Inhibition of the synthesis of proteins needed for Epstein-Barr virus replication by antisense RNA against the zta gene

Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication.     

Regulation of pp60c-src expression in rat and mouse fibroblasts by an inducible antisense gene: effects on serum regulation of growth and polyoma virus middle T function.

Expression of antisense c-src RNAs in rat and mouse fibroblasts had a dramatic effect on the function of polyoma virus middle T (mT). Antisense c-src RNA decreased the amount of mT:pp60c-src complexes in de novo virus-infected cells and prevented expression of the transformed phenotype in rat F111 cells. Expression of antisense c-src RNA in infected NIH3T3 cells also reduced the formation of mT:pp60c-src complexes but did not affect the ability of polyoma virus to carry out a productive infection. Further analysis of the effects of antisense c-src RNA in uninfected cells revealed that pp60c-src is required for cell growth. When pp60c-src synthesis was reduced, F111 cells stopped proliferating and showed decreased S6 phosphorylation in response to serum. However, F111 cells expressing reduced pp60c-src could be efficiently transformed by v-rasHa, even in the presence of low serum. Thus, pp60c-src appears to function as a component of a signal transduction pathway which regulates cell proliferation in response to serum.     

反义RNA在基因治疗中的应用

由于反义RNA作为封闭基因表达的有效手段具有特异性强、安全性高、操作简单、靶基因范围广等特点,已被广泛应用于基因治疗肿瘤和病毒相关疾病的研究,反义RNA治疗肿瘤可以通过抑制癌基因的表达、封闭融合癌基因、抑制肿瘤细胞的耐药性、调节细胞因子的表达量等途径;反义RNA治疗病毒相关疾病多集中在艾滋病上,其手段主要是反义封闭TAR。反义RNA作为基因治疗的新途径具有良好的前景,但在设计上和应用上还存在一些急待解决的问题。     

Antisense RNA inhibits splicing of pre-mRNA in vitro.

Antisense RNAs complementary to human beta-globin pre-mRNA or to a chimeric globin/adenovirus E2a pre-mRNA specifically and efficiently inhibit pre-mRNA splicing in vitro. The level of inhibition depends on the length, position and concentration of the antisense RNA relative to the pre-mRNA substrate. Antisense RNAs complementary to sequences greater than 80 nucleotides downstream of the globin 3' splice site inhibit at least as efficiently as those extending across the splice sites. Thus splicing is sensitive to perturbations involving exon sequences some distance from the splice sites. Inhibition is mediated by factors which affect the annealing of antisense and substrate RNAs. Direct analysis of RNA duplex formation demonstrates the presence of an activity in HeLa cell nuclear extract which promotes the rapid annealing of complementary RNAs in an ATP-independent manner. Both annealing and inhibition are greatly reduced when antisense RNA is added to the splicing reaction greater than or equal to 5 min after substrate. This result may reflect a transition between an open structure, in which annealing of antisense RNA with pre-mRNA is facilitated, and a closed complex in which pre-mRNA is sequestered at an early stage of spliceosome assembly.