Construction and analysis of adenovirus/HIV-1 rev recombinants

The human immunodeficiency virus Rev protein is required for the cytoplasmic accumulation and probably the translational utilisation of mRNAs encoding the late viral structural proteins. Rev function is mediated by direct binding of the protein to a c/5-acting RNA sequence, the Rev-responsive element (RRE), carried by these mRNAs. Human adenovirus type 5 (Ad5) also encodes a protein, E1B 55K, required for the cytoplasmic accumulation of late viral mRNAs. Rev and E1B SSK both regulate gene expression post-transcriptionally and directly or indirectly act on the mRNA transport pathway. To explore the potential functional analogy between these two regulatory proteins, the components of the Rev/RRE system were introduced into AdS. By inserting the RRE into Ad5 late region L3 (expression of which is normally dependent on E1B 55K), and a rev expression cassette into early region E1A, a system was set up where the action of Rev could be directly compared with that of E1B 55K. A series of six recombinant adenoviruses was constructed which, together with two viruses already available, contained the rev gene and/or the RRE and/or the Ad5 E1B SSK gene in all possible combinations. Expression of functional Rev from the appropriate recombinants was confirmed by a CAT reporter gene assay. The eight viruses were used to study the effects of Rev and the RRE on the expression of Ad5 late RNAs and proteins. It was shown that the Rev/RRE system can detectably increase the cytoplasmic accumulation of certain Ad5 mRNAs in the absence of E1B 55K. Surprisingly, these included some mRNAs in which the RRE, although present in the primary transcripts, was removed from the mature species. A mechanism was proposed in which Rev/RRE action in this system may commit an RNA to a pathway of facilitated nuclear export before excision of the RRE during processing