应用假病毒技术研究HIV-1复制抑制剂

建立以HIV-1复制为靶点的细胞水平假病毒药理筛选模型，并运用该模型筛选化合物库中随机选取的化合物。细胞水平的HIV-1假病毒药理筛选模型是应用水泡性口膜炎病毒的外壳糖蛋白(vesicular stomatitis virus glycoprotein，VSV-G)包装HIV-1核心建立的，简称VSVG/HIV模型。细胞被VSVG/HIV感染后，病毒携带的报告基因的表达水平(荧光素酶活性或GFP阳性细胞比例)反映了HIV-1的复制水平。对HIV-1复制有抑制作用的化合物应用VSVG/MLV模型对其特异性地进一步检测。被感染细胞中报告基因的表达水平与病毒稀释度呈显著的剂量依赖效应。阳性药物齐多夫定(zidovudine，AZT)、拉米夫定(lamivudine，3TC)、去羟基苷(didanosine，DDI)可剂量依赖性地抑制HIV-1的复制，其IC₅₀分别为48.5 nmol·L^-1、 0.13 μmol·L^-1和1.73 μmol·L^-1，与文献报道一致。在随机筛选的500个化合物中有3个化合物可以抑制HIV-1的复制，IC₅₀分别为1.92、 5.38和3.39 μmol·L^-1，而对MLV的复制无影响。VSVG/HIV假病毒系统是一种安全、有效的针对HIV-1复制的药理筛选模型。当将此模型与VSVG/MLV模型联合使用时，可判断化合物作用的特异性。实验表明：3个化合物，2-甲硫基-5-(4-甲基苯并)酰胺基-1,3,4-噻二唑(化合物A)、N-(3-羟基苯基)-2-(4-异丁基苯基)丙酰胺(化合物B)和N-(4-甲基吡啶基)-4-甲基苯磺酰胺(化合物C)可以特异性抑制HIV-1的复制。     

Lack of MERS Coronavirus Neutralizing Antibodies in Humans,Eastern Province,Saudi Arabia

We used a lentiviral vector bearing the viral spike protein to detect neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) in persons from the Eastern Province of Saudi Arabia. None of the 268 samples tested displayed neutralizing activity, which suggests that MERS-CoV infections in humans are infrequent in this province.     

Retrograde Transgene Expression via Neuron-Specific Lentiviral Vector Depends on Both Species and Input Projections

For achieving retrograde gene transfer, we have so far developed two types of lentiviral vectors pseudotyped with fusion envelope glycoprotein, termed HiRet vector and NeuRet vector, consisting of distinct combinations of rabies virus and vesicular stomatitis virus glycoproteins. In the present study, we compared the patterns of retrograde transgene expression for the HiRet vs. NeuRet vectors by testing the cortical input system. These vectors were injected into the motor cortex in rats, marmosets, and macaques, and the distributions of retrograde labels were investigated in the cortex and thalamus. Our histological analysis revealed that the NeuRet vector generally exhibits a higher efficiency of retrograde gene transfer than the HiRet vector, though its capacity of retrograde transgene expression in the macaque brain is unexpectedly low, especially in terms of the intracortical connections, as compared to the rat and marmoset brains. It was also demonstrated that the NeuRet but not the HiRet vector displays sufficiently high neuron specificity and causes no marked inflammatory/immune responses at the vector injection sites in the primate (marmoset and macaque) brains. The present results indicate that the retrograde transgene efficiency of the NeuRet vector varies depending not only on the species but also on the input projections.     

Efficient Pseudotyping of Different Retroviral Vectors Using a Novel,Codon-Optimized Gene for Chimeric GALV Envelope

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C_4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C_4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 10⁶ 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.     

Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer

Background/Purpose

Postnatal gene transfer in respiratory epithelium has been inefficient, particularly in submucosal gland cells, the target cells for cystic fibrosis transmembrane regulator (CFTR) gene transfer in cystic fibrosis. The authors hypothesized that fetal tracheobronchial epithelium may be more receptive to gene transfer in that precursor cells of the respiratory epithelium may be more accessible.

Methods

Vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vector was first tested in human fetal tracheal organ culture then validated in a human fetal tracheal xenograft model in severe combined immunodeficiency (SCID) mice. Between 7 × 10⁷ and 1.6 × 10⁸ transducing units of lentiviral vector encoding the transgene LacZ under the control of the human CMV promoter were administered onto the lumenal surface of the xenografts (n = 6). Biopsy specimens were taken from the xenografts at one month (n = 1), 2 months (n = 2), 3 months (n = 1), 6 months (n = 4), and 9 months (n = 1) after vector administration. Analysis of transgene expression was performed on X-gal stained sections.

Results

Transgene expression was observed in 20.2% to 99% of the surface epithelial cells (mean, 70.8 ± 32.3% SD) and in 28.4% to 99% of the submucosal gland cells (mean, 68.5 ± 26.2% SD) out to 9 months after vector administration in the tracheal xenografts, equivalent to 63 weeks postconceptual age. No staining was seen in the controls.

