Human metapneumovirus (HMPV) is a negative-strand, non-segmented ssRNA virus of the Paramyxoviridae family and a major cause of acute respiratory tract infections in children, elderly and immunocompromised populations worldwide (van den Hoogen et al., 2002; van den Hoogen et al., 2003). HMPV and the closely-related respiratory syncytial virus (RSV) constitute respectively the Metapneumovirus and Pneumovirus genera of the Pneumivirinae subfamily. These viruses share conserved replication strategies and similar genome organizations with other members of the Mononegavirales order, which includes numerous important human pathogens such as measles, rabies, and Ebola virus. The HMPV genome encodes nine proteins, three of which are necessary and sufficient for viral replication, namely the nucleoprotein (N), the RNA-dependent RNA polymerase (L) and its essential cofactor, the phosphoprotein (P). The genomic RNA is encapsidated by the N protein, which acts as a template for the L protein that is responsible for both replication and transcription. The replicase is highly processive, and generates a complete, encapsidated positive-sense antigenome, which is in turn used as a template for the synthesis of genomic RNA. The transcriptase produces capped and polyadenylated monocistronic mRNAs using a sequential stop and restart mechanism in which the polymerase responds to cis-acting signals located in intergenic regions (Sutherland et al., 2001).
Members of the Pneumovirinae subfamily harbour highly conserved 9-10 nucleotide transcription promoters (‘gene start’) and semi-conserved 12-13 nucleotide ‘gene end’ (GE) signals with the consensus sequence 5′-AGUUAnnnAAAAA-3′ (positive sense), which direct polyadenylation and release of nascent mRNA and are critical for polymerase processivity (Harmon et al., 2001; Sutherland et al., 2001). Termination of each gene is required to allow transcription of the next gene downstream, and the propensity of the polymerase to dissociate from its template results in transcriptional attenuation at each gene junction (Fearns and Collins, 1999).
HMPV M2-1 is a basic protein of 187 amino acids that is required for virus infectivity in vivo, but has been found to be dispensable for recovery or growth of recombinant virus in tissue culture (Buchholz et al., 2005). The closely-related M2-1 protein from avian metapneumovirus, which shares 85% sequence identity with HMPV M2-1, has been shown to increase minigenome expression by at least 100-fold (Naylor et al., 2004). These observations are in contrast with studies of RSV M2-1, which was shown to be absolutely essential for transcription of full-length viral mRNAs (Collins et al., 1996; Fearns and Collins, 1999). M2-1 proteins from HMPV and RSV are amongst the most conserved within the Pneumivirinae and share 38% overall sequence identity, suggesting similar functional roles. In addition, M2-1 shares structural and functional similarity with Ebola virus VP30 (Blondot et al., 2012). Recently, the crystal structure of RSV M2-1 has been solved by X-ray crystallography, revealing a tight, disk-like tetrameric assembly (Tanner et al., 2014), which contrasts with previous solution studies that suggested a non-globular, extended tetramer (Esperante et al., 2011).
M2-1 is recruited to the viral transcription complex by the intrinsically-disordered P protein (Khattar et al., 2001; Derdowski et al., 2008). The recruitment of M2-1 occurs through an interaction with the M2-1 core domain (Tran et al., 2009; Blondot et al., 2012), resulting in the formation of a high-affinity, non-globular complex (Esperante et al., 2012), which in RSV is controlled by phosphorylation of Thr108 of the P protein (Asenjo et al., 2006). Studies on RSV have shown that M2-1 functions as both an intragenic and intergenic transcription antitermination factor in Pneumoviruses, allowing synthesis of complete viral mRNAs and inhibiting transcription termination at the GE signal with various efficiencies, resulting in an increased proportion of polycistronic read-through mRNAs (Hardy and Wertz, 1998; Hardy et al., 1999). Interestingly, NMR studies have shown that the RSV M2-1 core domain preferentially recognizes poly-A tails of viral mRNAs (Blondot et al., 2012), and this property has been confirmed with full-length protein using fluorescence anisotropy (Tanner et al., 2014). On the basis of this data, it has been suggested that M2-1 likely binds nascent mRNA transcripts, thus preventing premature termination through stabilization of the transcription complex and inhibition of RNA secondary structure formation (Blondot et al., 2012; Tanner et al., 2014). As structural data are lacking for HMPV M2-1, its role in HMPV transcription antitermination has remained elusive.
In this study, we report X-ray crystallographic structures of the HMPV M2-1 at resolutions ranging from 2.0 to 2.5 Å. Unlike the disk-like assembly reported for RSV, our HMPV M2-1 structures revealed dissociation of one protomer core domain from the tetramer interface. Solution small angle X-ray scattering (SAXS) and atomistic coarse-grained molecular dynamics (MD) simulations demonstrated that M2-1 behaves as a dynamic, modular protein in equilibrium between open and closed forms. Crystallographic studies of M2-1 bound to nucleic acids reveal how the M2-1 zinc finger specifically recognizes RNA. Finally, SAXS and electron microscopy showed that interaction with GE signals induces the closed conformation of M2-1, a process which is coupled with concentration-dependent aggregation in solution. Our results provide a structural basis for the recognition of GE signals by M2-1, and the prevention of premature mRNA termination.
