Respiratory syncytial virus M2-1 protein requires phosphorylation for efficient function and binds viral RNA 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 RNA-dependent 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 cytoplasmic inclusions and interacts with the N protein, as assayed by coimmunoprecipitation with a monoclonal antibody (MAb) to M2-1 (12, 15). There is a conserved Cys3His1 motif, predicted to bind zinc, at the amino terminus of the M2-1 protein, and maintenance of the predicted zinc coordinating residues is essential for the function of the protein (15). Mutation of the cysteine or histidine residues of this motif results in a loss of M2-1 phosphorylation, loss of the M2-1–N interaction, and loss of M2-1 transcriptional activity (15). Although, it has not been directly demonstrated that this motif in M2-1 binds zinc, it is apparent that maintenance of the motif is required for activity. The M2-1 protein in RS virus-infected cells exists in different phosphorylation states, resulting in its migration as at least two bands by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (15, 23, 30); the slower-migrating species contains the majority of the phosphorylated form, and the faster-migrating species lacks significant phosphorylation. The sites of M2-1 phosphorylation were recently reported as threonine 56 and serine 58 (9). The contribution of phosphorylation to the function of M2-1 has not been investigated. As with other negative-strand viruses, transcription of RS viral genes is obligatorily sequential. The RdRp enters at or near the 3′ end of the genome, and genes are transcribed in order from the 3′ to the 5′ end of the genome (10). Attenuation occurs at each gene junction, resulting in a gradient of mRNA synthesis such that genes nearest the 3′ end of the genome are transcribed more abundantly than those near the 5′ end (2, 10). The junctions between RS virus genes contain semiconserved gene end and conserved gene start sequences separated by nonconserved intergenic sequences of various lengths (3). The gene end sequence contains the signals for the RS virus RdRp to polyadenylate and terminate synthesis of the mRNA (3, 19, 21). However, the efficiency of termination at the various RS virus gene junctions differs (14). The conserved gene start sequence signals initiation of a new mRNA (20). In order to initiate synthesis of the new mRNA, the RdRp must first terminate synthesis of the upstream message (17, 21). The M2-1 protein functions as both a transcriptional processivity factor and an antiterminator (4, 16). The M2-1 processivity function prevents intragenic termination, allowing the synthesis of full-length mRNAs (11), and the antitermination function causes the RdRp to ignore the semiconserved gene end sequence (14, 16), resulting in the synthesis of polycistronic RNAs observed in infection. The relative contributions of processivity and antitermination to the function of M2-1 have not been clearly discriminated. Both of these features of M2-1 may be necessary to allow deeper access of the RS virus RdRp on the genomic template, which is potentially important, given that pneumovirus genomes are longer and contain more genes than those of most paramyxovirus members. In addition, the various RS virus genes respond differently to the M2-1 protein (11, 14). This differential sensitivity may represent a mechanism by which the virus further regulates the expression of certain gene products. In the work reported here, we first determined the number and location of phosphorylated residues in the M2-1 protein, and our results show that serines 58 and 61 are phosphorylated, in contrast to a previous report (9). We next examined the effect of eliminating phosphorylation, by site-specific mutagenesis of individual sites or sites in combination, on the function of the M2-1 protein as a transcriptional antiterminator and on its interaction with the N protein. Our data show that phosphorylation is important for the M2-1 protein function in transcription. Further, we show that the M2-1 protein binds viral mRNAs in infected cells and that the interaction of the M2-1 and N proteins in transfected cells is mediated by RNA.