©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction between the Nucleocapsid Protein and the Phosphoprotein of Human Parainfluenza Virus 3
MAPPING OF THE INTERACTING DOMAINS USING A TWO-HYBRID SYSTEM (*)

Hong Zhao (1), Amiya K. Banerjee (1) (2)(§)

From the (1) Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106 and the (2) Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 NH-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH-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 NH-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.


INTRODUCTION

The ribonucleoprotein (RNP)() 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 (1, 2) . 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 (3) . 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 LP complex interacts with the NP-RNA template to transcribe the genomic RNA into six distinct mRNAs (4) . On the other hand, during the replication step in vivo, NP forms a soluble complex with P, and the resulting NPP complex interacts with the transcribing RNP to switch transcription reaction to replication (5) . The requirements of the formation of both the LP complex and the NPP 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 (6, 7, 8) . Recently, this has also been demonstrated in Sendai virus, a paramyxovirus, by expression of the recombinant proteins in the cell (5, 9) . 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 (1, 2) . 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 (10) . A basic protein, designated C, is synthesized by translation of an alternate +1 open reading frame of the P mRNA (11) . 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 NPP complex, which is the essential component involved in the switch from transcription to replication. We used a modified two-hybrid system (12) 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 (13) . 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 METHODS

Plasmids

pGAL4, 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) (14) . 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.) (15) . pGALVP16, originally named pSGVP490, which encodes GAL4 DNA binding domain fused with VP16 transactivating domain was provided by M. Ptashne and has been described elsewhere (16) . 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 (17) . The internal control plasmid, pRSV--gal, which contains the -galactosidase gene under Rous sarcoma virus promoter control, was obtained from the Promega Company. pVPN of VSV and pGALP of VSV were described previously (13, 18).

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 (10) 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 Assay

COS-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--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 10 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 (19). 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 -galactosidase activity (19) .

Western Blot Analyses of Expressed Proteins

A 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.


RESULTS

Association of the HPIV-3 NP and P in Vivo

A 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 [C]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 (P ) was transfected with pVP16, or when pVPNP (NP ) 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 [C]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 [C]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 -galactosidase activity, which measured the efficiency of transfection. The CAT activity in each fraction was calculated based upon -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 1: The association of NP and P of HPIV-3 in vivo. COS-1 cells were co-transfected with the indicated plasmids and the reporter plasmid. The resulting cell extracts were then assayed for CAT activity as described under ``Materials and Methods.'' 2.0 µg of cell extract was used for each reaction. The percentage of acetylation (% Acetyl.) was quantitated by using a PhosphorImager and standardized by -galactosidase activity. P, pGALP of HPIV-3; NP, pVPNP of HPIV-3; P, pGALP of VSV; NV, pVPN of VSV.




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.



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 (N ). 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 (13) . 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 NP

Next, 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 (10C) reduced the CAT activity by 40% while 20C 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. 40C and 100C 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 (410C), leaving only an NH-terminal 193-amino acid fragment. Further deletions from the COOH-terminal end, i.e. 510C and 540C, resulted in a slight decrease in interaction, but the truncated proteins continued to interact strongly. However, the deletion mutant 570C lost completely its capacity to interact with NP. Thus, an NH-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 NH-terminal fragment that interacted more than the wild type is 200C (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.



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 NH-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 P350C (250 and 240% of the wild type, respectively), confirming that the NH-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.




Figure 5: The requirement of the NH terminus of P for the association with NP. A, schematic representation of NH-terminal deletion mutants of P fused to the GAL4 DNA-binding region. The number of amino acids deleted from the NH-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.



Fig. 4 also shows that the P/D protein, which is synthesized by an RNA editing mechanism (10) although it contains the NH-terminal 241 amino acids, interacts poorly with NP compared with P350C 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 NH-terminal region of P for its association with NP. As shown in Fig. 5, removal of 20 amino acids from the NH terminus (20N) decreases the CAT activity by more than 80%. Further removal of amino acids (40N and 80N) 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 NH-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 P

The 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 NPC or PC 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.



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 Vivo

Finally, we studied the domain of NP responsible for interaction with P. By systematic NH- and COOH-terminal deletions of NP, we made several VPNP mutant chimeras. As shown in Fig. 7 , deletions of the COOH-terminal 20 (20C) and 80 (80C) amino acids did not reduce CAT activity significantly. However, deletion of an additional 80 amino acids (160C) totally abrogated the interaction. Similarly, deletion of the NH-terminal 20 amino acids (20N) did not reduce the CAT activity, but removal of 40 (40N) and 80 (80N) amino acids from the NH 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 NH 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 NH- 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.



All NP mutants were tested by Western blot analysis. The expression levels are consistent with that of the wild-type NP (data not shown).


