Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses
Ligand-directed gene targeting to mammalian cells by pseudotype baculoviruses
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DOI：
10.1128/jvi.79.6.3639-3652.2005
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发表时间：
2005-03-01
影响因子：
5.4
通讯作者：
Matsuura, Y
中科院分区：
文献类型：
--
作者：
Kitagawa, Y;Tani, H;Matsuura, Y
加州链球绦虫多核型多角体病毒(AcMNPV)可感染多种哺乳动物细胞和昆虫细胞，便于将其作为病毒载体导入哺乳动物细胞。糖蛋白gp64是出芽的AcMNPV囊膜的主要成分，参与病毒通过受体介导的内吞作用和随后的膜融合进入细胞。我们研究了伪型杆状病毒颗粒瞬时携带感兴趣的配体而不是gp64作为一种配体导向的基因传递到靶细胞的潜在产生。然而，在将携带绿色荧光蛋白基因的gp64缺失的伪型杆状病毒在表达gp64的昆虫细胞中扩增时，我们观察到将gp64基因整合到病毒基因组中的复制能力强的病毒的高频出现。为了避免产生复制能力强的逆转株，我们用重组杆状病毒在表达gp64的细胞中不进行进一步扩增，制备了伪型杆状病毒。我们构建了携带水疱性口炎病毒G蛋白(VSVG)或麻疹病毒受体(CD46或SLAM)的cDNA的gp64零重组杆状病毒。VSVG伪型杆状病毒有效地将报告基因导入多种哺乳动物细胞系，而CD46和SLAM伪型杆状病毒则允许配体受体导向的报告基因转导表达麻疹病毒包膜糖蛋白的靶细胞。特异性抗体可抑制伪型杆状病毒介导的基因转导。这些结果表明，伪型杆状病毒可能应用于配基导向的基因导入靶细胞。
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells.