Davis A R, Nayak D P, Lofgren J
J Virol. 1978 Jun;26(3):603-14. doi: 10.1128/JVI.26.3.603-614.1978.
Treatment of normal guinea pig embryo cells with 5-bromodeoxyuridine (BUdR) activates endogenous guinea pig retrovirus. In this report the effect of BUdR treatment upon the level of endogenous retroviral RNA in normal guinea pig embryo cells was determined by using hybridization of viral complementary DNA (cDNA) to cellular RNA. We found that 0.0075% (120 copies per cell) of total RNA of untreated cells was virus-specific, whereas 0.32% (5,120 copies per cell) of total cellular RNA obtained from cells 48 h after BUdR treatment was virus-specific. Thus, BUdR causes an approximately 40-fold amplification of virus-specific RNA after 48 h of treatment. Several lines of evidence favor the hypothesis that the amplification of virus-specific RNA observed after BUdR treatment involves enhancement of transcription rather than an alteration of post-transcriptional processing. At different times after BUdR treatment, similar increases in virus-specific RNA concentration occur in both nucleus and cytoplasm. After 48 h of BUdR treatment, nuclear virus-specific RNA increased 99-fold, from 29 copies per cell to 2,880 copies per cell, whereas cytoplasmic virus-specific RNA increased 47-fold from 85 copies per cell to 4,000 copies per cell. Decay rates of virus-specific RNA in the presence of actinomycin D were similar in the presence or absence of BUdR, indicating that BUdR does not stabilize virus-specific RNA. In BUdR-treated cells the t1/2 of virus-specific RNA was 170 min either in the continued presence of BUdR or after the removal of BUdR, and 150 min in untreated cells. The size distribution of nuclear virus-specific RNA sequences, after denaturation with dimethyl sulfoxide, was similar in untreated and BUdR-treated cells, suggesting similar nuclear processing of viral RNA in both untreated and BUdR-treated cells. The accumulation of nuclear precursors to 38S virus-specific RNA was not observed at steady-state levels in untreated or BUdR-treated cells. Similar species of virus-specific RNA (14S 24S, 38S, and 70S) were present in the total cellular RNA of untreated and BUdR-treated cells. Additionally, virus-specific RNA was present in purified polyribosomes of untreated cells. Finally, direct analysis of the amount of radiolabeled virus-specific RNA in nuclear RNA pulse-labeled for 30 min with [3H]uridine was performed by the method of Coffin et al. (J. Mol. Biol. 86:373-396, 1977) for quantitative determination of pulse-labeled virus-specific RNA. It was found that labeled virus-specific RNA comprised 0.0035 to 0.004% of the total pulse-labeled nuclear RNA of cells treated for 48 h with BUdR. This 50-fold increase in radiolabeled virus-specific RNA may full- account for the 40-fold increase in steady-state levels of virus-specific RNA observed after 48 h of BUdR treatment.
用5-溴脱氧尿苷（BUdR）处理正常豚鼠胚胎细胞可激活内源性豚鼠逆转录病毒。在本报告中，通过病毒互补DNA（cDNA）与细胞RNA杂交，确定了BUdR处理对正常豚鼠胚胎细胞内源性逆转录病毒RNA水平的影响。我们发现，未处理细胞的总RNA中0.0075%（每细胞120个拷贝）是病毒特异性的，而在BUdR处理48小时后获得的细胞总RNA中，0.32%（每细胞5120个拷贝）是病毒特异性的。因此，BUdR处理48小时后可使病毒特异性RNA扩增约40倍。多条证据支持这样的假说，即BUdR处理后观察到的病毒特异性RNA扩增涉及转录增强而非转录后加工改变。在BUdR处理后的不同时间，细胞核和细胞质中病毒特异性RNA浓度均出现类似增加。BUdR处理48小时后，细胞核病毒特异性RNA增加了99倍，从每细胞29个拷贝增加到2880个拷贝，而细胞质病毒特异性RNA增加了47倍，从每细胞85个拷贝增加到4000个拷贝。在放线菌素D存在的情况下，病毒特异性RNA的衰减率在有或没有BUdR时相似，表明BUdR不会使病毒特异性RNA稳定。在BUdR处理的细胞中，无论继续存在BUdR还是去除BUdR，病毒特异性RNA的半衰期均为170分钟，而未处理细胞中为150分钟。用二甲基亚砜变性后，未处理细胞和BUdR处理细胞中细胞核病毒特异性RNA序列的大小分布相似，表明未处理细胞和BUdR处理细胞中病毒RNA的细胞核加工相似。在未处理或BUdR处理的细胞中，未观察到38S病毒特异性RNA的细胞核前体在稳态水平上的积累。未处理细胞和BUdR处理细胞的总细胞RNA中存在类似种类的病毒特异性RNA（14S、24S、38S和70S）。此外，未处理细胞的纯化多核糖体中存在病毒特异性RNA。最后，采用科芬等人（《分子生物学杂志》86:373 - 396，1977）的方法，对用[³H]尿苷脉冲标记30分钟的细胞核RNA中放射性标记的病毒特异性RNA量进行直接分析，以定量测定脉冲标记的病毒特异性RNA。发现用BUdR处理48小时的细胞中，标记的病毒特异性RNA占总脉冲标记细胞核RNA的0.0035%至0.004%。放射性标记的病毒特异性RNA增加50倍可能充分解释了BUdR处理48小时后观察到的病毒特异性RNA稳态水平增加40倍的现象。
