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• W1516686388 abstract "A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression. A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression. INTRODUCTIONThe ribonucleoprotein (RNP)1( 1The abbreviations used are: RNPribonucleoproteinPphosphoproteinLlarge proteinHPIVhuman parainfluenza virus type 3NPnucleocapsid proteinVSVvesicular stomatitis viruskbkilobase(s)PCRpolymerase chain reactionGALPGal fused to PGALCGal fused to CCATchloramphenicol acetyltransferaseVP16virion protein of herpes simplex virus.) complexes of human parainfluenza virus type 3 (HPIV-3) contain a single negative strand genome RNA (15.4 kb) complexed with at least three viral proteins (1Banerjee A.K. Barik S. De B.P. Pharmacol & Ther. 1991; 51: 47-70Crossref PubMed Scopus (75) Google Scholar, 2Galinski M.S. Adv. Virus Res. 1991; 39: 129-162Crossref PubMed Scopus (41) Google Scholar). The nucleocapsid protein (NP) is the most abundant component that encapsidates the genomic RNA to form the NP-RNA template and maintains the structural integrity and the template function of the RNA genome. The other two proteins associated with the NP-RNA template constitute the RNA polymerase complex, which consists of the large protein (L) and the phosphoprotein (P). L (251 kDa) is likely to be the RNA polymerase, whereas P (90 kDa) is an auxiliary protein essential for the function of L (3Sanchez A. Banerjee A.K. Virology. 1985; 143: 45-54Crossref PubMed Scopus (21) Google Scholar). The three RNP-associated proteins play important roles in the life cycle of the virus. During the transcriptive phase, in vivo or in vitro, the L⋅P complex interacts with the NP-RNA template to transcribe the genomic RNA into six distinct mRNAs (4De B.P. Galinski M.S. Banerjee A.K. J. Virol. 1990; 64: 1135-1142Crossref PubMed Google Scholar). On the other hand, during the replication step in vivo, NP forms a soluble complex with P, and the resulting NP⋅P complex interacts with the transcribing RNP to switch transcription reaction to replication (5Horikami S.M. Curran J. Kolakofsky D. Moyer S.A. J. Virol. 1992; 66: 4901-4908Crossref PubMed Google Scholar). The requirements of the formation of both the L⋅P complex and the NP⋅P complex in transcription and replication, respectively, have been shown in vitro and in vivo for vesicular stomatitis virus (VSV), a prototype negative strand RNA virus (6Banerjee A.K. Barik S. Virology. 1992; 188: 417-428Crossref PubMed Scopus (131) Google Scholar, 7Pattnaik A.K. Wertz G.W. J. Virol. 1990; 64: 2948-2957Crossref PubMed Google Scholar, 8Peluso R.W. Moyer S.A. Virology. 1988; 162: 369-376Crossref PubMed Scopus (91) Google Scholar). Recently, this has also been demonstrated in Sendai virus, a paramyxovirus, by expression of the recombinant proteins in the cell (5Horikami S.M. Curran J. Kolakofsky D. Moyer S.A. J. Virol. 1992; 66: 4901-4908Crossref PubMed Google Scholar, 9Curran J. Homann H. Buchholz C. Rochat S. Neubert W. Kolakofsky D. J. Virol. 1993; 67: 4358-4364Crossref PubMed Google Scholar). The precise mechanism and the roles played by the respective complexes in the switch from transcription to replication remain unclear.For many paramyxoviruses, including HPIV-3, the P mRNA has the capacity to encode multiple proteins (1Banerjee A.K. Barik S. De B.P. Pharmacol & Ther. 1991; 51: 47-70Crossref PubMed Scopus (75) Google Scholar, 2Galinski M.S. Adv. Virus Res. 1991; 39: 129-162Crossref PubMed Scopus (41) Google Scholar). For HPIV-3, a protein, designated P/D, is synthesized, in addition to the wild-type P, by an RNA editing mechanism by incorporating Gly residues at a specific site (10Galinski M.S. Troy R.M. Banerjee A.K. Virology. 1992; 186: 543-550Crossref PubMed Scopus (73) Google Scholar). A basic protein, designated C, is synthesized by translation of an alternate +1 open reading frame of the P mRNA (11Luk D. Sanchez A. Banerjee A.K. Virology. 1986; 153: 318-325Crossref PubMed Scopus (37) Google Scholar). The precise functions of these proteins and their interactions, if any, with L, P, and NP during the life cycle of the virus remain to be determined.To gain insight into the replication process of HPIV-3, we have studied the formation of the NP⋅P complex, which is the essential component involved in the switch from transcription to replication. We used a modified two-hybrid system (12Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4822) Google Scholar) to study the nature of the interaction between these two proteins in an in vivo context as well as map the interacting domains. Using a GAL4/VP16-based three-plasmid transfection system we have been able to identify the interacting domains of N and P of VSV (13Takacs A.M. Das T. