RETROVIRAL ANTI-SENSE RNA--CELLULAR AND VIRAL RESPONSES

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Anti-sense RNA provides a means for specifically blocking the expression of a given gene through formation of a duplex with the target RNA. This technique has also been used to block replication of retroviruses containing RNA complementary to cellular RNA. Thus, this technique offers a potential therapeutic strategy for specifically blocking expression of an oncogenic viral gene product and/or replication of an oncogenic retrovirus. Use of this technology, however, has been limited because the mechanisms by which complementary RNA blocks gene expression or retroviral replication are poorly understood. A better understanding of mechanisms by which anti-sense RNA blocks gene expression and retroviral replication may permit more effective applications of this technology. A model system using retroviral vectors capable of expressing RNA complementary to cellular sequences has been developed. Vectors producing RNA complementary to different regions of the same target mRNA have been isolated. Those vectors producing anti-sense transcripts which are effective in blocking expression of the target mRNA are approximately 100 x more potent than many previously reported, they are complementary to a region of the mRNA which has previously not been considered to be a good target, and they apparently block expression of the complementary mRNA by a mechanism which has not been described previously. The purpose of this proposal is to exploit this model to define the mechanism(s) by which this anti-sense system blocks expression of its compementary cellular mRNA and to determine what effect the complementary cellular RNA has on replication of the retrovirus. Studies are described in which a series of retroviral constructs will be used to 1) define more precisely the regions of the complementary RNAs participating in duplex formation in vivo and in vitro, 2) effects of different RNA transcripts upon duplex formation and stability, and 3) mechanisms by which effective anti-sense constructs block translation of complementary cellular mRNA's. Experiments using this same model are described which will address the following questions: 1) Are cells which produce RNA complementary to that of the retrovirus less susceptible to infection by this retrovirus? If so, what factors affect the extent of this resistance? 2) Do complementary cellular RNAs adversely affect packaging and release of viruses capable of infecting other cells?