Peng H, Callison D E, Li P, Burrell C J
National Centre for HIV Virology Research, University of Adelaide, South Australia.
AIDS. 1997 Apr;11(5):587-95. doi: 10.1097/00002030-199705000-00006.
To construct retroviral vectors expressing sense or antisense RNA targeted at HIV reverse transcription intermediates, and to test the anti-HIV properties of these constructs in transduced T cells.
Five double-copy retroviral vectors were constructed, in which the expression of the sense or antisense RNA corresponding to HIV minus- or plus-strand strong-stop DNA was driven by the human tRNA(met) promoter.
The templates for the sense or antisense RNA were polymerase chain reaction-cloned from HIV pNL43 into a murine leukaemia virus-based vector and corresponding defective virions were packaged in PA317 cells. Human Jurkat T cells transduced with these vectors were challenged with HIV and monitored for viral RNA, viral DNA and p24 production for 23 weeks.
Intracellular expression of HIV sense RU5 sequences (RNA complementary to minus-strand strong-stop DNA) enhanced HIV replication in T cells. Expression of HIV sense or antisense U3RU5 sequences (identical or complementary to plus-strand strong-stop DNA) conferred long-term inhibition of HIV replication, despite continuous presence of viral challenge in the transduced cell cultures.
Plus-strand strong-stop DNA as an intermediate in the early process of viral reverse transcription can be explored as an additional target for anti-HIV gene therapy.
构建表达针对HIV逆转录中间体的正义或反义RNA的逆转录病毒载体，并在转导的T细胞中测试这些构建体的抗HIV特性。
构建了五种双拷贝逆转录病毒载体，其中对应于HIV负链或正链强终止DNA的正义或反义RNA的表达由人tRNA(met)启动子驱动。
将正义或反义RNA的模板通过聚合酶链反应从HIV pNL43克隆到基于鼠白血病病毒的载体中，并在PA317细胞中包装相应的缺陷病毒颗粒。用这些载体转导的人Jurkat T细胞用HIV进行攻击，并监测病毒RNA、病毒DNA和p24的产生情况，持续23周。
HIV正义RU5序列（与负链强终止DNA互补的RNA）的细胞内表达增强了T细胞中HIV的复制。尽管在转导的细胞培养物中持续存在病毒攻击，但HIV正义或反义U3RU5序列（与正链强终止DNA相同或互补）的表达赋予了对HIV复制的长期抑制作用。
正链强终止DNA作为病毒逆转录早期过程中的一种中间体，可被探索作为抗HIV基因治疗的另一个靶点。