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10375-10379Crossref PubMed Scopus (90) Google Scholar). Here we report that NP and P of HPIV-3 interact very strongly in vivo, and by mutational studies, we identified the interacting domains. Additionally, a domain within P has been characterized that seems to act as a negative regulator in its interaction with NP.MATERIALS AND METHODSPlasmidspGAL4, originally called pSG424, which encodes the DNA-binding domain of GAL4 under the SV40 ori/early promoter control, was provided by M. Ptashne (Harvard University) (14Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (470) Google Scholar). pVP16, originally named pAASVVP16, which encodes the VP16 transactivating domain of herpesvirus under the SV40 ori/early promoter control, was provided by H. Vasavada (A. Miles Co.) (15Vasavada H.A. Ganguly S. Germino F.J. Wang Z.X. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10686-10690Crossref PubMed Scopus (48) Google Scholar). pGALVP16, originally named pSGVPΔ490, which encodes GAL4 DNA binding domain fused with VP16 transactivating domain was provided by M. Ptashne and has been described elsewhere (16Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (971) Google Scholar). The reporter plasmid, pG5B-CAT, which contains five copies of the GAL4 binding site, the E1b TATA promoter, and the CAT gene, was also provided by M. Ptashne (Harvard University) and has been described previously (17Martin K.J. Lillie J.W. Green M.R. Nature. 1990; 346: 147-152Crossref PubMed Scopus (140) Google Scholar). The internal control plasmid, pRSV-β-gal, which contains the β-galactosidase gene under Rous sarcoma virus promoter control, was obtained from the Promega Company. pVPN of VSV and pGALP of VSV were described previously (13Takacs A.M. Das T. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10375-10379Crossref PubMed Scopus (90) Google Scholar, 18Takacs A.M. Perrine K.G. Barik S. Banerjee A.K. New Biol. 1991; 3: 581-591PubMed Google Scholar).pGALP (HPIV-3) was constructed by amplifying the entire P gene of HPIV-3 by using pPET3aP as template and the oligonucleotide primers containing either a BamHI site (the 5′ primer) or a SacI site (the 3′ primer). The purified and digested 1.8-kb PCR product was ligated with BamHI/SacI-digested pGAL4. pGALP/D was made similarly except that pPG7 (10Galinski M.S. Troy R.M. Banerjee A.K. Virology. 1992; 186: 543-550Crossref PubMed Scopus (73) Google Scholar) was used as a template. The P mutant plasmids were constructed via PCR using plasmid pPET3aP as template and oligonucleotide primers that spanned the portion of the P gene of interest and contained either a BamHI site or SacI site. To ensure that the fused P is in frame, two extra nucleotides were added between the BamHI site and the first codon of P at the 5′ primer. The linker joining GAL4 and all P gene sequences encodes the following peptide: Pro-Glu-Phe-Pro-Gly-Ile-Leu.pVPNP was made by amplifying the entire NP gene of HPIV-3 by using pPET3aNP as template and the oligonucleotide primers containing either an EcoRI site (the 5′ primer) or a ClaI site (the 3′ primer). The purified and digested 1.6-kb PCR product was ligated with EcoRI/ClaI-digested pVP16. The mutant pVPNP plasmids were also made by PCR using plasmid pPET3aNP as a template and oligonucleotide primers that spanned the portion of the NP gene of interest and contained either an EcoRI or a ClaI site. The linker joining all VP16 and NP sequences encodes the following peptide: Glu-Phe-Ala.pGALC was made by amplifying the entire C gene of HPIV-3 by using pPET3aC (obtained by inserting a full-length C gene into pPET3a plasmid) as template and the oligonucleotide primers containing either a BamHI site (5′ primer) or a SacI site (3′ primer). The purified and digested 0.6-kb PCR product was ligated with BamHI/SacI-digested pGAL4. The linker joining the GAL4 and C sequences encodes the same peptide as is present between the GAL4 and P sequences.Similarly, pVPC