Deletion and substitution analysis defines regions and residues within thephosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein
Sk. Khattar et al., Deletion and substitution analysis defines regions and residues within thephosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein, VIROLOGY, 285(2), 2001, pp. 253-269
The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a mu
ltifunctional protein that plays a central role in transcription and replic
ation of the viral genomic RNA. To investigate the domains and specific res
idues involved in different activities of the P protein, we generated a tot
al of 22 deletion and 17 point mutants of the P protein. These mutants were
characterized using an intracellular BRSV-CAT minigenome replication syste
m for the ability to (1) direct minigenome transcription, (2) direct minige
nome replication, and (3) form complexes with nucleocapsid protein (N) and
large polymerase protein (L). These studies revealed that all the regions o
f P protein except amino acids 41-80 are essential for minigenome transcrip
tion and replication. Interestingly, amino acids 41-60 appeared to contain
sequences that negatively regulate transcription and replication. Analysis
of the N- or C-terminal ends indicated that deletion of up to 3 amino acids
from the N- or C-terminus completely ablated the replication, while leavin
g substantial residual transcription. Single amino acid substitutions withi
n the N-terminal 4 or C-terminal 13 amino acids showed that substitution at
position 2, 4, 234, 236, 238, 240, or 241 was highly inhibitory to both tr
anscription and replication, whereas substitution at position 3 was highly
inhibitory to replication while leaving substantial residual transcription.
Substitution of serine residues at the C-terminus indicated that loss of p
hosphorylation sites did not appear to have any effect on transcription and
replication. Coimmunoprecipitation of P-N and P-L complexes with P-specifi
c antiserum revealed that substitution mutations at the N- or C;terminus di
d not affect binding to N and L proteins, except that substitution mutation
at C-terminus position 234, 236, 238, 240, or 241 affected binding to N pr
otein by 10-fold. (C) 2001 Academic Press.