The interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV–CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161–180 amino acids and a C-terminal region of 221–241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121–160. The data suggest that N and L protein binding domains on the P protein do not overlap.
AsenjoA.,
VillanuevaN.2000; Regulated but not constitutive human respiratory syncytial virus (HRSV) P protein phosphorylation is essential for oligomerization. FEBS Letters 467:279–284
BarrJ.,
EastonA. J.1995; Characterization of the interaction between the nucleoprotein and phosphoprotein of pneumonia virus of mice. Virus Research 39:221–235
BuchholzU. J.,
FinkeS.,
ConzelmannK.-K.1999; Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter. Journal of Virology 73:251–259
ByrappaS.,
GavinD. K.,
GuptaK. C.1995; A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase.. Genome Research5404–407
ChenikM.,
ChebliK.,
GaudinY.,
BlondelD.1994; In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein. Journal of General Virology 75:2889–2896
ChenikM.,
SchnellM.,
ConzelmannK. K.,
BlondelD.1998; Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. Journal of Virology 72:1925–1930
CollinsJ. K.,
TeegardenR. M.,
MacveanD. W.,
SmithG. H.,
FrankG.,
SalmanS.1988; Prevalence and specificity of antibodies to bovine respiratory syncytial virus in sera from feedlot and range cattle. American Journal of Veterinary Research 49:1316–1319
CollinsP. L.,
HillM. G.,
CristinaJ.,
GrosfeldH.1996; Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative strand RNA virus. Proceedings of the National Academy of Sciences, USA 93:81–85
CurranJ.,
BoeckR.,
KolakofskyD.1991; The Sendai virus P gene expresses both an essential protein and an inhibitor of RNA synthesis by shuffling modules via mRNA editing. EMBO Journal 10:3079–3085
Garcia-BarrenoB.,
DelgadoT.,
MaleroJ.1996; Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusion. Journal of Virology 70:801–808
HorikamiS. M.,
CurranJ.,
KolakofskyD.,
MoyerS. A.1992; Complexes of Sendai virus NP–P and P–L proteins are required for defective interfering particle genome replication in vitro. Journal of Virology 66:4901–4908
KhattarS. K.,
YunusA. S.,
CollinsP. L.,
SamalS. K.2000; Mutational analysis of the bovine respiratory syncytial virus nucleocapsid protein using a minigenome system: mutations that affect encapsidation, RNA synthesis, and interaction with the phosphoprotein. Virology 270:215–228
LerchR. A.,
StottE. J.,
WertzG. W.1989; Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs. Journal of Virology 63:833–840
LingR.,
DavisP. J.,
QingzhongY.,
WoodC. M.,
PringleC. R.,
CavanaghD.,
EastonA. J.1995; Sequence and in vitro expression of the phosphoprotein gene of avian pneumovirus. Virus Research 36:247–257
MallipeddiS. K.,
SamalS. K.,
MohantyS. B.1990; Analysis of polypeptides synthesized in bovine respiratory syncytial virus-infected cells. Archives of Virology 115:23–36
MallipeddiS. K.,
LupianiB.,
SamalS. K.1996; Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P interaction using a two-hybrid system. Journal of General Virology 77:1019–1023
SamalS. K.,
PasteyM. K.,
McPhillipsT. H.,
MohantyS. B.1993; Bovine respiratory syncytial virus nucleocapsid protein expressed in insect cells specifically interacts with the phosphoprotein and the M2 protein. Virology 193:470–473
SlackM. S.,
EastonA. J.1998; Characterization of the interaction of the human respiratory syncytial virus phosphoprotein and nucleocapsid protein using the two-hybrid system. Virus Research 55:167–176
SmallwoodS. K.,
RyanK. W.,
MoyerS. A.1994; Deletion analysis defines a carboxyl-proximal region of Sendai virus P protein that binds to the polymerase L protein. Virology 202:154–163
TakacsA. M.,
DasT.,
BanerjeeA. K.1993; Mapping of interacting domains between nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system. Proceedings of the National Academy of Sciences, USA 90:10375–10379
van der PoelW. H.,
BrandA.,
KrampsJ. A.,
Van OirshotJ. T.1994; Respiratory syncytial virus infections in human beings and cattle. An epidemiological review. Journal of Infection 29:215–228
YunusA. S.,
CollinsP. L.,
SamalS. K.1998; Sequence analysis of functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans -acting requirements for RNA replication. Journal of General Virology 79:2231–2238
ZhaoH.,
BanerjeeA. K.1995; Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Journal of Biological Chemistry 21:12485–12490