Chevrier-Miller M, Morange M, Arrigo A P, Pinto M
Ecole Normale Supérieure, CNRS-URA 1302, Paris, France.
Biochem Biophys Res Commun. 1996 Nov 21;228(3):695-703. doi: 10.1006/bbrc.1996.1719.
Bacteria and eukaryotic cells respond to cold stress by inducing and enhancing the synthesis of specific arrays of proteins. We describe here cold-induced enhancement of expression for two reporter genes; luciferase and beta-galactosidase, both under the control of HIV-1 LTR sequences, observed in mouse fibroblasts and human HeLa cells respectively. Increased expression of luciferase in fibroblasts when shifted to 25 degrees C was detectable at 30 degrees C but was not observed following cold shock at 4 degrees C. To sustain the cold-induced effect, cells had to be kept at subphysiological temperature. The observed enhancement of luciferase activity did not result from a particular site of integration of the reporter gene and was evident whether cold-stressed cells were stationary or growing. Cold-induced expression of luciferase was evidenced at the protein level, enzymatic activity and RNA level, furthermore, active transcription and translation were required for overexpression. The cold effect which has been generalized with the reporter gene beta-galactosidase appears to be a process involving, at least in part, the HIV-1 LTR sequences and might correspond to an increase in the half-life of mRNA. The cold-dependent enhanced expression of luciferase and beta-galactosidase reported here, together with data describing the activation of HIV-1 LTR by hyperthermia, point out the particular temperature sensitivity of these regulatory sequences. This potential thermal modulation may be useful in the comprehension of regulatory processes in latency and reactivation of viral expression during HIV-1 infection.
细菌和真核细胞通过诱导和增强特定蛋白质阵列的合成来应对冷应激。我们在此描述了在小鼠成纤维细胞和人HeLa细胞中分别观察到的，在HIV-1长末端重复序列（LTR）控制下的两种报告基因（荧光素酶和β-半乳糖苷酶）的冷诱导表达增强。当转移到25摄氏度时，成纤维细胞中荧光素酶表达的增加在30摄氏度时可检测到，但在4摄氏度冷休克后未观察到。为了维持冷诱导效应，细胞必须保持在亚生理温度。观察到的荧光素酶活性增强并非源于报告基因整合的特定位点，并且无论冷应激细胞是静止的还是生长的，这种增强都很明显。荧光素酶的冷诱导表达在蛋白质水平、酶活性和RNA水平上都得到了证实，此外，过表达需要活跃的转录和翻译。用报告基因β-半乳糖苷酶普遍观察到的冷效应似乎是一个至少部分涉及HIV-1 LTR序列的过程，可能对应于mRNA半衰期的增加。此处报道的荧光素酶和β-半乳糖苷酶的冷依赖性增强表达，以及描述热疗激活HIV-1 LTR的数据，指出了这些调控序列对温度的特殊敏感性。这种潜在的热调节可能有助于理解HIV-1感染期间病毒表达潜伏期和再激活的调控过程。