was constructed by amplifying the entire C gene of HPIV-3 by using pPET3aC as template and the oligonucleotide primers containing either an XbaI site (5′ primer) or a ClaI site (3′ primer). To create an XbaI site in the pVP16 vector, the pVP16 was digested with EcoRI, filled with Klenow, fused with an 8-mer linker containing an XbaI site, and finally digested with XbaI and ClaI. The purified and digested 0.6-kb PCR product was ligated with the corresponding XbaI/ClaI-digested pVP16. The linker joining the Vp16 and C sequences encodes the following peptide: Glu-Phe-Ser-Arg.All plasmid constructs were confirmed by restriction enzyme digestion and by dideoxy sequencing across each junction region and mutant region.Plasmid Transfection and CAT AssayCOS-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/ml of penicillin, and 100 units/ml of streptomycin. For transfection, 0.5 μg of pRSV-β-gal, 0.8 μg of the test and reporter plasmids were mixed with 20 μg of Lipofectamine (Life Technologies, Inc.) in OPTI-MEM medium (Life Technologies, Inc.) for at least 15 min and then added to a 60 × 35-mm dish containing 5 × 105 cells subcultured the previous day. After 6 h the transfection solution was aspirated away, and the cells were grown in 4 ml of Dulbecco's modified Eagle's medium with serum for 48 h. Cells were harvested, and 2 μg of cell extract was assayed for CAT activity (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After autoradiography of the separated acetylated chloramphenicol forms, the spots were quantitated using a PhosphorImager (Molecular Dynamics). 10 μg of cell extract was assayed for β-galactosidase activity (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Western Blot Analyses of Expressed ProteinsA rabbit polyclonal antibody to GAL4 (amino acids 1-147) was provided by M. Ptashne (Harvard University). A rabbit polyclonal antibody to HPIV-3 was kindly provided by Ranjit Ray (St. Louis University). The expression levels of all the VPNP (wild-type as well as mutant) chimeras in the transfected cell extracts were determined by immunoblotting with the antibody to HPIV-3 using gene screen membrane (DuPont) following the manufacturer's protocol. The expression levels of GALP and GALC chimeras in the transfected cell extracts were similarly assayed by the antibody against GAL4.RESULTSAssociation of the HPIV-3 NP and P in VivoA GAL4/VP16-based three-plasmid system was used to study the association of NP and P in vivo. Plasmids were constructed that encoded the following fused proteins: the amino terminus of P of HPIV-3 fused to the GAL4 DNA-binding region and the amino terminus of NP of HPIV-3 fused to the VP16 transactivating domain. When these two plasmids along with the CAT reporter plasmid were co-transfected into COS-1 cells, any association of NP and P in vivo would activate transcription of the CAT gene via the VP16 transactivating region. This could then be assayed by the conversion of [14C]chloramphenicol to its acetylated forms.Fig. 1 shows the results of the three-plasmid transfections. When the reporter plasmid was transfected (CAT alone), when pGALP (PP) was transfected with pVP16, or when pVPNP (NPP) was transfected with pGAL4, no CAT activity was detected. pGALVP16 (GALVP), which produced a high level of CAT activity, served as positive control. These results indicate that either NP fused to VP16 or P fused to GAL4 alone cannot activate the CAT gene by binding to the promoter or to any proteins associated with the promoter. However, when both pGALP and pVPNP were co-transfected with the reporter plasmid there was an extremely high level of CAT gene expression, which was shown by more than 90% conversion of [14C]chloramphenicol to its acetylated forms. This result demonstrates the high affinity interaction between NP and P in vivo. Since the CAT activity from NP and P co-transfection is so high, we titrated the cell extract used in the assay to determine the amount that would result in CAT activity within the linear range. Fig. 2 shows that cell extracts containing as little as 5.0 μg of protein resulted in almost complete conversion of [14C]chloramphenicol to its acetylated forms. Depending on the individual cell extract, only 1.0-2.0 μg of protein equivalent of cell extract was needed to obtain CAT activity within the linear range. This concentration of cell extract was