Coupled translation of the second open reading frame of M2 mRNA is sequence dependent and differs significantly within the subfamily Pneumovirinae

Abstract

Coupled translation, first described in the M2 gene of pneumovirus respiratory syncytial virus (RSV), is an alternative mechanism of translational initiation in which the ribosomes which translate the first (M2-1) open reading frame (ORF) move a short distance upstream after termination and reinitiate translation from a second (M2-2) overlapping ORF. Here, we show that the same mechanism occurs in two closely related viruses, avian pneumovirus (APV) and pneumonia virus of mice (PVM), although with markedly different efficiencies. To identify the reasons for the variation in efficiency of coupled expression between RSV and APV, we used chimeric M2-1 genes containing different lengths of the M2-1 ORF from each virus. An essential component allowing coupled expression in the chimeras was a segment from the RSV M2-1 coding region containing a high degree of secondary structure. Additional sequences at the 5' end of the RSV M2-1 ORF also promoted coupled translation when the region with high levels of secondary structure was present. These data indicate that at least two distant parts of the mRNA transcript, together with a suitable overlapping region, are involved in the coupling process. Replacement of the last 102 nucleotides of the RSV M2-1 ORF with the equivalent APV sequence showed identical levels of coupled translation. Thus, the overlapping region can direct the ribosome back onto the start codon of the second ORF while the upstream coding sequence of the M2-1 ORF determines the levels of coupled expression.

FIG. 1.

(A) Organization of selected pneumovirus M2 transcripts of RSV strain A2, PVM strain 15, and APV strain CVL/14, adapted from reference . The superscripts refer to the nucleotide positions of the first residues of the start and stop codons of the ORFs in the mRNAs. (B) Sequence of the overlap in the M2 transcript of RSV, PVM, and APV ORFs. Start codons for the second ORFs are underlined and italicized, and the stop codons of the M2-1 ORFs are shown in boldface.

FIG. 2.

(A) Schematic representation of the reporter gene constructs made to evaluate coupled translation in PVM. Transcription from the plasmids were under the control of the bacteriophage T7 promoter, as indicated. (B) Sequence alignment of the overlap regions in the PVM M2-1/CAT constructs. The CAT ORF is cloned in frame using the XbaI site (italics) to the M2-2 start codon(s) within the overlap. Positions of the M2-2 ORF start codons are underlined and in italics, and the positions of the M2-1 ORF stop codons are shown in boldface. Mutated residues are in lowercase. (C) Levels of CAT protein expression of each mutant relative to the pPVMWC positive control.

FIG. 3.

(A) Schematic representation of the reporter gene constructs made to evaluate coupled translation in APV. (B) Sequence alignment of the overlap regions in the APV M2-1/CAT constructs. The CAT ORF is cloned in frame using the XbaI site (italics) to the M2-2 start codon(s) within the overlap. Positions of the M2-2 ORF start codons are underlined and in italics, and the positions of the M2-1 ORF stop codons are shown in boldface. Mutated residues are in lowercase. Additional sequences downstream of the XbaI site in pAPVStop2 are shown with the additional mutation removing the in-frame stop codon highlighted. (C) Levels of CAT protein expression of each mutant relative to the pAPVWC positive control.

FIG. 4.

Schematic representation of (A) chimeric APV M2-2-CAT protein (plasmid pAPVM2-2CAT) and (B) chimeric RSV/APV M2-1 with overlapping CAT ORFs made to evaluate coupled translation in full-length APV M2 reporter constructs.

FIG. 5.

(A) Schematic representation of the RSV-APV M2-1 chimeric proteins. The numbers indicate the nucleotide positions of the junction regions of the chimeric genes (above RSV and below APV). The position of the 3′ end of the region identified as being important for coupled expression in the RSV M2 gene (11) is indicated with an arrow, when present. The M2-2 ORF fused to the CAT gene ORF is also shown. (B) Levels of CAT protein expression of each mutant relative to the RSV pWildCAT positive control.