<p>The mechanisms underlying mRNA capping in non-segmented negative-strand RNA viruses, except rhabdoviruses, remain elusive. Here, we demonstrate that the RNA-dependent RNA polymerase (RdRp), composed of the large protein (L) and the phosphoprotein (P), of human respiratory syncytial virus (HRSV, a pneumovirus) catalyzes capping through GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers 5'-monophosphorylated RNA (pRNA) from 5'-triphosphorylated RNA to GDP via a putative covalent L-pRNA intermediate. The HR motif (motif D) on the catalytic loop of the L PRNTase domain is critical for capping, whereas the GxxT sequence in motif B is dispensable, unlike in rhabdoviruses. The capping activity is inhibited at the intermediate formation step by JNJ-8003, an antiviral compound that binds a cleft between motifs D and E. Pneumovirus-specific residues in the HRSV counterpart of the rhabdovirus priming-capping loop are not essential for either capping or de novo RNA synthesis initiation, although a di-lysine sequence is required for efficient capping. The L-P complex preferentially methylates the cap core at the guanine-N7 position and then methylates cap 0 at the 2'-O position, forming cap 1, in direct contrast to the methylation order observed in rhabdoviruses. Thus, the L enzymatic domains exhibit common and virus-specific properties in cap formation among pneumoviruses and rhabdoviruses.</p>
