Egger Denise, Bienz Kurt
Institute for Medical Microbiology, University of Basel, CH-4000 Basel, Switzerland.
J Virol. 2002 Nov;76(21):10960-71. doi: 10.1128/jvi.76.21.10960-10971.2002.
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.
在小核糖核酸病毒复制过程中，基因重组频繁发生。为探究参与重组的细胞内位点和结构，用1型脊髓灰质炎病毒Mahoney株和2型脊髓灰质炎病毒Sabin株感染HeLa细胞。通过荧光原位杂交和共聚焦显微镜对两种基因组进行定位。杂交时，使用型特异性荧光核糖探针来观察同一基因组区域，与此同时，通过型特异性逆转录-聚合酶链反应和测序证明该区域发生了重组。杂交分析表明，超过85%的复制复合体包含1型和2型RNA序列，它们在横向距离50纳米或更小的范围内排列。细胞的连续感染排除了高比例混合复制复合体是由于输入病毒聚集所致的可能性。随着时间推移对输入基因组RNA的观察显示，病毒基因组迁移到相对较少的、明显且可能是特定的核周位点，复制即在此处开始。最早的重组RNA链可在RNA复制开始时同时检测到。复制起始位点数量有限可能是观察到混合复制复合体优先形成的原因，每个复合体容纳几条亲代RNA链，从而允许重组发生。
