The M2–2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription

Abstract

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.