Hirsch I, Roubal J, Vonka V
Intervirology. 1976;7(3):155-75. doi: 10.1159/000149949.
Newly synthesized herpes simplex virus type 1 DNA yielded a heterogeneous sedimentation profile in neutral sucrose gradients, with the main peak occurring at approximately 40S. Components sedimenting slower than virion DNA and a rapidly sedimenting intracellular HSV DNA were also observed. Both the low-molecular weight and the rapidly sedimenting components seemed to be precursors of virion DNA: they almost completely disappeared after a 60-min chase of a 3-min pulse of 3H-thymidine, and were converted into DNA which cosedimented with virion 32P-labeled DNA. However, sedimentation analysis in alkaline sucrose gradients showed that a 60-min period was insufficient for completing the maturation of HSV DNA. Cleavage of parental DNA molecules was observed in neutral sucrose gradients after infection with 3H-thymidine-labeled virions. No evidence for the formation of covalently closed circles during the replication process was obtained. The presence of single-stranded regions in the replicative form of HSV DNA was revealed. Some of the short-pulse (30 sec) labeled HSV DNA (26.1%) was eluted from hydroxylapatite columns with the properties of single-stranded DNA, and 22% of its trichloroacetic acid precipitability was susceptible to single-strand specific S1 nuclease treatment. Pulse-chase experiments indicated that the life-time of this single-stranded component in nascent DNA was probably not longer than 3 min. A small proportion of single-stranded regions, however, survived for longer periods. Almost all of the newly synthesized short-pulse-labeled HSV DNA exhibited an affinity for nitrocellulose filters. This affinity, which was S1 nuclease-sensitive, gradually decreased with prolongation of the time of the chase. After chasing the pulse for 1 h, the attachment of newly synthesized DNA was comparable with virion DNA.
新合成的单纯疱疹病毒1型DNA在中性蔗糖梯度中呈现出异质沉降图谱，主峰出现在约40S处。还观察到沉降速度比病毒体DNA慢的组分以及快速沉降的细胞内单纯疱疹病毒DNA。低分子量组分和快速沉降组分似乎都是病毒体DNA的前体：在3H-胸腺嘧啶脉冲标记3分钟后进行60分钟的追踪后，它们几乎完全消失，并转化为与病毒体32P标记DNA共沉降的DNA。然而，碱性蔗糖梯度沉降分析表明，60分钟的时间不足以完成单纯疱疹病毒DNA的成熟。在用3H-胸腺嘧啶标记的病毒体感染后，在中性蔗糖梯度中观察到亲本DNA分子的切割。在复制过程中未获得共价闭环形成的证据。揭示了单纯疱疹病毒DNA复制形式中存在单链区域。一些短脉冲（30秒）标记的单纯疱疹病毒DNA（26.1%）以单链DNA的特性从羟基磷灰石柱上洗脱，其22%的三氯乙酸沉淀性对单链特异性S1核酸酶处理敏感。脉冲追踪实验表明，新生DNA中这种单链组分的寿命可能不超过3分钟。然而，一小部分单链区域存活时间更长。几乎所有新合成的短脉冲标记的单纯疱疹病毒DNA都对硝酸纤维素滤膜具有亲和力。这种对S1核酸酶敏感的亲和力随着追踪时间的延长而逐渐降低。在对脉冲进行1小时的追踪后，新合成DNA的附着情况与病毒体DNA相当。
