Berberich S L, Macias M, Zhang L, Turek L P, Stoltzfus C M
Program in Genetics, University of Iowa, Iowa City.
J Virol. 1990 Sep;64(9):4313-20. doi: 10.1128/JVI.64.9.4313-4320.1990.
Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.
劳氏肉瘤病毒是一种禽逆转录病毒，可使哺乳动物细胞发生转化但不能在其中复制。为了确定RNA剪接差异在多大程度上导致了这种缺乏有效感染的情况，将源自劳氏肉瘤病毒布拉格A株的克隆前病毒DNA转染到小鼠NIH 3T3细胞中，并通过核糖核酸酶保护法，使用跨越三个主要剪接位点的串联反义核糖探针，将病毒RNA与转染的鸡胚成纤维细胞的病毒RNA进行比较。与鸡胚成纤维细胞相比，NIH 3T3细胞中的病毒RNA水平较低，但RNA的剪接效率更高。未剪接与剪接RNA水平之比的差异并非由于NIH 3T3细胞中未剪接RNA的稳定性增加所致。尽管鸡胚成纤维细胞中src和env mRNA的水平相等，但NIH 3T3细胞中剪接的病毒mRNA几乎全是src。在NIH 3T3细胞中，env mRNA通过使用位于env编码序列内的隐蔽5'剪接位点和正常的src 3'剪接位点进行进一步加工，形成双剪接mRNA。该mRNA与src mRNA相同，只是在src剪接连接处插入了一段来自env基因5'端的159个核苷酸的序列。还存在少量单剪接RNA，其中仅剪接掉了隐蔽5'和src 3'剪接位点之间的区域。NIH 3T3细胞中病毒env mRNA的异常加工可能部分解释了这些细胞对劳氏肉瘤病毒有效感染的不敏感性。
