Ramezani A, Joshi S
Department of Medical Genetics and Microbiology, University of Toronto, Ontario, Canada.
Antisense Nucleic Acid Drug Dev. 1996 Fall;6(3):229-35. doi: 10.1089/oli.1.1996.6.229.
Moloney murine leukemia virus (MMLV)-derived pUCMoTiN-based retroviral vectors were engineered to allow constitutive and Tat (trans-activator of transcription)-inducible expression of five hammerhead ribozymes targeted against highly conserved sequences within the group antigen (Gag), protease (Pro), reverse transcriptase (RT), tat, and envelope (Env) coding regions of human immunodeficiency virus type-1 (HIV-1) RNA. Amphotropic retroviral vector particles were used to infect a human CD4+ lymphocyte-derived MT4 cell line. The pool of stable MT4 transductants expressing these ribozymes were each tested for their susceptibility to HIV-1 infection. RzTat conferred no protection to MT4 cells. RZGag and RzRT completely inhibited virus multiplication for 6 days. RzPro and RzEnv conferred the best protection, as they completely inhibited virus production for 12 and 15 days, respectively. No correlation was found between the degree of HIV-1 resistance conferred and the ability of these ribozymes to cleave their target RNA in vitro. From RzPro-expressing HIV-1-infected cells following virus escape, RzPro and target RNA sequences were amplified and checked for cleavage in vitro. The ribozyme expressed in these cells was shown to cleave the corresponding target RNA. Thus, a mutation in the ribozyme or target RNA does not seem to be the mechanism underlying virus escape.
基于莫洛尼鼠白血病病毒（MMLV）的pUCMoTiN逆转录病毒载体经过改造，可实现针对人类免疫缺陷病毒1型（HIV-1）RNA的群抗原（Gag）、蛋白酶（Pro）、逆转录酶（RT）、反式激活因子（tat）和包膜（Env）编码区域内高度保守序列的5种锤头状核酶的组成型表达和tat诱导型表达。嗜异性逆转录病毒载体颗粒用于感染人CD4+淋巴细胞衍生的MT4细胞系。对表达这些核酶的稳定MT4转导子群体分别进行HIV-1感染敏感性测试。RzTat对MT4细胞没有保护作用。RZGag和RzRT在6天内完全抑制病毒增殖。RzPro和RzEnv提供了最佳保护，因为它们分别在12天和15天内完全抑制病毒产生。在赋予HIV-1抗性的程度与这些核酶在体外切割其靶RNA的能力之间未发现相关性。从病毒逃逸后表达RzPro的HIV-1感染细胞中扩增RzPro和靶RNA序列，并在体外检查切割情况。这些细胞中表达的核酶显示可切割相应的靶RNA。因此，核酶或靶RNA中的突变似乎不是病毒逃逸的潜在机制。