used in all subsequent CAT assays. Each fraction was also tested for β-galactosidase activity, which measured the efficiency of transfection. The CAT activity in each fraction was calculated based upon β-galactosidase activity. In a separate series of experiments (data not shown) the expression levels of P and NP chimeras were measured by Western blot analyses (see “Materials and Methods”). The extent of protein synthesis in each reaction was similar.Figure 2:Titration of CAT activity from the transfected cell extract. COS-1 cells were co-transfected with pGALP, pVPNP, and the reporter plasmid. Aliquots of cell extracts as indicated were used for CAT assay as described in the text. CE, cell extract.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that the CAT activity is due to a specific NP and P interaction, the HPIV-3 P was replaced with P of VSV (P<itinf;V). Similarly, HPIV-3 NP was replaced by nucleocapsid protein(N) of VSV (NV). When the heterologous plasmids were co-transfected, no CAT activity was detected in either case. As expected, VSV P- and N-containing appropriate plasmids produced high CAT activity (13Takacs A.M. Das T. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10375-10379Crossref PubMed Scopus (90) Google Scholar). These results clearly show that NP of HPIV-3 does not interact with P of VSV, or vice versa. Thus, the interaction between NP and P is highly specific for HPIV-3.Domains of P Involved in Its Interaction with NPNext, we tested different P mutants to investigate the domains of P involved in the interaction with NP. As shown in Fig. 3, deletion of the COOH-terminal 10 amino acids (Δ10C) reduced the CAT activity by 40% while Δ20C reduced the CAT activity by 70%, suggesting that the COOH terminus of P is involved in its association with NP. Interestingly, further deletions of amino acids from the COOH terminus, e.g. Δ40C and Δ100C and so on, resulted in a gradual increase in CAT activity. The highest CAT activity (more than 250% of the wild type) resulted when 410 amino acids were removed from the COOH terminus (Δ410C), leaving only an NH2-terminal 193-amino acid fragment. Further deletions from the COOH-terminal end, i.e. Δ510C and Δ540C, resulted in a slight decrease in interaction, but the truncated proteins continued to interact strongly. However, the deletion mutant Δ570C lost completely its capacity to interact with NP. Thus, an NH2-terminal P fragment as short as 63 amino acids long interacted with the NP more strongly (greater than 2-fold) than the wild type. The largest NH2-terminal fragment that interacted more than the wild type is Δ200C (Fig. 3), i.e. 403 amino acids long. These data strongly suggest that a domain is present in P, which resides between amino acids 63 and 403, that negatively regulates its interaction with NP.Figure 3:Domains of P involved in the association with NP. A, schematic representation of P showing the COOH-terminal deletion mutants of the 603-amino acid P fused to the GAL4 DNA-binding region. The number of amino acids deleted from the COOH-terminal end of P is indicated. B, CAT activity is shown from the co-transfection of pGALP mutants with pVPNP and the reporter plasmid. 2.0 μg of cell extract was used in each reaction. Average CAT activity from at least three experiments of each P mutant was determined by CAT assay and expressed as a percentage of that of wild-type P, representing 100% CAT activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To further confirm that the removal of such a regulatory domain from P indeed increases the association of P with NP, we constructed a plasmid (PAB, Fig. 4) that contains 230 NH2-terminal amino acids linked in-frame with the COOH-terminal 20 amino acids of P. As shown in Fig. 5, the PAB interacts with NP as efficiently as the PΔ350C (250 and 240% of the wild type, respectively), confirming that the NH2-terminal one-third of P interacts more strongly (more than 2.5-fold) than the wild-type P. The deleted portion of P thus appears to act as the negative regulatory domain in its association with NP.Figure 4:Association of NP with internally deleted P and P/D. A, schematic representation is shown of the various P mutants fused to the GAL4 DNA-binding region. The number of amino acids deleted from P is described under “Results.” B, CAT activity of the co-transfection of pGALP mutants with pVPNP and the reporter plasmid is shown. 