Miyakawa Kei, Fujita Rika, Katane Masumi, Kubo Yoshinao, Amanuma Hiroshi
Discovery Research Institute, RIKEN, Wako, Saitama 351-0192, Japan.
Department of AIDS Research, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.
J Gen Virol. 2008 Dec;89(Pt 12):3137-3143. doi: 10.1099/vir.0.2008/006031-0.
There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1alpha) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 10(4) c.f.u. ml(-1). Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 10(6) c.f.u. ml(-1). The SDF-1alpha ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3-51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.
人们已经进行了各种尝试来改变鼠白血病病毒载体的细胞进入受体嗜性。我们最近报道了嗜亲性莫洛尼鼠白血病病毒载体的成功重新靶向。该载体（S3-D84K）包含一种病毒包膜（Env）蛋白，在其第79位脯氨酸处插入了全长（68个氨基酸）的基质细胞衍生因子-1α（SDF-1α）。S3-D84K载体通过CXC趋化因子受体4（CXCR4）转导某个人类细胞系，滴度约为10⁴ c.f.u. ml⁻¹。在此，发现S3-D84K载体通过CXCR4转导另一个人类细胞系，滴度接近10⁶ c.f.u. ml⁻¹。S3-D84K Env蛋白的SDF-1α配体以不同方式进行了修饰。一种是进行C末端截短（截短3 - 51个氨基酸），同时有或没有从半胱氨酸到甘氨酸的改变；另一种是在不截短的情况下对形成二硫键的半胱氨酸进行从半胱氨酸到丙氨酸的改变。检测了7种截短和3种丙氨酸突变嵌合Env蛋白的病毒体掺入情况，并检测了展示突变蛋白的逆转录病毒载体的CXCR4结合和重新靶向转导情况。两种突变载体通过CXCR4的转导滴度不高于S3-D84K载体，而其他突变载体由于嵌合Env蛋白的病毒体掺入缺陷或无法与CXCR4结合，通过CXCR4对细胞的转导作用极小。这些结果表明，作为靶向配体，可能折叠成嵌合Env蛋白内一个独特结构域的全长序列是更可取的。
