Mutations in Conserved Domain I of the Sendai Virus L Polymerase Protein Uncouple Transcription and Replication

Abstract

To begin to map functional domains of the Sendai P-L RNA polymerase complex we wanted to characterize the P binding site on the Sendai L protein. Analysis of in vitro and in vivo P-L polymerase complex formation with carboxyl-truncations of the L protein showed that the N-terminal half of the protein was required. Site-directed mutagenesis of the Sendai virus L gene was employed to change amino acids within a highly conserved region of the N-terminal domain I from amino acids (aa) 348-379 singly or in pairs from the Sendai to the corresponding measles L sequence or to alanine. The mutant L proteins coexpressed with the viral P and NP proteins in mammalian cells were assayed for their ability to form the P-L complex and to synthesize RNA in vitro and showed a variety of defective phenotypes. While most of the mutant L proteins still formed the P-L polymerase complex, a change from serine to arginine at aa 368 and a three-amino-acid insertion at aa 379 virtually abolished both complex formation and RNA synthesis. Changes of aas 370 and 376-377 in the L protein gave only small decreases in viral RNA synthesis. Substitutions at either aas 349-350 or aas 354-355 and a three-amino-acid insertion at aa 348 in the L protein yielded enzymes that catalyzed significant transcription, but were defective in DI RNA replication, thus differentially affecting the two processes. Since DI leader RNA, but not genome RNA, was still synthesized by this class of mutants, the defect in replication appears to be in the ability of the mutant enzyme to package newly synthesized nascent RNA. Single changes at aas 362, 363, and 366 in the L protein gave enzymes with severely decreased overall RNA synthesis, although some leader RNA was synthesized, suggesting that they cannot transcribe or replicate past the leader gene. These studies have identified a region in conserved domain I critical for multiple functions of the Sendai virus L protein.

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

“…For Sendai virus (SeV), CRI is implicated in recruitment of N protein during replication and interaction with P protein (8) and CRII appears to play a role in template binding (27,38). Whether these regions of L mediate the same function in VSV has not been determined.…”

The Phosphoprotein (P) Binding Site Resides in the N Terminus of the L Polymerase Subunit of Sendai Virus

“…Alignment of the L proteins of viruses of the order Mononegevirales showed six domains of relatively high conservation, designated I to VI from the N terminus to the C terminus of the protein, which were proposed to specify the essential activities common to all L proteins (14, 15). Recent characterization of Sendai virus L mutants in each of the six domains suggests that multiple domains contribute to the different steps in viral RNA synthesis, since mutants in different domains gave the same defective phenotypes (2,3,7,11,16). Viral RNA synthesis is downregulated by the C proteins encoded from the P gene (12).…”

“…The L proteins of paramyxoviruses are all over 2,000 aa, and studies to begin mapping the P binding site on L showed a general location in the N-terminal quarter or half of the protein (2,10,13). Of the mutants characterized in the various domains in the N-terminal half of Sendai virus L only one, S368R in domain I, abolished binding to Sendai virus P (2,7,16).…”

The complete Mokola virus genome sequence: structure of the RNA-dependent RNA polymerase.

“…They were either neutral, or inhibited or enhanced the polymerase function, or inhibited transcription without altering replication. Similarly, by mimicking the measles virus L protein around the conserved GHP motif of block I (position 372) (Chandrika et al, 1995), it was hoped to give the Sendai virus L protein the ability to bind the measles virus P protein (Horikami et al, 1994). However, uncoupling between transcription and replication was obtained.…”