DISCUSSION

It 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 (1, 5, 20) . P acts as a transcription factor when it complexes with L; phosphorylation plays an important role in this process in the VSV system (21, 22, 23) . P also complexes with NP to possibly keep the latter protein in a replication-competent form (8) and facilitates the LP 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 (13) , and more recently in rabies virus (24) , both NH- 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 (25, 26) , whereas the NH-terminal domain acts as a chaperone for NP during chain assembly of genome replication (27) . The COOH-terminal domain of Sendai virus NP appears to be specifically involved in its interaction with P (28) . The domains in P involved in the interaction with L in the VSV system appear to be located both at the NH- and COOH-terminal domain of P (29, 30) .()

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 NH-terminal 63-amino acid fragment of P is capable of interacting with NP more than two times as efficiently than the wild type (Fig. 3). Since removal of 20 amino acids from the NH-terminal end of P decreased its interaction with NP to 30% (Fig. 4), it seems that the NH-terminal domain between amino acids 1 and 63 contains both the essential and highly interactive domain for P. Thus, it seems that the structure of the HPIV-3 P protein is quite unique. In the wild-type configuration P down-regulates a highly interactive domain within the protein, which stretches from the NH-terminal penultimate amino acid to 403. A negative regulatory domain seems to be located within the COOH-terminal 200-amino acid stretch, since removal of this domain significantly increases the interaction of the remaining P with the NP. It is interesting to note that within this domain removal of the COOH-terminal 20 amino acids (Fig. 3) significantly decreases the interaction with NP. Thus, the COOH-terminal domain spanning 200 amino acids perhaps contains both negative and positive elements that regulate its interaction with NP. The precise structure of such a domain in the function of P remains to be elucidated. Clearly, the presence of such an interactive/regulatory domain in P seems to be unique for HPIV-3, since a domain with similar characteristic is not present in the VSV P (13) . In the prototype Sendai virus system, two noncontiguous domains at the COOH-terminal end of P have been identified that interact with the viral RNP (25, 26) . The L binding site has recently been shown to reside between the two RNP binding domains (20, 31) . Assuming a similar situation exists in the HPIV-3 P, the putative regulatory domain, interestingly, would cover both the RNP-binding and the L-binding domains. It remains to be determined whether binding of L and RNP within this domain could influence the interaction of the highly reactive free NH-terminal P protein with the soluble NP for genome replication (27) . It is noteworthy that in the VSV system the NH-terminal domain of P is also a transactivator that can complement functionally in trans the COOH-terminal end of P bound to the NH-RNA template and L in transcription (32) . Whether the highly NP-interactive NH-terminal domain is similarly active in transcription in the HPIV-3 P remains to be determined.

The in vivo interaction studies also demonstrated that P/D, although it contains NH-terminal superinteracting domain, interacts with NP less efficiently than the wild-type P (Fig. 4). This clearly indicates that the frameshifted region at the COOH-terminal end exerts a negative effect on the remaining NH-terminal highly NP-P interacting domain. The precise role of the P/D in the life-cycle of the virus remains unclear. In Sendai virus, however, the edited V protein has been shown to have a regulatory role in viral replication (20) , although it does not seem to interact with L (20, 31) . It is noteworthy that in the HPIV-3 system, in addition to P and P/D, a preterminated NH-terminal P (Pt) is synthesized as a result of the editing reaction and is found to be present in HPIV-3-infected cells (10) . Since Pt contains the NH-terminal 242 amino acid residues, it is expected that it would interact with NP more strongly than the wild-type P. An interesting possibility is whether such protein would regulate replication reaction by binding more tightly with NP. We have also shown that the HPIV-3 C protein does not interact with P or NP (Fig. 5), although it appears to be present in the purified virion (3) .

Our results in the HPIV-3 system show that both NH- and COOH-terminal ends of P are needed for its interaction with NP; the NH-terminal end seems to be more important than the COOH-terminal end. These results are similar to that for the VSV and rabies P (13, 24) . However, the domain of NP involved in interaction with P is quite different in HPIV-3 and VSV. Unlike VSV, the interacting domain lies at the NH-terminal domain of HPIV-3 NP (Fig. 7). In the Sendai system, using a protein-blotting technique, Homann et al.(28) have observed an important role of the COOH-terminal domain of NP for P interaction. Interestingly, deletion of an internal 20 residues in the hydrophobic part of NP completely abolished its binding to P. Thus, it seems that different negative strand RNA viruses have evolved in a manner such that different domains of P and NP interact with each other to carry out the precise function of the PNP complex in the replication reaction.

Finally, it is important to note that the various mutant proteins, both P and NP, used in the experiments have the potential to undergo conformational changes caused by specific mutations. Thus, the actual interactive site(s) within the protein may not be located within the deleted domains. The change of conformation of the mutant proteins may directly effect the interacting site(s) present elsewhere in the proteins. Detailed studies along these lines would certainly shed light to understand the role of the PNP complex and the structure of the interacting domains of the two proteins involved in the genome RNA replication.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant AI-32027 (to A. K. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, Research Inst., The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-0625; Fax: 216-444-0512; E-mail: banerja@ccsmtp.ccf.org.

The abbreviations used are: RNP, ribonucleoprotein; P, phosphoprotein; L, large protein; HPIV, human parainfluenza virus type 3; NP, nucleocapsid protein; VSV, vesicular stomatitis virus; kb, kilobase(s); PCR, polymerase chain reaction; GALP, Gal fused to P; GALC, Gal fused to C; CAT, chloramphenicol acetyltransferase; VP16, virion protein of herpes simplex virus.

Takacs, A., and Banerjee, A. K., Virology, in press.


ACKNOWLEDGEMENTS

We thank Drs. Bishnu P. De, Tapas Das, and Adrienne M. Takacs for valuable comments on the manuscript and advice throughout the project.


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