Identification of a single amino acid change in the human respiratory syncytial virus L protein that affects transcriptional termination.
Infectious Human respiratory syncytial virus (HRSV) with an aberrant RNA synthesis pattern was recovered from a cDNA clone. The virus displayed increased levels of polycistronic readthrough mRNAs resulting from failure of the polymerase to terminate transcription efficiently at the gene ends. An asparagine (N) to aspartic acid (D) change at amino acid 1049 in the large (L) polymerase protein was found to be responsible for the readthrough phenotype. Virus encoding N at position 1049 displayed an RNA synthesis pattern indistinguishable from the A2 strain of HRSV. We compared the transcriptional activities of polymerases that encoded an N or D at position 1049 (L(1049N) or L(1049D)) by using dicistronic subgenomic replicons and found that the alteration of amino acid 1049 specifically affected transcriptional termination but had no effect on genome replication. L(1049N) recognized and terminated transcription at each of the naturally occurring gene junctions with differing efficiencies but at significantly higher efficiency than L(1049D). The abilities of the polymerases to recognize the cis-acting gene end signals required for termination were compared by examining the effect of single nucleotide changes at positions 1 to 8 of the M gene end and were found to be similar. This work identifies a single amino acid position in the polymerase that specifically affects the ability of the polymerase to terminate transcription, but which does not affect genome replication or interaction with the M2-1 protein. This work shows the critical importance of the previously defined cis-acting signals for termination, confirms differential termination at the varied gene junctions, and shows that the polymerase responds to the cis-acting sequences similarly, whether it has N or D at position 1049.
Cartee
Tara L
TL
Department of Microbiology, University of Alabama School of Medicine, Birmingham, Alabama 35294, USA.
Megaw
A George
AG
Oomens
A G P
AG
Wertz
Gail W
GW
D019943:Amino Acid Substitution; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D014764:Viral Proteins