Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors
Examining human T-lymphotropic virus type 1 infection and replication by cell-free infection with recombinant virus vectors
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DOI：
10.1128/jvi.75.18.8461-8468.2001
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发表时间：
2001-09-01
影响因子：
5.4
通讯作者：
Morse, BA
中科院分区：
文献类型：
--
作者：
Derse, D;Hill, SA;Morse, BA
开发了一种利用基于重组人T细胞白血病病毒1型（HTLV-1）的载体的灵敏和定量无细胞感染试验，以分析病毒复制周期中的早期事件。以前的困难与病毒的低感染性和有限的表达，阻止了清楚地了解这些事件。通过用三种质粒转染细胞来产生病毒原液：（i）编码HTLV-1结构和调节蛋白的包装质粒，（ii）含有萤火虫荧光素酶或增强型黄色荧光蛋白基因的HTLV-1转移载体，和（iii）包膜表达质粒。通过将靶细胞暴露于过滤的上清液来启动单轮感染，并通过测定细胞提取物中的荧光素酶活性或通过流式细胞术计数转导的细胞来定量。逆转录酶抑制剂3 ' -叠氮基-3' -脱氧胸苷（AZT）的作用和包装载体中整合酶基因的突变分别表明，转导依赖于重组病毒基因组的逆转录和整合。在该HTLV-1复制系统中，AZT的50%抑制浓度确定为30 nM。用水泡性口炎病毒G蛋白或HTLV-1包膜假型化的HTLV-1颗粒的稳定性是逆转录病毒的典型特征，在37 ℃下表现出约3.5小时的半衰期。重组HTLV-1病毒粒子的特异性感染性比类似的HIV-1粒子低至少3个数量级，尽管两者都是具有相同包膜的假型。因此，HTLV-1的低感染性在很大程度上取决于核心颗粒的性质和后处理的效率。
A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3 ' -azido-3 ' -deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37 degreesC. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes.