Biologisches Institut, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
J Virol. 2011 Nov;85(22):12001-12. doi: 10.1128/JVI.05567-11. Epub 2011 Aug 10.
Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially.
具有小型环状单链 DNA 基因组的双生病毒在植物细胞核内通过使用各种双链 DNA（dsDNA）中间体进行复制：独特的开环和共价闭环以及异质线性 DNA。它们的 DNA 可能在胞嘧啶残基处部分甲基化，如先前通过亚硫酸氢盐测序和随后的 PCR 检测到的那样。为了确定环状分子的甲基化模式，利用亚硫酸氢盐处理和随后的滚环扩增研究了番茄黄卷叶病毒（TYLCSV）和鸭跖草花叶病毒的 DNA。两种病毒的产物进行鸟枪法测序后，亚硫酸氢盐反应后 C 保持率随机分布为 50%。然而，用未甲基化的单链噬菌体 DNA 作为对照，得到了相同的 C 保持率。仅在 TYLCSV 的 C2/C3 启动子内的一小段 DNA 显示出 C 的超保护，保护率超过平均值加 1 个标准差的阈值。同样，使用甲基化敏感的限制性内切酶表明，双生病毒通过滚环或重组依赖的复制模式非常有效地逃避甲基化沉默。相比之下，通过使用甲基胞嘧啶特异性抗体检测甲基化碱基的尝试仅在异质线性 dsDNA 中检测到甲基化 DNA，并且甲基化依赖的限制性内切酶表明病毒的异质线性 dsDNA 优先甲基化。
