de Mars M, Cizdziel P E, Murphy E C
Department of Tumor Biology, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston 77030.
J Virol. 1988 Jun;62(6):1907-16. doi: 10.1128/JVI.62.6.1907-1916.1988.
Murine sarcoma virus ts110 (MuSVts110) is a conditionally transformation-defective MuSV mutant lacking 1,487 bases found in its wild-type parent, MuSV-349 (MuSV-124). Expression of the MuSVts110 v-mos gene product, P85gag-mos, requires splicing of the viral transcript to align the gag and mos genes in frame. However, this splice event is restricted to growth temperatures of 33 degrees C or lower. No splicing of the viral RNA, no production of P85gag-mos, and, hence, no cell transformation is observed at growth temperatures above 33 degrees C. To determine whether thermosensitive splicing is an intrinsic property of To determine whether thermosensitive splicing is an intrinsic property of MuSVts110 RNA specified by the 1,487-base deletion or a result of a cellular defect, we examined an "equivalent" or MuSVts110 DNA (designated ts32 DNA) constructed by combining wild-type MuSV-124 DNA fragments with a synthetic oligonucleotide to yield an otherwise wild-type viral DNA containing the same 1,487-base deletion as authentic MuSVts110. As observed in control cells (6m2 cells) infected with the authentic MuSVts110 virus, NIH 3T3 cells transfected with ts32 DNA appeared morphologically transformed when grown at 33 degrees C, but were converted to a more normal, flattened shape within a few hours of a shift to 39 degrees C. In concert with these morphological changes, both the processing of the ts32 RNA transcripts and the production of ts32 p85gag-mos kinase were found to be optimal at growth temperatures from 28 to 33 degrees C, but dramatically reduced at 37 to 41 degrees C. Like authentic P85gag-mos, the ts32 P85gag-mos kinase activity was rapidly inactivated by brief exposure to 39 degrees C. These results suggested that the MuSVts110 equivalent is functionally indistinguishable from authentic MuSVts110 and that the novel temperature-sensitive splicing of MuSVts110 transcripts is specified by an intrinsic property of the viral RNA.
鼠肉瘤病毒ts110（MuSVts110）是一种条件性转化缺陷型MuSV突变体，在其野生型亲本MuSV - 349（MuSV - 124）中缺失1487个碱基。MuSVts110的v - mos基因产物P85gag - mos的表达需要对病毒转录本进行剪接，以使gag和mos基因读框对齐。然而，这种剪接事件仅限于33℃或更低的生长温度。在高于33℃的生长温度下，未观察到病毒RNA的剪接、P85gag - mos的产生，因此也未观察到细胞转化。为了确定热敏剪接是由1487个碱基缺失所指定的MuSVts110 RNA的固有特性还是细胞缺陷的结果，我们检查了一种“等效”的MuSVts110 DNA（命名为ts32 DNA），它是通过将野生型MuSV - 124 DNA片段与合成寡核苷酸组合构建而成的，从而产生一种在其他方面为野生型的病毒DNA，其包含与 authentic MuSVts110相同的1487个碱基缺失。正如在用 authentic MuSVts110病毒感染的对照细胞（6m2细胞）中所观察到的那样，用ts32 DNA转染的NIH 3T3细胞在33℃生长时出现形态转化，但在转移到39℃后的几小时内转变为更正常、扁平的形状。与这些形态变化一致，发现ts32 RNA转录本的加工和ts32 p85gag - mos激酶的产生在28至33℃的生长温度下最佳，但在37至41℃时显著降低。与 authentic P85gag - mos一样，ts32 P85gag - mos激酶活性通过短暂暴露于39℃而迅速失活。这些结果表明，MuSVts110等效物在功能上与 authentic MuSVts110无法区分，并且MuSVts110转录本新的温度敏感性剪接是由病毒RNA的固有特性所指定的。
