The M2-2 Protein of Human Respiratory Syncytial Virus is a Regulatory Factor Involved in the Balance Between RNA Replication and Transcription

The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory "switch" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.