Department of Viral and Cellular Genetics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
J Virol. 2014 Mar;88(6):3505-15. doi: 10.1128/JVI.02761-13. Epub 2014 Jan 8.
Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells.
One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.
使用禽源 Rous 肉瘤病毒（RSV）转化啮齿类动物细胞，为研究病毒在非允许细胞中的整合和表达开辟了新途径。我们感兴趣的是：（i）RSV 转化的哺乳动物细胞与禽类细胞融合以拯救病毒时，伴随的分子变化；（ii）逆转录病毒基因产物对这一过程的增强作用。RSV 转化的仓鼠 RSCh 细胞系的特征是仅产生少量的 env mRNA、无包膜糖蛋白和少量未加工的 Gag 蛋白。病毒未剪接基因组 RNA 从核内运出受阻，稳定性下降。DF-1 细胞与 RSCh 细胞融合导致 env mRNA、包膜糖蛋白和加工的 Gag 以及病毒样颗粒形成。DF-1 细胞的蛋白合成抑制抑制了拯救病毒的步骤。此外，在 RSCh 细胞中还发现了新的异常剪接 env mRNA 种类。最后，我们证明，通过补充 env 基因和高度表达的 gag 基因，可以显著提高病毒拯救效率，而通过辅助病毒感染，则可以最大程度地提高病毒拯救效率。总之，在 RSV 转化的仓鼠细胞与鸡成纤维细胞融合后，Env 和 Gag 的合成增加，并且提供的两种蛋白质均可增强 RSV 的拯救作用。我们的结论是，鸡成纤维细胞产生了一些非允许的啮齿动物细胞中 RSV 复制所需的因子，特别是 Env 和 Gag 的合成。
