Functional dissection of the C-terminal domain of rabies virus RNA polymerase L protein

The rabies virus large (L) protein interacts with its cofactor phosphoprotein (P protein) to function as an RNA-dependent RNA polymerase (RdRp). The C-terminal domain (CTD) of the L protein plays a critical role in P protein binding. We previously reported that the highly conserved NPYNE sequence in the hydrophilic region of the CTD (positions 1929-1933 of the L protein [L1929-1933]) is important for both P protein binding and RdRp function. To elucidate the functional role of the CTD in detail, we examined the importance of each of the hydrophilic residues in the NPYNE sequence (underlined). A rabies virus mutant with Ala substitutions in these hydrophilic residues showed low replication capacity. Comprehensive analyses of a revertant of the mutant virus and a series of L protein mutants revealed that Asn at L1929 is crucial for both P protein binding and RdRp function. Analyses of the L protein mutants also showed that Asn at L1932 and Glu at L1933 have roles in RdRp function and P protein binding, respectively. Furthermore, we demonstrated that the NPYNE sequence is essential for stabilizing the L protein through the L-P interaction. In a previously determined L protein structure, all of the hydrophilic residues in the NPYNE sequence form the first alpha-helix in the CTD. Therefore, our findings indicate that this helix is important for P protein-binding ability, RdRp function, and stabilization of the L protein, thereby contributing to efficient viral replication.