Covey S N, Turner D S
Department of Virus Research, John Innes Institute, John Innes Centre, Colney, Norwich, U.K.
J Gen Virol. 1993 Sep;74 ( Pt 9):1887-93. doi: 10.1099/0022-1317-74-9-1887.
Cauliflower mosaic virus (CaMV) nucleic acids accumulate in the cell in different structural conformations related to their roles in gene expression, replication and virion assembly. We have characterized changes in the population CaMV DNA and RNA replication products which occur following culture of infected turnip leaves under conditions where callus proliferates. After only 5 days in culture, a significant increase in the level of genome-length and subgenomic supercoiled (SC) DNA forms was observed by two-dimensional (2D) gel electrophoresis. Open circular (OC) molecules, corresponding to these SC DNAs, with mobilities consistent with the presence of a single break in each strand, were also detected after 5 days culture. By 10 days culture, the proportion of OC molecules with only one break per double-stranded molecule had increased. After 34 days culture, SC DNA with a range of sizes predominated in the unencapsidated DNA fraction. The change in pattern of OC and SC DNA forms during callus proliferation suggests a possible precursor/product relationship involving generation of deleted molecules from gap-containing virion DNA-like molecules followed by sequential repair of the gaps to produce SC DNA. Moreover, heterogeneity in the mobility of OC DNAs in the neutral dimension of 2D electrophoresis, a feature exhibited by twisted CaMV virion DNA, changed during the time-course suggesting that untwisting occurs during gap repair. Although the relative abundance of SC DNA increased during callus proliferation, CaMV polyadenylated 35S and 19S transcripts declined together with immediate reverse transcription products. We suggest that cellular changes during callus growth lead to a decline in authentic CaMV transcripts in the cytoplasm resulting in cessation of synthesis of viral products and progeny DNA genomes. In consequence, pre-existing virion DNAs return to the nucleus, possibly as a result of a relaxation in a cytoplasmic control mechanism, where they are assembled into various forms of SC DNA. The presence of CaMV SC DNAs in replicating cells might also enhance illegitimate integration into host chromosomes, as hybridization of CaMV DNA to high M(r) DNA was observed.
花椰菜花叶病毒（CaMV）核酸以不同的结构构象在细胞中积累，这些构象与其在基因表达、复制和病毒粒子组装中的作用相关。我们已经对感染芜菁叶在愈伤组织增殖条件下培养后，群体CaMV DNA和RNA复制产物的变化进行了表征。培养仅5天后，通过二维（2D）凝胶电泳观察到基因组长度和亚基因组超螺旋（SC）DNA形式的水平显著增加。培养5天后，还检测到与这些SC DNA相对应的开环（OC）分子，其迁移率与每条链中存在一个单链断裂一致。培养10天后，每个双链分子仅含一个断裂的OC分子比例增加。培养34天后，一系列大小的SC DNA在未包装的DNA组分中占主导地位。愈伤组织增殖过程中OC和SC DNA形式模式的变化表明，可能存在一种前体/产物关系，涉及从含缺口的病毒粒子DNA样分子产生缺失分子，随后依次修复缺口以产生SC DNA。此外，二维电泳中性维度中OC DNA迁移率的异质性，这是扭曲的CaMV病毒粒子DNA所表现出的特征，在时间进程中发生了变化，表明在缺口修复过程中发生了解旋。虽然在愈伤组织增殖过程中SC DNA的相对丰度增加，但CaMV多聚腺苷酸化的35S和19S转录本与即时逆转录产物一起下降。我们认为，愈伤组织生长过程中的细胞变化导致细胞质中真实的CaMV转录本减少，从而导致病毒产物和子代DNA基因组的合成停止。因此，先前存在的病毒粒子DNA可能由于细胞质控制机制的放松而返回细胞核，在那里它们被组装成各种形式的SC DNA。在复制细胞中CaMV SC DNA的存在也可能增强非法整合到宿主染色体中，因为观察到CaMV DNA与高分子量（M(r)）DNA杂交。
