Molecular basis for naturally occurring elevated readthrough transcription across the M-F junction of the paramyxovirus SV5

Abstract

Transcription of the paramyxovirus RNA genome is thought to involve a sequential stop-start mechanism whereby monocistronic mRNAs are produced by polyadenylation and termination of 3' upstream gene followed by reinitiation at the downstream start site. For a number of paramyxoviruses, transcription across the M-F gene junction results in the synthesis of high levels of a dicistronic M-F readthrough RNA. In cells infected with the paramyxovirus SV5, 15% or less of the transcripts from the viral P, M, SH, HN, and L genes were detected as readthrough products with the 3' proximal gene. By contrast, approximately 40% of the SV5 F mRNA was detected as a dicistronic M-F transcript. A comparison of the individual SV5 gene junctions showed that elevated M-F readthrough transcription correlate with the M gene end having the shortest U tract for directing polyadenylation and a gene end sequence that differs from the consensus sequence. We have tested the hypothesis that elevated M-F readthrough transcription results from an inefficient termination signal at the end of the M gene. A reverse genetics system was established whereby SV5 transcription was reconstituted in transfected cells using cDNA-derived polymerase components and dicistronic minigenomes that encoded either the SV5 M-F or the SH-HN gene junction. Chimeric SV5 minigenomes were constructed to contain exchanges of a 10 base gene end sequence and the U tract from the M-F (approximately 40% readthrough) and SH-HN (approximately 15% readthrough) junctions. Northern blot analysis of RNA synthesized from these altered templates showed that, in the context of the M-F intergenic region, increasing the length of the M gene end U tract from four residues to six or eight U residues did not decrease M-F readthrough transcription. In contrast, chimeric minigenomes that contained the 10 base region from the end of the SH gene directed very efficient gene termination and a corresponding decrease in readthrough transcription. Mutational analysis showed that a single G to A substitution located five bases 3' to the M gene U tract was sufficient to convert the M gene end region to an efficient signal for polyadenylation-termination. These results demonstrate a role for the gene end region located immediately 3' to the U tract as a major determinant of transcription termination in the paramyxovirus genome. The possible role of M-F readthrough transcription in the paramyxovirus growth cycle is discussed.