2.0 μg of cell extract was used for each reaction. Average CAT activity from at least three experiments was determined by CAT assay and expressed by a percentage of that of wild-type P, representing 100% CAT activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5:The requirement of the NH2 terminus of P for the association with NP. A, schematic representation of NH2-terminal deletion mutants of P fused to the GAL4 DNA-binding region. The number of amino acids deleted from the NH2-terminal end of P is indicated. B, CAT activity of the co-transfection of pGALP mutants with pVPNP and the reporter plasmid is shown. 2.0 μg of cell extract was used in each reaction. Average CAT activity from at least three experiments of each P mutant was determined by CAT assay and expressed as a percentage of that of wild-type P, representing 100% CAT activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 also shows that the P/D protein, which is synthesized by an RNA editing mechanism (10Galinski M.S. Troy R.M. Banerjee A.K. Virology. 1992; 186: 543-550Crossref PubMed Scopus (73) Google Scholar) although it contains the NH2-terminal 241 amino acids, interacts poorly with NP compared with PΔ350C or PAB and even less than the wild-type P (70% of the wild type). Thus, it seems that the frameshifted COOH-terminal portion of the P/D down-modulates the interaction between P/D and NP.Next, we studied the requirement of the NH2-terminal region of P for its association with NP. As shown in Fig. 5, removal of 20 amino acids from the NH2 terminus (Δ20N) decreases the CAT activity by more than 80%. Further removal of amino acids (Δ40N and Δ80N) resulted in total abrogation of CAT activity. These data, coupled with the COOH-terminal deletion data (Fig. 3), indicate that both ends of P are needed for its interaction with NP. However, the NH2-terminal domain seems to be more critical than the COOH-terminal domain since further deletion from the latter end increases its interaction with NP.Interaction of C with NP or PThe same three-plasmid system was used to study the possible interaction between NP and C or P and C in vivo. Plasmids were constructed that encode the C protein fused to either the GAL4 DNA-binding region or the VP16 transactivating domain. As shown in Fig. 6, when pGALC or pVPC were co-transfected with VPNP or GALP, respectively, along with reporter plasmid, no CAT activity was detected, even when 5-fold excess of the cell extracts compared with pGALP and pVPNP were used. These results indicate that the C protein of HPIV-3 does not interact with P or NP of HPIV-3 in this system.Figure 6:Association of NP⋅C or P⋅C of HPIV-3 in vivo. COS-1 cells were co-transfected with the indicated plasmids and the reporter plasmid. The resulting cell extracts were assayed for CAT activity as described under “Materials and Methods.” P, pGALP of HPIV-3; NP, pVPNP of HPIV-3; VPC, VP16 fused to C; GALC, Gal fused to C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All P mutants, P/D, and C were tested by Western blot analyses to confirm their extent of expression. The expression levels are quite consistent, comparable with that of the wild-type P, and easily detected by Western blot analyses (data not shown).Domains of NP Involved in Its Interaction with P in VivoFinally, we studied the domain of NP responsible for interaction with P. By systematic NH2- and COOH-terminal deletions of NP, we made several VPNP mutant chimeras. As shown in Fig. 7, deletions of the COOH-terminal 20 (Δ20C) and 80 (Δ80C) amino acids did not reduce CAT activity significantly. However, deletion of an additional 80 amino acids (Δ160C) totally abrogated the interaction. Similarly, deletion of the NH2-terminal 20 amino acids (Δ20N) did not reduce the CAT activity, but removal of 40 (Δ40N) and 80 (Δ80N) amino acids from the NH2 terminus reduced the CAT activity by more than 95%. These results indicate that both ends of NP are needed for P interaction but that the NH2 terminus of NP seems to be critical for its association with P.Figure 7:Domain of NP required for the association with P in vivo. A, schematic representation of the NP protein showing the systematic NH2- and COOH-terminal deletion mutants fused to the VP16 transactivating domain. The number of amino acids deleted from both ends of the 515-amino acid NP are indicated. B, CAT activity from the co-transfection of pVPNP mutants with pGALP and the reporter plasmid. 