Sun L Q, Warrilow D, Wang L, Witherington C, Macpherson J, Symonds G
R.W. Johnson Pharmaceutical Research Institute-Sydney, New South Wales, Australia.
Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9715-9. doi: 10.1073/pnas.91.21.9715.
Several hammerhead ribozymes targeted to different sites within the retroviral packaging (psi) sequences of the Moloney murine leukemia virus (Mo-MLV) and the human immunodeficiency virus type 1 (HIV-1) were designed and shown to cleave target RNA in vitro at the chosen sites. The engineered ribozymes, as well as antisense sequence complementary to the Mo-MLV psi packaging region, were cloned into the 3' untranslated region of the neomycin-resistance gene (neo). This was coupled to the simian virus 40 early promoter within the pSV2neo vector. For the ribozymes against the Mo-MLV psi site, the constructs were transfected into Mo-MLV-infected and virus-producing mouse NIH 3T3 cells. With the exception of one of the single ribozymes (the one least effective in cutting target RNA in vitro), all of the constructs effectively (70-80%) suppressed retrovirus production. These results demonstrate a direct correlation between in vitro cleavage and in vivo ribozyme-mediated virus suppression. In addition, a ribozyme targeted to the HIV-1 psi packaging site was engineered into the same vector and transfected into the human T-cell line SupT1. The transfectants were cloned and then challenged with HIV-1. When compared to vector-transfected control cells, a significant reduction in HIV-1 production was observed as measured by p24 and syncytia formation assays. This study demonstrates a feasible approach to the suppression of retrovirus replication by targeting the psi packaging site with hammerhead ribozymes.
设计了几种针对莫洛尼鼠白血病病毒（Mo-MLV）和人类免疫缺陷病毒1型（HIV-1）逆转录病毒包装（ψ）序列内不同位点的锤头状核酶，并证明它们能在体外选定的位点切割靶RNA。将工程化核酶以及与Mo-MLV ψ包装区域互补的反义序列克隆到新霉素抗性基因（neo）的3'非翻译区。这与pSV2neo载体中的猿猴病毒40早期启动子相连。对于针对Mo-MLV ψ位点的核酶，将构建体转染到受Mo-MLV感染并产生病毒的小鼠NIH 3T3细胞中。除了一种单链核酶（在体外切割靶RNA效果最差的那种）外，所有构建体均有效地（70%-80%）抑制了逆转录病毒的产生。这些结果证明了体外切割与体内核酶介导的病毒抑制之间存在直接相关性。此外，将一种针对HIV-1 ψ包装位点的核酶构建到同一载体中，并转染到人T细胞系SupT1中。对转染子进行克隆，然后用HIV-1进行攻击。通过p24和多核体形成试验测定，与载体转染的对照细胞相比，观察到HIV-1产生显著减少。这项研究证明了一种通过用锤头状核酶靶向ψ包装位点来抑制逆转录病毒复制的可行方法。
