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Deletion and substitution analysis defines regions and residues within the phosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein.

Abstract：

:The phosphoprotein (P) of bovine respiratory syncytial virus (BRSV) is a multifunctional protein that plays a central role in transcription and replication of the viral genomic RNA. To investigate the domains and specific residues involved in different activities of the P protein, we generated a total of 22 deletion and 17 point mutants of the P protein. These mutants were characterized using an intracellular BRSV-CAT minigenome replication system for the ability to (1) direct minigenome transcription, (2) direct minigenome replication, and (3) form complexes with nucleocapsid protein (N) and large polymerase protein (L). These studies revealed that all the regions of P protein except amino acids 41-80 are essential for minigenome transcription 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 leaving substantial residual transcription. Single amino acid substitutions within 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 transcription 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 phosphorylation sites did not appear to have any effect on transcription and replication. Coimmunoprecipitation of P-N and P-L complexes with P-specific antiserum revealed that substitution mutations at the N- or C-terminus did 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 protein by 10-fold.

journal_name

Virology

journal_title

Virology

authors

Khattar SK,Yunus AS,Collins PL,Samal SK

doi

10.1006/viro.2001.0960

subject

Has Abstract

pub_date

2001-07-05 00:00:00

pages

253-69

issue

eissn

0042-6822

issn

1096-0341

pii

S0042-6822(01)90960-2

journal_volume

285

pub_type

杂志文章

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Deletion and substitution analysis defines regions and residues within the phosphoprotein of bovine respiratory syncytial virus that affect transcription, RNA replication, and interaction with the nucleoprotein.