PROSITE documentation PDOC52108

Mononegavirales large (L) protein polyribonucleotidyl transferase (PRNTase or capping) domain profile

Non-segmented negative-strand (NNS) RNA viruses belonging to the order Mononegavirales are highly diversified eukaryotic viruses, including significant human pathogens, such as rabies (rhabdovirus), Nipah (paramyxovirus), respiratory syncytial (pneumovirus), and Ebola (filovirus). Although these NNS RNA viruses manifest various morphological and biological characteristics, the structures of their genomes and multifunctional RNA-dependent RNA polymerase (RdRp) large (L) proteins are quite similar. Therefore, their basic strategies for genome transcription and replication are expected to be similar. Their multifunctional, L polymerase proteins, carried within the virions, have biochemical properties that distinguish them from most other RNA polymerases of viruses or of their hosts. In addition to their RdRp activity, NNS RNA virus L proteins catalyze an unusual sequence of mRNA capping reactions, and the RdRp itself polyadenylates the viral message. NNS RNA viruses produce 5'-capped and 3'-polyadenylated mRNAs resembling higher eukaryotic mRNAs [1,2,3,4,5,6,7,8,9].

NNS RNA virus L acts as an all-in-one viral mRNA assembly machinery. The single-polypeptide L protein catalyzes all pre-mRNA modifications co-transcriptionally in the following order: (i) 5'-capping (polyribonucleotidylation of GDP) to form a GpppA cap core structure, (ii) 2'-O-methylation of GpppA into GpppAm, (iii) guanine-N(7)-methylation of GpppAm into m(7)GpppAm (cap 1), (iv) 3'-polyadenylation to yield a poly(A) tail. It is made of five domains: three-an RdRp domain (see <PDOC50507>), an mRNA capping or GDP polyribonucleotidyl transferase (PRNTase) domain, and a methyltransferase (MTase) domain (see <PDOC51555>)-with assigned enzymatic activity and two-a connector or connecting domain (CD) between the PRNTase and MTase domains and a C-terminal domain (CTD)-that appear to have largely organizational roles. The L PRNTase domain is responsible for the addition of a 5' cap to nascent viral mRNAs. The mechanism of mRNA capping with L proteins is distinct from that with the eukaryotic mRNA capping enzyme. The L PRNTase (EC 2.7.7.88) domain transfers 5'-monophospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme (L)-pRNA intermediate to yield the cap on RNA, whereas eukaryotic guanylyltransferase (GTase) transfers GMP from GTP to 5'-diphospho-RNA (ppRNA) via a covalent enzyme-GMP intermediate. In addition, the PRNTase domain contains a priming loop, which is thought to facilitate de novo initiation. The PRNTase priming loop undergoes temporal conformational changes that regulate initiation, elongation, and mRNA capping during RNA synthesis. The loop from the PRNTase domain projects into the catalytic site of the RdRp domain, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L [1,2,3,4,5,6,7,8,9].

The PRNTase domain is located across from the RdRp domain (shell-like arrangement). The largely α-helical, N-terminal half, which abuts the polymerase domain, is well ordered. The C-terminal half has several poorly defined segments, including the loop that bears the HR sequence (see <PDB:5A22>). The PRNTase domain possesses five collinear motifs, R-x(3)-W-x(3,8)-phi-x-G-x-zeta-x-[PA] (motif A), [YW]-phi-G-S-x-T (motif B),W (motif C), H-R (motif D), and zeta-x-x-phi-x-[FY]-Q-x-x-phi (motif E) (phi, hydrophobic; zeta, hydrophilic amino acids), which are conserved in NNS RNA viral L proteins. The different conserved motifs are clustered around the center of the cavity formed between the RdRp and PRNTase domains and are located in close proximity to build the active site of the PRNTase domain. Two motifs, B and D, form the catalytic pocket of the capping domain. Motif B that contains the GxxT sequence participates in guanosine nucleotide binding. Motif D (also referred to as the His-Arg or HR motif) consists of two catalytic residues that are critical for cap formation. The His in motif D serves as a catalytic amino acid residue, in which the N(epsilon2) position of the imidazole ring is covalently linked to the 5'-terminal α-phosphorus atom of 5'-monophospho-RNA (pRNA) with a phosphoamide bond during the intermediate formation. The pRNA linked to the His is specifically transferred to GDP, resulting in the release of GpppRNA from the PRNTase domain [1,2,3,4,5,6,7,8,9].

The profile we developed covers the entire L protein PRNTase domain.