Conclusions

Excellent gene transfer in human fetal tracheal xenografts after VSV-G pseudotyped lentiviral vector administration, which may result from more accessible target precursor cells during development, suggests the feasibility of fetal gene therapy for the treatment of congenital airway disease such as cystic fibrosis.     

Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus

Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.     

Prospects for gene therapy using haemopoietic stem cells

Gene therapy has thus far promised much and delivered little. Much of this has been due to deficiencies in the reagents and methodologies employed in early clinical trials. Recent technological advances in vectors and haemopoietic stem cell manipulation, coupled with improved pre-clinical assays of gene transfer and expression in re-populating stem cells give cause for greater optimism. Here we review these advances and indicate areas requiring further development before clinical gene therapy in the haemopoietic system becomes a widely applicable treatment modality.     

A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type– and Rab11-FIP1C–dependent incorporation into virions

Lentiviruses such as HIV-1 encode envelope glycoproteins (Env) with long cytoplasmic tails (CTs) that include motifs mediating interactions with host-cell–trafficking factors. We demonstrated recently that Rab11-family interacting protein 1C (FIP1C) is required for CT-dependent incorporation of Env into HIV-1 particles. Here, we used viruses bearing targeted substitutions within CT to map the FIP1C-dependent incorporation of Env. We identified YW₇₉₅ as a critical motif mediating cell-type–dependent Env incorporation. Disruption of YW₇₉₅ reproduced the cell-type–dependent particle incorporation of Env that had previously been observed with large truncations of CT. A revertant virus bearing a single amino acid change near the C terminus of CT restored wild-type levels of Env incorporation, Gag–Env colocalization on the plasma membrane, and viral replication. These findings highlight the importance of YW₇₉₅ in the cell-type–dependent incorporation of Env and support a model of HIV assembly in which FIP1C/RCP mediates Env trafficking to the particle assembly site.Lentiviruses such as HIV encode envelope glycoproteins (Envs) with long cytoplasmic tails (CTs) of 150 amino acids or more, whereas avian and murine retroviruses generally encode CTs of 20–30 residues. The reasons for this difference are not entirely clear, but may be attributable to interactions with host-trafficking pathways that define the specificity of Env incorporation into viral particles. A large number of tyrosine- and dileucine-based motifs are present in the HIV-1 Env CT, some of which have been shown to interact with factors involved in vesicular trafficking. The membrane-proximal Yxxϕ motif (Y₇₁₂) has been well studied and serves as a docking site for the μ-subunit of the clathrin adaptor AP-2 (1, 2). Disruption of this motif enhances cell-surface Env concentration, yet somewhat paradoxically reduces Env incorporation into particles and particle infectivity (3–6). Disruption of YW₈₀₂ has also been shown to reduce Env incorporation and infectivity (5, 7). The C-terminal dileucine LL₈₅₅ motif interacts with the AP-1 (8) or AP-2 (9) clathrin adaptor proteins and plays a role in endocytosis and in determining the cell-surface levels of Env. We recently performed a systematic mutagenesis of tyrosine- and dileucine-based motifs in the Env CT that confirmed the importance of Y₇₁₂ on cell-surface levels of Env (3). This study also illuminated an important region of the CT-spanning residues 795–803, in which disruption of YW or LL motifs had dramatic effects on viral replication.In an important advance to our understanding of the role of the Env CT, Murakami and Freed demonstrated that the incorporation of Env into viral particles was cell-type–dependent and that this incorporation in most T-cell lines and macrophages requires an intact long cytoplasmic tail (10). They demonstrated that Env incorporation in 293T cells did not require the long CT, nor was the CT absolutely required for incorporation in particles produced from HeLa or MT-4 cells. However, Env incorporation into particles produced from other T-cell lines and macrophages was severely impaired by CT truncation, and productive replication of virus bearing an Env with a truncated tail was possible only in MT-4 cells. This study strongly implicated host factors in the CT-dependent incorporation of Env.We recently reported that Rab11-FIP1C (FIP1C) (also known as Rab coupling protein or RCP) and Rab14 are required for Env incorporation and that the effect of FIP1C was dependent upon the Env CT (11). This suggested to us that the cell-type–dependent findings reported by Murakami and Freed (10) may be related to FIP1C-mediated transport of Env mediated through motifs on the Env CT. To test this hypothesis, we examined a panel of viruses bearing mutations of tyrosine- and dileucine-based motifs for their ability to redistribute FIP1C to the plasma membrane. We identified YW₇₉₅ as a critical motif that is required for CT-dependent FIP1C redistribution out of the endosomal recycling compartment. Remarkably, the disruption of YW₇₉₅ completely recreated the pattern of cell-type dependence on Env incorporation previously observed with CT truncation, and FIP1C depletion had no effect on the level of incorporation of this mutant Env. A downstream second-site revertant was derived that restored Env incorporation and dependence on FIP1C for particle incorporation, suggesting that YW₇₉₅ and FIP1C mediate Env incorporation in a cell-type–specific manner.