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

Abstract

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.H uman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children and the immunocompromised. HRSV is a pneumovirus of the Paramyxoviridae family of the order Mononegavirales-the nonsegmented negative-strand RNA viruses. Its genome encodes 10 genes that are each transcribed by an RNA-dependant RNA polymerase (RdRp) into single mRNAs. During transcription, the RdRp uses a single promoter in the 3′ leader region (Le) of the genome (1) and responds to gene start and gene end sequences flanking each gene, directing initiation and termination of mRNA transcription, respectively (2). During genome replication, the RdRp bypasses these signals to synthesize a full-length antigenome. The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3, 4).M2-1 prevents premature transcription termination both intra-and intergenically (5, 6). 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). Based on mutational analysis and a partial M2-1 structure determined using NMR (12, 13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exh...

RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription

“…4B). Cooperative binding of RNA by VP30 would not be too surprising, considering that the VP30 hexamer may expose multiple RNA binding patches rather than a single composite RNA-binding interface, similar to the VP30-related tetrameric M2-1 transcription factor of respiratory syncytial virus (RSV), which presents several positively charged patches that are spread over an extended surface area (27). As the aforementioned previous study (15) reported a substantially increased K d for the R40A mutant using an RNA substrate derived from the antigenomic strand (nt 36 to 80), we additionally tested binding of the VP30 arginine mutants to the antigenomic RNA (nt 56 to 158) probe.…”

“…The almost complete loss of RNA binding affinity observed for the hexamerization-defective VP30_5LA mutant indicated that a VP30 hexamer is required to assemble a composite interaction array for RNA substrates, similar to the functionally related tetrameric M2-1 protein exposing an array of positively charged patches spread over the protein's surface (27). Examples of similar architectural concepts may include the bacterial Hfq hexamer and eukaryal Lsm proteins that "chaperone" RNA-RNA and RNAprotein interactions (35).…”

Predictive and comparative analysis of Ebolavirus proteins

“…In addition, a scan of PDB sequences with the conserved pattern C-x (8)-C-x(4)-C-x(3)-H using ScanProsite 74 reveals exactly the same motif in CCCH zinc fingers (PDB id: 2d9n). All the CCCH zinc fingers belongs to one homologous group in the ECOD database, 48 and this family contains the N-terminal domain of the transcription antiterminator M2-1 from another Mononegavirales, Pneumovirus (4C3B 75 and 4CS7, 76 alignment shown in Fig. S1).…”

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

“…In addition, the M2-1 protein binds directly to P (27). The binding of P and RNA to M2-1 was found to be mutually exclusive because of partially overlapping interaction surfaces (26,28). Although A73 and N88 are away from the RNA/P binding interface, they could possibly be on the path of the exiting nascent RNA molecule.…”

“…The M2-1 protein also increases the synthesis of polycistronic read-through mRNAs as noted. It likely binds nascent mRNA cotranscriptionally and prevents termination by the viral polymerase (26). In addition, the M2-1 protein binds directly to P (27).…”