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.

Increased Readthrough Transcription across the Simian Virus 5 M-F Gene Junction Leads to Growth Defects and a Global Inhibition of Viral mRNA Synthesis

“…6C) that has little similarity to the SV5 gene end consensus sequence 3Ј-A/UUUCU 4 to 7 (32). Based on our previous results (33), it is possible that the combination of a new gene end and a foreign intergenic region (Rep22) creates a signal on the nucleocapsid template or in the nascent mRNA chain which is efficiently recognized for termination by the SV5 polymerase.…”

“…In cells infected with WT SV5, ϳ40% of the F mRNA is detected by Northern blotting in an M-F dicistronic form (32). M-F readthrough transcription has been reported to be even higher in the case HPIV-2 (ϳ60%; Parks et al, unpublished), HPIV-1 (ϳ80% [4]), and SV41 (100% [43]).…”

“…However, recent evidence suggests that cis-acting signals at the gene junctions may also contribute to controlling the relative level of mRNA synthesis from a particular viral gene (11,12,14,33). Our mutational analysis has focused on the M-F intergenic region, since it has been proposed that this junction may represent a critical site for transcription control (20,32,33). In this study, we have tested the hypothesis that M-F readthrough transcription serves as a mechanism to control viral transcription and growth.…”

The Aberrant Gene-End Transcription Signal of the Matrix M Gene of Human Parainfluenza Virus Type 3 Downregulates Fusion F Protein Expression and the F-Specific Antibody Response In Vivo

“…For instance, in the case of parainfluenza virus type 5, a single nucleotide within the M-GE sequence located 5 positions upstream of the oligo(U) tract enhances M-F readthrough transcription (26). For Sendai virus (SeV), the GS signal of the F gene has a reduced efficiency, resulting in the reduced transcription of the F gene as well as of downstream genes (27).…”

“…For Sendai virus (SeV), the GS signal of the F gene has a reduced efficiency, resulting in the reduced transcription of the F gene as well as of downstream genes (27). Interestingly, simian virus 41 completely lacks any apparent M-GE signal, and the M gene is transcribed exclusively as an M-F bicistronic mRNA with reduced monocistronic F transcription (26,28). HPIV1, on the other hand, has several cis-acting elements, including the M-F intergenic sequence, the F-GS signal, and a long upstream NTR in the F gene, which together attenuate M gene transcription termination, promote higher levels of M-F readthrough, and result in reduced expression levels of F protein in infected cells (29).…”