GENETIC CONSEQUENCES OF PACKAGING 2 RNA GENOMES IN ONE RETROVIRAL PARTICLE - PSEUDODIPLOIDY AND HIGH-RATE OF GENETIC-RECOMBINATION
GENETIC CONSEQUENCES OF PACKAGING 2 RNA GENOMES IN ONE RETROVIRAL PARTICLE - PSEUDODIPLOIDY AND HIGH-RATE OF GENETIC-RECOMBINATION
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DOI：
10.1073/pnas.87.4.1556
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发表时间：
1990-02-01
影响因子：
11.1
通讯作者：
TEMIN, HM
中科院分区：
文献类型：
--
作者：
HU, WS;TEMIN, HM
逆转录病毒在每个病毒粒子中包含两个完整的病毒基因组RNA。建立了一个在单轮复制中研究病毒粒子与两种不同基因组RNA产物的系统。采用含有新霉素抗性基因(Neo)和潮霉素B磷酸转移酶基因(Hygro)的脾坏死病毒剪接载体。从该载体中衍生出两个移码突变体，使得neo和hygro基因在不同的载体中失活。因此，每个载体只对一种选择产生抗性。将含有移码突变的载体分别扩增并混合感染DSDH辅助细胞。分离双抗细胞克隆，并用这些克隆产生的病毒感染D17细胞。该方案允许含有两个不同基因组RNA(杂合子)的病毒粒子完成一轮逆转录病毒复制。测定了抗单选择和双选择的后代的分子本质及其比例。我们的数据表明，每个感染性杂合病毒粒子只产生一个前病毒。逆转录病毒的重组率是每千个碱基每复制周期2.2%。重组前病毒是杂合病毒粒子的后代。
Retroviruses contain two complete viral genomic RNAs in each virion. A system to study in a single round of replication the products of virions with two different genomic RNAs was established. A spleen necrosis virus-based splicing vector containing both the neomycin-resistance gene (neo) and the hygromycin B phosphotransferase gene (hygro) was used. Two frameshift mutants were derived from this vector such that the neo and the hygro genes were inactivated in separate vectors. Thus, each vector confers resistance to only one selection. The vectors with frameshift mutations were separately propagated and were pooled to infect DSDh helper cells. Doubly resistant cell clones were isolated, and viruses produced from these clones were used to infect D17 cells. This protocol allowed virions containing two different genomic RNAs (heterozygotes) to complete one round of retroviral replication. The molecular nature of progeny that conferred resistance to single or double selection and their ratio were determined. Our data demonstrate that each infectious heterozygous virion produces only one provirus. The rate of retroviral recombination is .apprxeq.2% per kilobase per replication cycle. Recombinant proviruses are progeny of heterozygous virions.