A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation
A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation
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DOI：
10.1073/pnas.93.21.11321
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发表时间：
1996-10-15
影响因子：
11.1
通讯作者：
Greaves, RF
中科院分区：
文献类型：
--
作者：
Mocarski, ES;Kemble, GW;Greaves, RF
人巨细胞病毒（CMV）复制开始于两种调节蛋白IE1（491aa）和IE2（579aa）的表达，IE1/ie2启动子-增强子控制下的差异剪接转录物产生IE2。将去除所有406个IE1（491 aa）特异性氨基酸的缺失突变工程化到病毒基因组中，并将该突变体（RC 303 Delta Acc）在表达IE1（491 aa）的人成纤维细胞系（ihfie1.3）上繁殖。RC303 Delta Acc在低感染复数（mois）下不能在正常人成纤维细胞上复制。在mois> 3个噬斑形成单位/细胞时，病毒复制和后代的产生与野生型相当。然而，当mois在0.01和1之间时，突变病毒在正常成纤维细胞上复制缓慢，这一模式表明启动生产性感染需要多次命中。RC303 Delta Acc的复制与表达IE2（579 aa）的能力相关，这与IE1（491 aa）在ie1/ie2启动子-增强子的正性自动调节中的作用一致，并且与表明病毒体反式激活因子在高moi条件下补偿IE1（491 aa）缺乏的数据一致。ie1缺陷型CMV应该是完全无毒的，这表明其作为CMV潜伏期正常位点的造血祖细胞的基因治疗载体的实用性。
Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 Delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 Delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois >3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 Delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.