Structural Phosphoprotein M2-1 of the Human Respiratory Syncytial Virus Is an RNA Binding Protein

Abstract

The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparent K d of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH 2 terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus of the Paramyxoviridae family, is the most common infectious agent in bronchiolitis and pneumonia requiring hospitalization of infants and young children. Immunocompromised and elderly people are also severely affected. No vaccine or specific antiviral treatment is available at present (11). The best way to control these infections, according to the characteristics of the human populations mainly affected, is the use of different agents as live attenuated vaccines (38), humanized neutralizing monoclonal antibodies (21), and antiviral compounds (27).The HRSV nucleocapsids are, as in all paramyxoviruses, structural components and functional units for replication and transcription processes (11). The nucleocapsid proteins are thus multifunctional, serving as ideal targets for the action of antiviral compounds and for conveying attenuation mutations. The design of both types of reagents would be aided by understanding nucleocapsid protein functions.HRSV nucleocapsids are composed of the negative-sense RNA genome (15,222 nucleotides long), tightly bound to the N protein, the large polymerase protein (L), the phosphoprotein (P), and probably the M2-1 protein (11, 16). The RNA polymerizing activity of the viral nucleocapsids is due to L protein, for which activity P protein is essential (41). N protein plays a histone-like function, since synthesis of viral RNA always occurs on ribonucleoprotein templates. P protein also acts as a chaperone of N protein, maintaining it competent for encapsidation (30). M2-1 protein enhances the processivity of the viral polymerase (9, 10) and its readthrough of intergenic junctions (17). These properties identified M2-1 protein as an elongation and antiterminator transcription factor, a constituent of the nucleocapsids. The protein is encoded by the M2 gene, present only in the pneumoviruses, which codes for an mRNA containing two open reading frames (ORFs). ORF1 encodes the 22K structural protein, also known as M2-ORF1 or M2-1 protein (194 amino acids). ORF2 encodes M2-2 protein (90 amino acids), detected in HRSV-infected cells, which has a regulatory function in the balance between viral transcription and replication (1,6,8).The M2-1 protein was long ago described as a structural membrane phosphoprotein that could be present ...

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

“…3c). This P protein variant does not facilitate M2-1 protein activity, although the M2-1 variant is essentially in an unphosphorylated form (Cuesta et al, 2000). It appears that P-M2-1 physical interaction, rather than the absence of M2-1 protein phosphorylation, is needed for M2-1 protein transcriptional cofactor activities, as suggested previously (Mason et al, 2003).…”

“…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). According to our interpretation, when M2-1 and P protein variants T105A, T105D, T108D, T105,108A and T105,108D were coexpressed, M2-1 protein phosphorylation was found, as indicated by its reduced electrophoretic mobility (Fig.…”

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

“…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). In this article, we present the crystal structure of full-length, tetrameric HRSV M2-1 protein in both wild type (WT) and phosphomimetic forms.…”

Deletion of M2 Gene Open Reading Frames 1 and 2 of Human Metapneumovirus: Effects on RNA Synthesis, Attenuation, and Immunogenicity

“…These activities of M2-1 are sensitive to mutations in the Cys/His motif (23). The HRSV M2-1 protein also has been shown to be an RNA-binding protein and binds to the N protein (9,15). Expression of the HRSV M2-1 protein, either from an added support plasmid or by promiscuous expression from the antigenome cDNA, appears to be essential for the recovery of recombinant HRSV from transfected cDNA, and it has not been possible to recover infectious HRSV in which the integrity of the M2-1 protein has been drastically disturbed by introduced mutations or by extensive deletion (10,12,28,34).…”