cells, and thus vector production was reduced. The SFV-1 vector in the presence of vesicular stomatitis virus envelope protein G (VSV-G) produced an insignificant level of transduction, indicating that foamy viruses could not be pseudotyped with VSV-G to generate high-titer vectors. The availability of stable packaging cell lines represents a step toward the use of an SFV-1 vector delivery system that will allow scaled-up production of vector stocks for gene therapy.     

Factors that influence VSV-G pseudotyping and transduction efficiency of lentiviral vectors-in vitro and in vivo implications

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.     

携带FMDV前导蛋白基因逆转录病毒载体的构建及其在牛肾细胞中的表达

以口蹄疫病毒株OA/58 RNA为模板, 反转录并扩增目的cDNA。通过分子克隆技术将前导蛋白编码序列Lab与逆转录病毒载体pBPSTR1连接, 将构建正确的重组载体命名为pBPSTR1-Lab。通过分别利用不同浓度的嘌呤霉素和四环素来确定最佳筛选浓度和最佳调控浓度, 结果显示嘌呤霉素的最佳筛选浓度为3 mg/mL, 四环素的最佳调控浓度为1 mg/mL。利用pBPSTR1-Lab和包装质粒pVSV-G双质粒瞬时转染Gp2-293包装细胞来获得重组逆转录病毒。利用重组逆转录病毒来感染牛肾细胞, 并连续筛选12天来获得阳性克隆。通过除去四环素来诱导目的基因在牛肾细胞中表达, 发现牛肾细胞病变死亡。经过PCR和蛋白质免疫印迹证实稳定表达前导蛋白的牛肾细胞系已经建立, 为今后研究前导蛋白致病机理提供了平台。     

携带FMDV前导蛋白基因逆转录病毒载体的构建及其在牛肾细胞中的表达

以口蹄疫病毒株OA/58 RNA为模板, 反转录并扩增目的cDNA。通过分子克隆技术将前导蛋白编码序列Lab与逆转录病毒载体pBPSTR1连接, 将构建正确的重组载体命名为pBPSTR1-Lab。通过分别利用不同浓度的嘌呤霉素和四环素来确定最佳筛选浓度和最佳调控浓度, 结果显示嘌呤霉素的最佳筛选浓度为3 mg/mL, 四环素的最佳调控浓度为1 mg/mL。利用pBPSTR1-Lab和包装质粒pVSV-G双质粒瞬时转染Gp2-293包装细胞来获得重组逆转录病毒。利用重组逆转录病毒来感染牛肾细胞, 并连续筛选12天来获得阳性克隆。通过除去四环素来诱导目的基因在牛肾细胞中表达, 发现牛肾细胞病变死亡。经过PCR和蛋白质免疫印迹证实稳定表达前导蛋白的牛肾细胞系已经建立, 为今后研究前导蛋白致病机理提供了平台。