SV40 deletion mutant (d1861) with agnoprotein shortened by four amino acids

d1861 is an SV40 deletion mutant which was thought to lack the agnoprotein coding region and was used to verify the role of agnoprotein in the life cycle of SV40. In the present study the region flanking the deletion was sequenced and, in contrast to the available information, it was found that d1861 lacks 12 nt in phase, downstream from the AUG start codon of agnoprotein (residues 347-358). Using the runoff protocol with viral transcriptional complexes (VTC), that in vitro elongate the in vivo preinitiated nascent RNA, it was found that in vivo the major initiation site for late transcription is at residue 325, the same as in wild type (WT). In comparison with WT, d1861 encodes information for agnoprotein shortened by four amino acids and it has been identified in d1861 infected cells. However, pulse-chase experiments indicated that the rate of synthesis of d1861 agnoprotein is slower than that of WT agnoprotein and that it has a turnover rate of 1 hr as compared to 3 hr of WT agnoprotein. The reduced rate of synthesis of d1861 agnoprotein can be explained by nuclease S1 analyses in which the major leader of d1861 16 S RNA, that encodes the agnoprotein, appeared in significantly lower amounts as compared to the major leader of WT 16 S RNA. Furthermore, analysis of the potential secondary structures at the 5' end of the leader of d1861 16 S RNA has revealed stable structures in which the start codon of agnoprotein is sequestered in a stem. The involvement of RNA secondary structures in regulating the synthesis of agnoprotein is discussed.