McSharry J J, Wagner R R
J Virol. 1971 Jan;7(1):59-70. doi: 10.1128/JVI.7.1.59-70.1971.
Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.
本文描述了用于生产纯度足以进行可靠化学分析的水疱性口炎（VS）病毒的方法。从感染细胞中释放的VS病毒粒子通过聚乙二醇沉淀、柱色谱、速率区带离心和平衡离心等连续步骤进行浓缩和纯化，纯化倍数至少为150倍。发现在L细胞中繁殖的印第安纳血清型（VS(Ind)病毒）含有3%的核糖核酸、64%的蛋白质、13%的碳水化合物和20%的脂质；胆固醇与磷脂的摩尔比为0.6或更高。薄层色谱显示没有异常的中性脂质或磷脂，气液色谱显示没有异常脂肪酸掺入VS病毒粒子。在L细胞中生长的抗原性不同的新泽西血清型（VS(NJ)病毒）显示出类似的脂质谱，只是中性脂质的比例比在L细胞中生长的VS(Ind)病毒中的比例更大。当比较在鸡胚细胞中生长的VS(Ind)和VS(NJ)病毒的脂质组成时，这种差异不太明显，但在任何一种细胞类型中生长的VS(NJ)病毒总是比VS(Ind)病毒含有更多除胆固醇外的中性脂质。在L细胞或鸡胚细胞中生长的VS(Ind)和VS(NJ)病毒的脂质组成与质膜的脂质组成比与全细胞的脂质组成更相似。一个一致的发现是，与未感染的全L细胞和鸡胚细胞或其质膜相比，两种VS病毒中磷脂酰乙醇胺和鞘磷脂的含量相对较高，而磷脂酰胆碱的含量相对较低。现有的分离质膜的方法不足以对VS病毒粒子的脂质与宿主细胞质膜的脂质进行确凿的比较。然而，获得的数据与两个假设一致：（i）VS病毒的脂质组成主要反映其成熟的膜位点，（ii）插入细胞膜的新合成病毒蛋白影响选择掺入病毒膜的磷脂和中性脂质的比例。
