Recombination of poliovirus RNA proceeds in mixed replication complexes originating from distinct replication start sites
Recombination of poliovirus RNA proceeds in mixed replication complexes originating from distinct replication start sites
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DOI：
10.1128/jvi.76.21.10960-10971.2002
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发表时间：
2002-11-01
影响因子：
5.4
通讯作者：
Bienz, K
中科院分区：
文献类型：
--
作者：
Egger, D;Bienz, K
基因重组在小核糖核酸病毒复制过程中频繁发生。为了探索重组所涉及的细胞内位点和结构，用脊髓灰质炎病毒1型Mahoney和2型Sabin感染HeLa细胞。通过荧光原位杂交和共聚焦显微镜对两个基因组进行定位。对于杂交，使用类型特异性荧光核糖核酸探针来可视化相同的基因组区域，其中，平行地，用类型特异性逆转录PCR和测序来证明重组。杂交分析表明，>85%的复制复合物含有1型和2型RNA序列，其横向距离为50 nm或更小。细胞的连续感染排除了高比例的混合复制复合物是由于输入病毒聚集的可能性。随着时间的推移，输入基因组RNA的可视化显示病毒基因组迁移到相对较少的不同的，因此可能是特定的，复制开始的核周位点。第一个重组RNA链可以检测伴随着RNA复制的开始。有限数量的复制起始位点可能是观察到的优先形成混合复制复合物的原因，每个复合物容纳几个亲本RNA链，从而允许重组。
Genetic recombination occurs frequently during replication of picornaviruses. To explore the intracellular site and structures involved in recombination, HeLa cells were infected with poliovirus type 1 Mahoney and type 2 Sabin. The two genomes were located by fluorescent in situ hybridization and confocal microscopy. For hybridization, type-specific fluorescent riboprobes were used to visualize the same genomic region where, in parallel, recombination was demonstrated with type-specific reverse transcription-PCR and sequencing. The hybridization analysis indicated that >85% of the replication complexes contained both type 1 and type 2 RNA sequences aligned at a lateral distance of 50 nm or less. Sequential infection of cells ruled out the possibility that the high percentage of mixed replication complexes was due to aggregation of input virus. Visualization of input genomic RNA over time showed that the viral genomes migrated to relatively few distinct, and thus presumably specific, perinuclear sites where replication started. The first recombinant RNA strands could be detected concomitantly with the onset of RNA replication. The limited number of start sites for replication may be the reason for the observed preferential formation of mixed replication complexes, each accommodating several parental RNA strands and thus allowing recombination.