Mortreux F, Leclercq I, Gabet A S, Leroy A, Westhof E, Gessain A, Wain-Hobson S, Wattel E
Unité 524 Institut National de la Santé et de la Recherche Médicale (INSERM), Lille, France.
J Natl Cancer Inst. 2001 Mar 7;93(5):367-77. doi: 10.1093/jnci/93.5.367.
Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma, shows intrapatient genetic variability. Although HTLV-1 can replicate via the reverse transcription of virion RNA to a double-stranded DNA provirus (the conventional manner for retroviruses), its predominant mode of replication is via the clonal expansion (mitosis) of the infected cell. This expansion is achieved by the viral oncoprotein Tax, which keeps the infected CD4 T lymphocyte cycling. Because Tax also interferes with cellular DNA repair pathways, we investigated whether somatic mutations of the provirus that occur during the division of infected cells could account for HTLV-1 genetic variability.
An inverse polymerase chain reaction strategy was designed to distinguish somatic mutations from reverse transcription-associated substitutions. This strategy allows the proviral sequences to be isolated together with flanking cellular sequences. Using this method, we sequenced 208 HTLV-1 provirus 3' segments, together with their integration sites, belonging to 29 distinct circulating cellular clones from infected individuals.
For 60% of the clones, 8%-80% of infected cells harbored a mutated HTLV-1 provirus, without evidence of reverse transcription-associated mutations. Mutations within flanking cellular sequences were also identified at a frequency of 2.8 x 10(-4) substitution per base pair. Some of these clones carried multiple discrete substitutions or deletions, indicating progressive accumulation of mutations during clonal expansion. The overall frequency of somatic mutations increased with the degree of proliferation of infected T cells.
These data indicate that, in vivo, HTLV-1 variation results mainly from postintegration events that consist of somatic mutations of the proviral sequence occurring during clonal expansion. The finding of substitutions in flanking sequences suggests that somatic mutations occurring after integration, presumably coupled with selection, help move the cellular clones toward a transformed phenotype, of which adult T-cell leukemia/lymphoma is the end point.
人类T细胞白血病病毒1型（HTLV-1）是成人T细胞白血病/淋巴瘤的病原体，存在患者体内的遗传变异性。虽然HTLV-1可以通过将病毒粒子RNA逆转录为双链DNA前病毒进行复制（逆转录病毒的传统复制方式），但其主要复制模式是通过受感染细胞的克隆扩增（有丝分裂）。这种扩增是由病毒癌蛋白Tax实现的，它使受感染的CD4 T淋巴细胞持续循环。由于Tax也会干扰细胞DNA修复途径，我们研究了在受感染细胞分裂过程中发生的前病毒体细胞突变是否可以解释HTLV-1的遗传变异性。
设计了一种反向聚合酶链反应策略，以区分体细胞突变和与逆转录相关的替代。该策略允许将前病毒序列与其侧翼细胞序列一起分离。使用这种方法，我们对来自受感染个体的29个不同循环细胞克隆的208个HTLV-1前病毒3'片段及其整合位点进行了测序。
对于60%的克隆，8%-80%的受感染细胞携带突变的HTLV-1前病毒，没有与逆转录相关突变的证据。侧翼细胞序列中的突变也以每碱基对2.8×10(-4)替代的频率被鉴定出来。其中一些克隆携带多个离散的替代或缺失，表明在克隆扩增过程中突变的逐渐积累。体细胞突变的总体频率随着受感染T细胞的增殖程度而增加。
这些数据表明，在体内，HTLV-1变异主要源于整合后事件，这些事件包括在克隆扩增过程中发生的前病毒序列的体细胞突变。侧翼序列中替代的发现表明，整合后发生的体细胞突变，可能与选择相结合，有助于使细胞克隆向转化表型发展，成人T细胞白血病/淋巴瘤就是其终点。
