Quantitative dynamics of HIV type 1 expression
Quantitative dynamics of HIV type 1 expression
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DOI：
10.1089/aid.1996.12.117
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发表时间：
1996-01-20
影响因子：
1.5
通讯作者：
Giacca, M
中科院分区：
文献类型：
--
作者：
Comar, M;Marzio, G;Giacca, M
开发了一种竞争性PCR和RT-PCR方法，用于定量感染的生物样品中的HIV-1核酸，特别是关于不同加工的病毒转录物的产生动力学的研究，该程序需要利用竞争质粒DNA（在DNA样品上）或从该质粒获得的体外转录产物（在RNA样品上）并且允许定量前病毒DNA、病毒基因组RNA和病毒单链和多链mRNA。此外，它允许将这些测量直接标准化为参考细胞基因的量（用于DNA定量）或参考细胞转录物（用于RNA定量）。该定量程序用于监测用佛波醇-12-肉豆蔻酸酯-13-乙酸酯刺激后潜伏感染的U1单核细胞系和实验感染的外周血淋巴细胞中HIV-1转录激活的动态。尽管两种实验系统之间存在生物学差异，但在两种情况下，感染性病毒的产生都伴随着未剪接病毒mRNA水平的显著增加（在U1细胞中上升高达20，000倍），并通过随后的不同剪接转录物类别丰度的转换，这些观察结果加强了感染控制也受到转录后事件的影响的概念，并提示对HIV-1的定量评价。1转录物类别丰度，以定义疾病进展的潜在标志物。
A competitive PCR and RT-PCR procedure was developed for the quantification of HIV-1 nucleic acids in infected biological samples, with particular reference to the study of the kinetics of production of differently processed viral transcripts, The procedure entails the utilization of a competitor plasmid DNA (on DNA samples) or of an in vitro transcription product obtained from this plasmid (on RNA samples) and allows the quantification of proviral DNA, viral genomic RNA, and viral single- and multispliced mRNAs, Furthermore, it permits the direct standardization of these measurements to the amount of a reference cellular gene (for DNA quantification) or of a reference cellular transcript (for RNA quantification). This quantification procedure was used to monitor the dynamics of HIV-1 transcriptional activation in the latently infected U1 monocytic cell line after stimulation with phorbol-12-myristate-13-acetate, and in experimentally infected peripheral blood lymphocytes. Despite the biological differences between the two experimental systems, in both cases production of infectious virus is accompanied by a remarkable increase in the levels of unspliced viral mRNAs (rising up to 20,000 fold in U1 cells) and by a consequent switch in the abundance of the differently spliced transcript classes, These observations reinforce the notion that the control of infection is subjected also to posttranscriptional events and prompts for quantitative evaluation of HIV-1 transcript class abundance in infected individuals to define potential markers for disease progression.