Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells.
Activity and cleavage site specificity of an anti-HIV-1 hairpin ribozyme in human T cells.
复制标题
人类 T 细胞中抗 HIV-1 发夹核酶的活性和切割位点特异性。
DOI：
10.1006/viro.1994.1626
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发表时间：
1994
期刊：
影响因子：
3.7
通讯作者：
Wong-Staal,F
中科院分区：
文献类型：
--
作者：
Yamada,O;Kraus,G;Leavitt,MC;Yu,M;Wong-Staal,F
用逆转录病毒载体转导人CD 4 + T细胞（Molt-4），所述逆转录病毒载体含有靶向HIV-1 RNA的rev/env编码区的发夹状核酶（HXB 2：8629-8644）。该靶序列在许多HIV-1克隆中是保守的，包括原型病毒HXB 2，但感染性克隆SF 2在切割位点处含有单核苷酸取代（从N*GUC到N*UUC）。用HXB 2或SF 2攻击稳定表达核酶或其失能对应物的细胞，并监测产生的p24抗原的量。虽然这种核酶在Molt 4细胞中有效抑制HXB 2的复制，但它对SF 2复制仅显示出边缘抑制作用。用基本上作为反义分子起作用的失能核酶转导的细胞观察到相同水平的病毒产生。通过逆转录-聚合酶链反应检测，核酶的表达在HXB 2或SF 2感染的细胞中是相当的。这些数据提供了体内证据，发夹状核酶的抗病毒活性严格依赖于靶RNA中切割位点的存在，并支持核酶在体内作为催化RNA而不是作为反义RNA的结论。
Human CD4+ T cells (Molt-4) were transduced with retrovital vectors containing a hairpin ribozyme which targets the rev/env coding region of HIV-1 RNA (HXB2: 8629-8644). This target sequence is conserved among many HIV-1 clones, including the prototype virus HXB2, but the infectious clone SF2 contains a single nucleotide substitution at the cleavage site (from N*GUC to N*UUC). Cells stably expressing the ribozyme or its disabled counterpart were challenged with HXB2 or SF2 and the amount of p24 antigen produced was monitored. While this ribozyme was effective in inhibiting the replication of HXB2 in Molt 4 cells, it showed only marginal inhibitory effect on SF2 replication. The same level of virus production was observed with cells transduced by the disabled ribozyme, which functions essentially as an antisense molecule. Expression of the ribozyme was comparable in HXB2- or SF2-infected cells as detected by reverse transcription-polymerase chain reaction. These data providein vivoevidence that the antiviral activity of the hairpin ribozyme is strictly dependent on the presence of the cleavage site in the target RNA and supports the conclusion that the ribozyme acts as catalytic RNA rather than as antisense RNAin vivo.