2.0 μg of cell extract was used in each reaction. Average CAT activity from at least three different experiments of each NP mutant was determined by CAT assay and expressed as a percentage of that of wild-type NP, representing 100% CAT activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All NP mutants were tested by Western blot analysis. The expression levels are consistent with that of the wild-type NP (data not shown).DISCUSSIONIt is becoming increasingly clear that P of negative strand RNA viruses, with linear single strand RNA as the genetic material, is an important auxiliary protein with direct roles in transcription and replication of the virus (1Banerjee A.K. Barik S. De B.P. Pharmacol & Ther. 1991; 51: 47-70Crossref PubMed Scopus (75) Google Scholar, 5Horikami S.M. Curran J. Kolakofsky D. Moyer S.A. J. Virol. 1992; 66: 4901-4908Crossref PubMed Google Scholar, 20Curran J. Pelet T. Kolakofsky D. Virology. 1994; 202: 875-884Crossref PubMed Scopus (115) Google Scholar). P acts as a transcription factor when it complexes with L; phosphorylation plays an important role in this process in the VSV system (21Barik S. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6570-6574Crossref PubMed Scopus (126) Google Scholar, 22Barik S. Banerjee A.K. J. Virol. 1992; 66: 1109-1118Crossref PubMed Google Scholar, 23Takacs A.M. Barik S. Das T. Banerjee A.K. J. Virol. 1992; 66: 5842-5848Crossref PubMed Google Scholar). P also complexes with NP to possibly keep the latter protein in a replication-competent form (8Peluso R.W. Moyer S.A. Virology. 1988; 162: 369-376Crossref PubMed Scopus (91) Google Scholar) and facilitates the L⋅P complex to switch from transcription to replication. The precise mechanism by which these complexes interact with each other and the N RNA template leading to the replication reaction remains unclear. In the well studied VSV system (13Takacs A.M. Das T. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10375-10379Crossref PubMed Scopus (90) Google Scholar), and more recently in rabies virus (24Fu Z.F. Zheng Y. Wunner W.H. Koprowski H. Deitzschold B. Virology. 1994; 200: 590-597Crossref PubMed Scopus (72) Google Scholar), both NH2- and COOH-terminal domains of P seem to be required for its interaction with N. In the Sendai virus, the COOH-terminal end appears to interact with the viral RNP (25Ryan K.W. Portner A. Virology. 1990; 174: 515-521Crossref PubMed Scopus (69) Google Scholar, 26Ryan K.W. Morgan E.M. Portner A. Virology. 1991; 180: 126-134Crossref PubMed Scopus (52) Google Scholar), whereas the NH2-terminal domain acts as a chaperone for NP during chain assembly of genome replication (27Curran J. Marq J. Kolakofsky D. J. Virol. 1995; 69: 849-855Crossref PubMed Google Scholar). The COOH-terminal domain of Sendai virus NP appears to be specifically involved in its interaction with P (28Homann H.E. Willenbrink W. Buchholz C.J. Neubert W.J. J. Virol. 1991; 65: 1304-1309Crossref PubMed Google Scholar). The domains in P involved in the interaction with L in the VSV system appear to be located both at the NH2- and COOH-terminal domain of P (29Chattopadhyay D. Banerjee A.K. Cell. 1987; 49: 407-414Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 30Emerson S.U. Schubert M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5655-5659Crossref PubMed Scopus (101) Google Scholar).2( 2Takacs, A., and Banerjee, A. K., Virology, in press.) In the present studies, using a two-hybrid system, we have demonstrated that NP and P of HPIV-3 interact very efficiently in vivo (Fig. 1). The interesting observation is that deletion mapping of P (603 amino acids) revealed the presence of a highly interactive domain spanning amino acid residues 63-403 (340 amino acids). An NH2-terminal 63-amino acid fragment of P is capable of interacting with NP more than two times as efficient" @default.
• W1516686388 created "2016-06-24" @default.
• W1516686388 creator A5010877809 @default.
• W1516686388 creator A5062923942 @default.
• W1516686388 date "1995-05-01" @default.
• W1516686388 modified "2023-09-30" @default.
• W1516686388 title "Interaction between the Nucleocapsid Protein and the Phosphoprotein of Human Parainfluenza Virus 3." @default.
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