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.

RNA Sequence Determinants of a Coupled Termination-Reinitiation Strategy for Downstream Open Reading Frame Translation in Helminthosporium victoriae Virus 190S and Other Victoriviruses (Family Totiviridae)

“…Stop-restart in pneumoviruses, however, appears to occur by a distinct mechanism. At present, the evidence suggests that a large RNA structure element in a region upstream of stop and restart codons is required for reinitiation of translation in pneumoviruses (15,16), but the nature of this structure and how it works to promote reinitiation remain unknown.…”

“…This strategy has been shown to promote the translation of downstream ORFs in the mRNAs of several different plus-or minus-stranded RNA mammalian viruses, including those that encode protein VP2 or VP10 of certain caliciviruses (30,33,34), the BM2 protein of influenza B virus (21, 40), and the M2-2 proteins of respiratory syncytial virus (RSV) and other pneumoviruses (1,15,16). One example of a plus-stranded RNA fungal virus that employs such translational coupling has also been described (17).…”

Viral and Host Factors in Human Respiratory Syncytial Virus Pathogenesis

“…1). Each gene is transcribed into a separate, capped, polyadenylated mRNA encoding a single viral protein, except in the case of the M2 mRNA, which contains two overlapping open reading frames that are expressed by a ribosomal stop-restart mechanism into two distinct proteins, M2-1 and M2-2 (52).…”

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination–reinitiation mechanism

“…Transfections of HEp2 cells were carried out in triplicate and repeated a minimum of three times and CAT protein detection by ELISA was carried out as described previously (Gould and Easton 2007). The antibodies used for direct detection of proteins in Western blots were for CAT #ab50151 and for GFP #ab290 (AbCAM).…”