Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
Hum Gene Ther. 2012 Mar;23(3):274-86. doi: 10.1089/hum.2011.011. Epub 2012 Jan 12.
The effective entry of retroviruses into target cells depends on the presence of viral envelope (Env) proteins and cognate cellular receptors, such as the murine cationic amino acid transporter-1 (mCAT-1) for the ecotropic murine leukemia virus (MLV-E). Here, we examined whether human cells internalize MLV-E or other retroviral pseudotypes irrespective of the presence of a specific receptor. Using fluorescently tagged Gag to monitor viral internalization, and treating cells with chloroquine or bafilomycin A1, we show that endocytosis is the main pathway for productive transduction with ecotropic particles, but endocytosis of retroviral particles itself does not depend on a suitable receptor or Env. Nonspecific endosomal uptake and lysosomal degradation occurred with all "illegitimate" envelope-receptor combinations tested: MLV particles pseudotyped with the ecotropic envelope or measles virus H and F proteins as well as "ecotropic" or "bald" HIV-1 particles. Kinetic studies in cell lines and primary human T lymphocytes showed the persistence of Gag-GFP signals for more than 10 days after exposure to retroviral vector particles, even in the absence of a suitable receptor. Further studies testing the Gag-mediated transfer of protein or retroviral mRNA revealed that nonspecific endocytosis prevented the release of functional particle-associated proteins and nucleic acids into the cytosol. We conclude that receptor-targeted retroviral particles are unlikely to escape nonspecific cellular uptake unless appropriate protective principles are discovered. Conversely, as lysosomal degradation was found to inactivate mRNA and proteins embedded into retroviral particles, receptor targeting is a useful strategy for both transient and permanent cell modification by retrovirus-like particles.
逆转录病毒有效进入靶细胞依赖于病毒包膜 (Env) 蛋白和同源细胞受体的存在，例如，用于亲嗜性鼠白血病病毒 (MLV-E) 的鼠阳离子氨基酸转运蛋白-1 (mCAT-1)。在这里，我们研究了人类细胞是否会内化 MLV-E 或其他逆转录病毒假型，而不管是否存在特定受体。我们使用荧光标记的 Gag 来监测病毒内化，并使用氯喹或巴弗洛霉素 A1 处理细胞，结果表明，胞吞作用是亲嗜性颗粒有效转导的主要途径，但逆转录病毒颗粒本身的内吞作用不依赖于合适的受体或 Env。我们测试了所有“非法”包膜-受体组合，发现非特异性内体摄取和溶酶体降解都发生了：用亲嗜性包膜或麻疹病毒 H 和 F 蛋白以及“亲嗜性”或“无包膜”HIV-1 颗粒假型化的 MLV 颗粒。在细胞系和原代人 T 淋巴细胞中的动力学研究表明，即使没有合适的受体，暴露于逆转录病毒载体颗粒后，Gag-GFP 信号持续存在超过 10 天。进一步研究测试 Gag 介导的蛋白质或逆转录病毒 mRNA 的转移表明，非特异性内吞作用阻止了功能性颗粒相关蛋白和核酸进入细胞质的释放。我们得出结论，除非发现适当的保护原则，否则受体靶向的逆转录病毒颗粒不太可能逃脱非特异性细胞摄取。相反，由于溶酶体降解发现会使嵌入逆转录病毒颗粒中的 mRNA 和蛋白质失活，因此受体靶向是通过类似于逆转录病毒的颗粒对细胞进行瞬时和永久修饰的有用策略。
