Chee-Sheung C C, Ginsberg H S
J Virol. 1982 Jun;42(3):932-50. doi: 10.1128/JVI.42.3.932-950.1982.
A temperature-sensitive, fiber-minus mutant of type 5 adenovirus, H5ts142, was biochemically and genetically characterized. Genetic studies revealed that H5ts142 was a member of one of the three apparent fiber complementation groups which were detected owing to intracistronic complementation. Recombination analyses showed that it occupied a unique locus at the right end of the adenovirus genetic map. At the nonpermissive temperature, the mutant made stable polypeptides, but they were not glycosylated like wild-type fiber polypeptides. Sedimentation studies of extracts of H5ts142-infected cells cultured and labeled at 39.5 degrees C indicated that a limited number of the fiber polypeptides made at the nonpermissive temperature could assemble into a form having a sedimentation value of 6S (i.e., similar to the trimeric wild-type fiber), but that this 6S structure was not immunologically reactive. When H5ts142-infected cells were shifted to the permissive temperature, 32 degrees C, fiber polypeptides synthesized at 39.5 degrees C were as capable of being assembled into virions as fibers synthesized in wild type-infected cells; de novo protein synthesis was not required to allow this virion assembly. In H5ts142-infected cells incubated at 39.5 degrees C, viral proteins accumulated and aggregated into particles having physical characteristics of empty capsids. These particles did not contain DNA or its associated core proteins. However, when the infected culture was shifted to 32 degrees C, DNA appeared to enter the empty particles and complete virions developed. The intermediate particles obtained had the morphology of adenoviruses, but they contained less than unit-length viral genomes as measured by their buoyant density in a CsCl density gradient and the size of their DNA as determined in both neutral and alkaline sucrose gradients. The reduced size of the intermediate particle DNA was demonstrated to be the result of incompletely packaged DNA molecules being fragmented during the preparative procedures. Hybridization of labeled DNA extracted from the intermediate particles to filters containing restriction fragments of the adenovirus genome indicated that the molecular left end of the viral genome preferentially entered these particles.
对5型腺病毒的温度敏感型、纤维缺失突变体H5ts142进行了生化和遗传学特征分析。遗传学研究表明，H5ts142是因顺反子内互补而检测到的三个明显的纤维互补组之一的成员。重组分析表明，它位于腺病毒遗传图谱右端的一个独特位点。在非允许温度下，该突变体产生稳定的多肽，但它们不像野生型纤维多肽那样进行糖基化。对在39.5℃培养和标记的H5ts142感染细胞提取物的沉降研究表明，在非允许温度下产生的有限数量的纤维多肽能够组装成沉降值为6S的形式（即类似于三聚体野生型纤维），但这种6S结构没有免疫反应性。当H5ts142感染的细胞转移到允许温度32℃时，在39.5℃合成的纤维多肽与野生型感染细胞中合成的纤维一样能够组装成病毒粒子；不需要从头合成蛋白质来进行这种病毒粒子组装。在39.5℃孵育的H5ts142感染细胞中，病毒蛋白积累并聚集形成具有空衣壳物理特征的颗粒。这些颗粒不含DNA或其相关的核心蛋白。然而，当感染的培养物转移到32℃时，DNA似乎进入空颗粒并形成完整的病毒粒子。获得的中间颗粒具有腺病毒的形态，但通过它们在CsCl密度梯度中的浮力密度和在中性及碱性蔗糖梯度中测定的DNA大小来衡量，它们所含的病毒基因组长度小于单位长度。中间颗粒DNA大小的减小被证明是由于在制备过程中未完全包装的DNA分子断裂所致。从中间颗粒中提取的标记DNA与含有腺病毒基因组限制性片段的滤膜杂交表明，病毒基因组的分子左端优先进入这些颗粒。
