Ward P, Berns K I
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, New York 10021, USA.
J Virol. 1996 Jul;70(7):4495-501. doi: 10.1128/JVI.70.7.4495-4501.1996.
Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstructural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine replication of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is 50-fold greater in extracts from Ad-infected cells than in extracts from uninfected cells. In addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated strands, whereas the majority of labeled full-length AAV DNA molecules synthesized in the uninfected extract have only one newly replicated strand. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the case of the Ad-infected cell extract is much more likely to result in the synthesis of a full-length AAV DNA molecule. Most of the newly replicated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provides a helper function related to elongation during replication by a single-strand displacement mechanism. In the assay using the uninfected HeLa cell extract, replication frequently stalls before reaching the end of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template. In support of this conjecture, replication in the uninfected cell extract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA. In addition, when shorter substrate molecules are used as the template in the uninfected HeLa cell assay, a greater proportion of the labeled full-length substrate molecules contain two newly replicated strands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad-infected cells.
此前我们描述了一种用于2型腺相关病毒（AAV2）DNA复制的体外测定方法。将AAV2非结构蛋白Rep68添加到未感染细胞的提取物中可支持线性双链AAV DNA的复制。在本报告中，我们研究了未感染或腺病毒（Ad）感染的HeLa细胞提取物中线性双链AAV DNA的复制情况。与未感染细胞的提取物相比，Ad感染细胞的提取物中放射性标记核苷酸掺入全长线性AAV DNA的量高出50倍。此外，在Ad感染的提取物中合成的大多数标记全长AAV DNA分子有两条新复制的链，而在未感染的提取物中合成的大多数标记全长AAV DNA分子只有一条新复制的链。两种测定中原始模板上的复制起始次数大致相同；然而，Ad感染细胞提取物中的复制更有可能导致全长AAV DNA分子的合成。使用未感染细胞提取物的测定中，大多数新复制的分子呈茎环结构形式。我们推测，Ad感染通过单链置换机制提供了与复制过程中延伸相关的辅助功能。在使用未感染的HeLa细胞提取物的测定中，复制在到达基因组末端之前经常停滞，导致新合成的链从模板上被置换，随后自身折叠，并以自身为模板通过反向末端重复序列进行复制。为支持这一推测，通过将放射性标记核苷酸掺入全长底物DNA来衡量，较短底物分子在未感染细胞提取物中的复制效率更高。此外，当在未感染的HeLa细胞测定中使用较短底物分子作为模板时，更大比例的标记全长底物分子含有两条新复制的链。在使用Ad感染细胞提取物的测定中，较短底物分子相对于全长底物分子没有复制优势。
