Detection and characterization of proteins encoded by the second ORF of the M2 gene of pneumoviruses

The nucleotide sequence of the M2 gene of pneumonia virus of mice (PVM) was determined. The sequence showed that the gene encoded a protein of 176 ami no acids with a predicted molecular mass of 20165 Da from a major ORF, whic h is smaller than the equivalent proteins encoded by human, bovine and ovin e respiratory syncytial (RS) viruses. The PVM M2 protein is conserved, havi ng 41% similarity to the equivalent human RS virus protein. In common with the M2 genes of the RS viruses and avian pneumovirus (APV), the PVM mRNA al so contained a second ORF (ORF2) that partially overlaps the first ORF and which is capable of encoding a 98 residue polypeptide. No significant seque nce identity could be detected between the putative M2 ORF2 proteins of PVM , APV and the RS viruses. The expression of the M2 ORF2 proteins of the pne umoviruses was investigated by using monospecific antisera raised against G ST fusion proteins. Western blot analysis demonstrated the presence of poly peptides encoded by M2 ORF2 of PVM and RS virus corresponding with those pr edicted by in vitro translation studies, but this was not the case for APV, The PVM polypeptide was present as three distinct products in vivo. The PV M and RS virus polypeptides were also detected in cells by immunofluorescen ce, which showed that both were present in the cytoplasm with a degree of l ocalization in inclusion bodies. No APV M2 ORF2 protein could be detected i n vivo. The RS virus M2 ORF2 polypeptide was shown to accumulate during inf ection and the potential implications of this are discussed.