Replication of African swine fever virus DNA in infected cells
Replication of African swine fever virus DNA in infected cells
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DOI：
10.1006/viro.1999.9704
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发表时间：
1999-05-10
期刊：
影响因子：
3.7
通讯作者：
Salas, J
中科院分区：
文献类型：
--
作者：
Rojo, G;García-Beato, R;Salas, J
我们通过原位杂交和放射自显影检查了非洲猪瘟病毒DNA在感染细胞薄切片中的超微结构定位。在病毒DNA合成的早期阶段，在感染的Vero细胞的细胞核中发现了病毒特异性DNA序列，形成了靠近核膜的密集灶。后来，病毒DNA只在细胞质中被发现。非洲猪瘟病毒感染巨噬细胞的电镜放射自显像显示，细胞核在早期也是病毒DNA复制的位点。这些结果进一步证明了在非洲猪瘟病毒DNA合成过程中存在核和细胞质阶段。另一方面，对感染巨噬细胞细胞核和细胞质中合成的复制中间体进行碱性蔗糖沉降分析发现，早期在细胞核中合成了类似6-12S的小片段DNA，而后期在细胞质中标记了类似37-49S的大片段DNA。脉冲追踪实验证明，这些片段是成熟的交联病毒DNA的前体。通过脉冲场电泳和限制性内切酶分析，在非洲猪瘟病毒DNA复制的中后期观察到二聚体串联体的形成，主要是头对头连接。我们的研究结果表明，非洲猪瘟病毒DNA的复制是通过一种从头开始的机制进行的，即合成小的DNA片段，然后将其转化为更大的分子。这些分子的连接或进一步延伸将产生一个具有二聚体末端的双单元串联体，可以通过位点特异性的切口、重排和连接来分解生成基因组DNA，正如Baroudy等人在从头开始模型中提出的那样（B. M. Baroudy， S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp）。定量生物学。47 723-729)用于牛痘病毒DNA的复制。(C) 1999学术出版社。
We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells by in situ hybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments (similar to 6-12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of similar to 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by a de novo start mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy at al. (B. M. Baroudy, S. Venkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47 723-729) for the replication of vaccinia virus DNA. (C) 1999 Academic Press.