Mutations affecting transcriptional termination in the p gene end of subacute sclerosing panencephalitis viruses

Abstract

Numerous mutations are found in subacute sclerosing panencephalitis (SSPE) viruses, and the M gene is the gene most commonly affected. In some SSPE viruses, such as the MF, Osaka-1, Osaka-2, and Yamagata-1 strains, translation of the M protein is complicated by a transcriptional defect that leads to an almost exclusive synthesis of dicistronic P-M mRNA. To understand the molecular mechanisms of this defect, we sequenced the P gene at the P-M gene junction for several virus strains and probed the involvement of several mutations in the readthrough region via their expression in measles virus minigenomes containing different sequences of the P-M gene junction and flanking reporter genes. The deletion of a single U residue in the U tract of the Osaka-1 strain (3'-UAAUAUUUUU-5') compared with the consensus sequence resulted in a marked reduction of the expression of the downstream reporter gene. In addition, the expression of the downstream gene was markedly decreased by (i) the substitution of a C residue in the U tract of the P gene end of the OSA-2/Fr/B strain of the Osaka-2 virus (3'-UGAUAUUCUU-5' compared with the sequence 3'-UGAUAUUUUU-5' from a sibling virus of the same strain, OSA-2/Fr/V), and (ii) the substitution of a G in the sequence of the P gene end of the Yamagata-1 strain at a variable site immediately upstream from the six-U tract (3'-UGAUGUUUUUU-5' instead of 3'-UGAUUUUUUUU-5'). Mutations at the P gene end can account for the readthrough transcription variation at the P-M gene junction, which directly affects M protein expression.

FIG. 1.

Structure of the minigenome. The P-M gene junction sequence of the Edmonston MV was mutagenized to reproduce the sequences of OSA-1/Fr/V, OSA-2/Fr/V, OSA-2/Fr/B, OSA-3/Bs/V, and the two Yamagata-1 strains.

FIG. 2.

Analysis of mRNA of reporter genes. After conversion to cDNA from oligo(dT)-selected RNA with an oligo(dT)-containing primer (minig-R), the 3′-end regions of the firefly (F) and Renilla (R) luciferase genes were amplified by PCR with the same reverse primer and the gene-specific primer (scheme). For a detail of each plasmid clone, see Fig. 3.

FIG. 3.

Transcriptional termination efficiency of different P-M gene junction sequences inserted between the firefly and Renilla luciferase genes. Efficiency (superscript a) is expressed as the percentage (mean of four experiments [plusmn] standard deviation) of Renilla luciferase activity relative to that of firefly luciferase. The plasmid clone and the corresponding sequences of the P gene end and P-M gene junction are presented at the left of each bar. Superscript b, clone of a very rare variant found in the OSA-1/Fr/V virus; superscript C, clone containing a single nucleotide back-mutation from clone 2K-125. Nucleotides different from the consensus sequence are boxed.