Ehrengruber Markus U, Goldin Alan L
Department of Microbiology and Molecular Genetics, University of California, Irvine, California, USA.
J Neurovirol. 2007 Aug;13(4):353-63. doi: 10.1080/13550280701393204.
Semliki Forest virus (SFV) vectors are widely used in neurobiological studies because they efficiently infect neurons. As with any viral vector, they possess a limited cloning capacity, so infection with different SFV vectors may be required to introduce multiple transgenes into individual cells. However, this approach is limited by superinfection exclusion. The authors examined marker expression in baby hamster kidney cells, mouse cortical neurons, and rat hippocampal neurons using different fluorophore-encoding vectors that are based on the wild-type SFV4 strain and on the less cytopathic SFV4(PD) mutant, which carries two point mutations in nonstructural protein 2. For every fluorophore tested, SFV4(PD) gave higher (up to 22-fold) expression compared to SFV4. In infections using two and three different vectors, SFV4 caused relatively few multifluorescent baby hamster kidney cells when applied at 0-s, 15-min, or 2-h intervals. In contrast, SFV4(PD) permitted significantly enhanced marker coexpression, resulting in 46% doubly and 21% triply fluorescent baby hamster kidney cells, and 67% to 8% doubly fluorescent cortical and hippocampal neurons. At 15-min or 2-h addition intervals, SFV4(PD) still permitted 23% to 36% doubly fluorescent baby hamster kidney cells. The increased efficiency of SFV4(PD) in coexpressing separate markers from different viral particles suggests that mutations in nonstructural protein 2 affect alphaviral superinfection exclusion. The results demonstrate that SFV4(PD) is well-suited to coexpress multiple proteins in neuronal and non-neuronal cells. This capability is particularly valuable to express the various components of heteromeric protein complexes, especially when the individual cDNAs cannot be combined into single SFV particles.
辛德毕斯病毒（SFV）载体因其能有效感染神经元而被广泛应用于神经生物学研究。与任何病毒载体一样，它们的克隆能力有限，因此可能需要用不同的SFV载体感染，才能将多个转基因导入单个细胞。然而，这种方法受到超感染排斥的限制。作者使用基于野生型SFV4毒株和细胞病变效应较小的SFV4(PD)突变体（其在非结构蛋白2中携带两个点突变）的不同荧光团编码载体，检测了幼仓鼠肾细胞、小鼠皮层神经元和大鼠海马神经元中的标记物表达。对于所测试的每种荧光团，与SFV4相比，SFV4(PD)的表达更高（高达22倍）。在使用两种和三种不同载体的感染中，当以0秒、15分钟或2小时的间隔应用时，SFV4产生的多荧光幼仓鼠肾细胞相对较少。相比之下，SFV4(PD)能显著增强标记物的共表达，产生46%的双荧光和21%的三荧光幼仓鼠肾细胞，以及67%至8%的双荧光皮层和海马神经元。在15分钟或2小时的添加间隔下，SFV4(PD)仍能产生23%至36%的双荧光幼仓鼠肾细胞。SFV4(PD)在共表达来自不同病毒颗粒的单独标记物方面效率的提高表明，非结构蛋白2中的突变影响了甲病毒的超感染排斥。结果表明，SFV4(PD)非常适合在神经元和非神经元细胞中共表达多种蛋白质。这种能力对于表达异源蛋白复合物的各种成分特别有价值，尤其是当单个cDNA不能组合成单个SFV颗粒时。
