Yamada O, Kraus G, Leavitt M C, Yu M, Wong-Staal F
Department of Medicine and Biology, University of California, San Diego 92093-0665.
Virology. 1994 Nov 15;205(1):121-6. doi: 10.1006/viro.1994.1626.
Human CD4+ T cells (Molt-4) were transduced with retroviral 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 NGUC to NUUC). 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 provide in vivo evidence 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 RNA in vivo.
用人CD4+ T细胞（Molt-4）用含有靶向HIV-1 RNA（HXB2：8629-8644）的rev/env编码区的发夹状核酶的逆转录病毒载体进行转导。该靶序列在许多HIV-1克隆中是保守的，包括原型病毒HXB2，但感染性克隆SF2在切割位点含有单个核苷酸取代（从NGUC到NUUC）。用HXB2或SF2攻击稳定表达核酶或其失活对应物的细胞，并监测产生的p24抗原量。虽然这种核酶在抑制Molt 4细胞中HXB2的复制方面有效，但它对SF2复制仅显示出微弱的抑制作用。用失活核酶转导的细胞观察到相同水平的病毒产生，失活核酶基本上起反义分子的作用。通过逆转录-聚合酶链反应检测，在HXB2或SF2感染的细胞中核酶的表达相当。这些数据提供了体内证据，表明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而非反义RNA起作用的结论。
