Garwes D J, Bountiff L, Millson G C, Elleman C J
Adv Exp Med Biol. 1984;173:79-93. doi: 10.1007/978-1-4615-9373-7_7.
During a search for established cell lines to produce large quantities of porcine transmissible gastroenteritis virus (TGEV), we observed bright immunofluorescent staining 6- 12h after infection of pig kidney derived LLC-PK1 line. Infectious virus yield was, however, 2 log10 lower than that from secondary adult pig thyroid (APT/2) cell cultures, although small plaques were visible by three days in cultures maintained under agarose, suggesting limited replication. Attempts to adapt TGEV to the LLC-PK1 cell line by 10 serial 20h passes were unsuccessful. Procedures to purify virions from infected LLC-PK1 cells produced less than 1% of the particles isolated from parallel APT/2 cultures. Examination of intracellular viral RNA in actinomycin-D treated cells revealed similar amounts of genomic RNA and the 4 major subgenomic species in both cell types, suggesting that there was no defect in viral RNA replication. In vitro translation of polyadenylated RNA from infected APT/2 and LLC-PK1 cells, followed by immune precipitation of the products, showed similar profiles of precursors to structural polypeptides, confirming the functional integrity of the viral messengers in the restrictive cell. Comparison of the viral polypeptides synthesised following infection of the two cell types showed that similar species were synthesised in both, corresponding to a group of 28-30,000 mol. wt. envelope glycopolypeptides, a 47,000 mol. wt. nucleoprotein and peplomer glycopolypeptides of about 200,000 mol. wt. The rate of viral polypeptide synthesis in LLC-PK1 cells was reproducibly higher than in APT/2, resulting in the earlier detection of bands and greater incorporation of isotope. Tunicamycin at 1 microgram/ml had a similar effect in both cells, preventing glycosylation of the 26,000 mol. wt. precursor of the envelope glycopolypeptides and synthesis of the 200,000 peplomer glycoprotein. Degradation of the nucleoprotein from 47,000 to 42,000 mol. wt. although detectable in both cells was more marked in the LLC-PK1 cultures. Phosphorylation of these proteins was readily demonstrated in both cells, although phosphorylation of host proteins and, to some extent, viral envelope proteins was considerably greater in the LLC-PK1. The significance of this finding with respect to virus maturation is being investigated.
在寻找能够大量生产猪传染性胃肠炎病毒（TGEV）的既定细胞系的过程中，我们观察到猪肾源LLC - PK1细胞系在感染后6 - 12小时出现明亮的免疫荧光染色。然而，传染性病毒产量比二代成年猪甲状腺（APT/2）细胞培养物低2个对数10，尽管在琼脂糖下培养三天后可见小斑块，这表明病毒复制有限。通过连续10次20小时传代试图使TGEV适应LLC - PK1细胞系未成功。从感染的LLC - PK1细胞中纯化病毒粒子的程序所产生的粒子不到从平行APT/2培养物中分离粒子的1%。对放线菌素 - D处理的细胞内病毒RNA的检测显示，两种细胞类型中基因组RNA和4种主要亚基因组种类的量相似，这表明病毒RNA复制没有缺陷。对感染的APT/2和LLC - PK1细胞中聚腺苷酸化RNA进行体外翻译，然后对产物进行免疫沉淀，结果显示结构多肽前体的图谱相似，证实了在限制性细胞中病毒信使的功能完整性。比较两种细胞类型感染后合成的病毒多肽表明，两种细胞中合成的种类相似，对应一组分子量为28 - 30,000的包膜糖多肽、一种分子量为47,000的核蛋白以及分子量约为200,000的纤突糖多肽。LLC - PK1细胞中病毒多肽的合成速率始终高于APT/2细胞，导致条带更早被检测到且同位素掺入量更大。1微克/毫升的衣霉素在两种细胞中具有相似的作用，可阻止分子量为26,000的包膜糖多肽前体的糖基化以及分子量为200,000的纤突糖蛋白的合成。核蛋白从分子量47,000降解到42,000，虽然在两种细胞中都可检测到，但在LLC - PK1培养物中更明显。尽管宿主蛋白以及在某种程度上病毒包膜蛋白的磷酸化在LLC - PK1细胞中明显更高，但在两种细胞中都很容易证明这些蛋白的磷酸化。正在研究这一发现对于病毒成熟的意义。
