DEFECTIVENESS OF AVIAN MYELOCYTOMATOSIS VIRUS MC29 - ISOLATION OF LONG-TERM NONPRODUCER CULTURES AND ANALYSIS OF VIRUS-SPECIFIC POLYPEPTIDE-SYNTHESIS
DEFECTIVENESS OF AVIAN MYELOCYTOMATOSIS VIRUS MC29 - ISOLATION OF LONG-TERM NONPRODUCER CULTURES AND ANALYSIS OF VIRUS-SPECIFIC POLYPEPTIDE-SYNTHESIS
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DOI：
10.1016/0042-6822(77)90017-4
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发表时间：
1977-01-01
期刊：
影响因子：
3.7
通讯作者：
VOGT, PK
中科院分区：
文献类型：
--
作者：
BISTER, K;HAYMAN, MJ;VOGT, PK
通过焦点和琼脂菌落测定分离了禽骨髓细胞瘤病毒 MC29-A 转化的鸡和鹌鹑细胞的非生产者 (NP) 克隆。几个鹌鹑 MC29 NP 克隆被开发成长期培养物。将它们连续培养6个月。以及大约35个段落。它们不释放可通过[3H]尿苷掺入或逆转录酶测定检测到的病毒颗粒。通过用禽白血病病毒（例如劳斯相关病毒1型或环颈雉病毒（RPV））重复感染NP细胞克隆，可以随时拯救转化病毒。通过免疫沉淀和随后的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析NP和重复感染的MC29转化细胞培养物的[35S]蛋氨酸脉冲标记的蛋白质提取物。在 NP 细胞中，不合成禽类 RNA 肿瘤病毒 (pr76) 结构蛋白的共同前体多肽。相反，观察到了分子量为 110,000-120,000 的主要多肽 (MC29-110K)。在重复感染的细胞中，MC29-110K 和 pr76 均被合成。 MC29-110K 多肽通过抗全病毒（PR RSV-B [布拉格毒株劳斯肉瘤病毒 B 亚组]）的抗血清和单特异性抗 p27 血清沉淀。它不被抗糖蛋白血清沉淀。脉冲追踪实验表明，MC29-110K 多肽的翻转速度与 pr76 相当。追踪后没有检测到主要结构蛋白（p27、p19、p15）。竞争性放射免疫测定表明，NP MC29 细胞的蛋白质提取物对 p19 和 p27 沉淀具有抑制活性，但对 p15 没有抑制活性。
Nonproducer (NP) clones of chicken and quail cells transformed by avian myelocytomatosis virus MC29-A were isolated in focus and agar colony assays. Several quail MC29 NP clones were developed into long-term cultures. They were kept in continuous culture for 6 mo. and about 35 passages. They do not release virus particles detectable by [3H]uridine incorporation or reverse transcriptase assay. Rescue of transforming virus is possible at any time by superinfection of the NP cell clones with avian leukosis viruses such as Rous associated virus type 1 or ring-necked pheasant virus (RPV). [35S]Methionine pulse-labeled protein extracts of NP and of superinfected MC29 transformed cell cultures were analyzed by immune precipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NP cells the common precursor polypeptide for the structural proteins of avian RNA tumor viruses (pr76) is not synthesized. Instead, a major polypeptide with MW of 110,000-120,000 was observed (MC29-110K). In superinfected cells, both MC29-110K and pr76 are synthesized. The MC29-110K polypeptide was precipitated by an antiserum against whole virus (PR RSV-B [Prague strain Rous sarcoma virus subgroup B]) and by a monospecific anti-p27 serum. It was not precipitated by an anti-glycoprotein serum. Pulse-chase experiments showed that the MC29-110K polypeptide turned over at a rate comparable to that of pr76. None of the major structural proteins (p27, p19, p15) could be detected after the chase. Competition radioimmunoassays demonstrated that protein extracts of NP MC29 cells contain inhibitory activity for precipitation of p19 and p27, but not for p15.