Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection

Abstract

The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1-N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.Respiratory syncytial (RS) virus is an important human pathogen that causes lower respiratory tract infection in children. RS virus is a member of the genus Pneumovirus in the family Paramyxoviridae. Members of this family are nonsegmented, negative-sense, single-stranded RNA viruses. The RS virus genome contains 10 genes, encoding at least 11 proteins (6, 7). Three of the RS virus gene products are essential for RNA replication: the nucleocapsid (N) protein, which encapsidates the viral genome, and the large polymerase protein (L) and the phosphoprotein (P), which comprise the viral RNAdependent RNA polymerase (RdRp) (13,32,36).In contrast to replication, efficient transcription of RS mRNAs requires an additional gene product, the M2-1 protein. The M2-1 protein is encoded by the first of two open reading frames (ORFs) of the M2 gene (5, 8). The second ORF of the M2 gene encodes the M2-2 protein. The M2-1 protein is a basic phosphoprotein of 194 amino acids that functions in transcription as a processivity factor and an antiterminator (4, 16). M2-1 colocalizes with the N protein in cytopl...

Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex

“…This variant has diminished transcriptional elongation and anti-termination capacities (50 %) compared with those of normal M2-1 protein (Fig. 3c), as occurred for the corresponding A2 strain M2-1 protein variant (Cartee & Wertz, 2001). The M2-1 T56,S58A variant was assayed with the P protein variant T105,108A (Fig.…”

“…Transiently expressed Long and A2 strain M2-1 proteins had an electrophoretic mobility lower than that of M2-1 protein from purified extracellular viral particles, due to their phosphorylation at T56 and S58 or at S58 and S61 (Cartee & Wertz, 2001) residues, respectively. Coexpression of M2-1 and P proteins leads to their interaction; the M2-1 protein is probably not phosphorylated in this interaction and its electrophoretic mobility increases (Cuesta et al, 2000).…”

“…The P protein residue at position 105 is probably T (it cannot be replaced by A or D), whereas T108 can be replaced by A but not by D, suggesting that both residues are involved in contact with the M2-1 protein and that phosphorylation of P protein T108 prevents it. Phosphorylation at T108 would control P protein interaction with the M2-1 protein and, therefore, the M2-1 regulatory activity on the L protein during viral transcription (Cartee et al, 2003).Transiently expressed Long and A2 strain M2-1 proteins had an electrophoretic mobility lower than that of M2-1 protein from purified extracellular viral particles, due to their phosphorylation at T56 and S58 or at S58 and S61 (Cartee & Wertz, 2001) residues, respectively. Coexpression of M2-1 and P proteins leads to their interaction; the M2-1 protein is probably not phosphorylated in this interaction and its electrophoretic mobility increases (Cuesta et al, 2000).…”

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

“…M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7,8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11).…”

“…M2-1 is phosphorylated at serines 58 and 61 (9,14), and its abrogation using individual phosphoablatant mutants S58A and S61A dramatically reduces M2-1 antitermination activity (9), indicating that phosphorylation is critical for M2-1 function. The RNA binding specificity of M2-1 is still debated, with antigenomic leader, viral mRNA, poly-A, or no sequence specificity having been proposed (9,11,12,17).…”