Katane Masumi, Fujita Rika, Takao Eiko, Kubo Yoshinao, Aoki Yasunobu, Amanuma Hiroshi
Molecular Cell Science Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198, Japan.
J Gene Med. 2004 Mar;6(3):260-7. doi: 10.1002/jgm.487.
To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1.
We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated.
All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion.
Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.
为了使用逆转录病毒载体进行基因的细胞特异性递送，有必要将其受体嗜性重定向到细胞特异性受体。此前，我们报道了一种含有人类基质细胞衍生因子-1α（SDF-1α）嵌合包膜蛋白（Env）（S3）的莫洛尼鼠白血病病毒（MLV）逆转录病毒载体，它获得了通过CXCR4（SDF-1α的同源受体）转导人类细胞的能力，同时保留了通过mCAT1转导小鼠细胞的能力。
我们构建了S3 Env蛋白衍生物的表达质粒；S3-D84K含有天冬氨酸-84到赖氨酸（D84K）的替换，S3-H8R-D84K含有D84K以及额外的组氨酸-8到精氨酸的替换，S3-D84K-RY含有D84K以及额外的谷氨酰胺-227到精氨酸和天冬氨酸-243到酪氨酸的替换，这些替换被认为可以抑制组氨酸-8功能的丧失。研究了这些衍生物的细胞表达、病毒体掺入和进入功能。
所有三种衍生物都掺入了病毒体。S3-D84K载体失去了亲嗜性，但能够以10³至10⁴集落形成单位（cfu）/毫升的滴度转导表达CXCR4的人类和小鼠细胞。S3-H8R-D84K载体虽然其Env蛋白能与CXCR4结合，但未显示出转导作用。S3-D84K-RY载体通过CXCR4的转导滴度略低于S3-D84K载体。这些结果表明，S3-D84K Env蛋白的组氨酸-8残基是不可或缺的，并且可能在结合后膜融合中具有完全功能。
在莫洛尼MLV Env蛋白的脯氨酸-79处插入配体已被证明是构建直接靶向逆转录病毒载体的一种有价值的策略，因为它允许形成无亲嗜性的重定向Env蛋白，并且不会破坏必需的组氨酸-8残基的功能。
