INTRODUCTION

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Human parainfluenza virus type 3 (HPIV3) is a paramyxovirus and a significant cause of lower respiratory illness, such as bronchiolitis and pneumonia in newborns and infants (4, 31). The nonsegmented negative-strand RNA genome of HPIV3 is 15,461 nucleotides long and is tightly encapsidated by the nucleocapsid protein N (68 kDa) to form a helical nucleocapsid (2, 20, 22). Associated with this are the two virus-encoded proteins, the large subunit L (257 kDa) of the RNA-dependent RNA polymerase complex and the phosphoprotein P (90 kDa), forming a ribonucleoprotein (RNP) complex (2, 20, 22). Consistent with its encapsidation function, N is present in abundance (2,600 molecules), whereas the L and P proteins are present in lesser amounts (30 and 300 molecules, respectively) in the RNP of a virion (30). The L and P proteins together constitute the RNA-dependent RNA polymerase complex (L-P) that transcribes the genomic RNA encapsidated by N protein but not the naked RNA (2, 20, 22). In the case of HPIV3, cellular actin is also required for mRNA synthesis both in vitro and in vivo (14, 15, 25). Moreover, the same RNA polymerase complex or a modified form appears to be involved in replication, a process that synthesizes full-length plus-strand genome RNA, which in turn serves as the template for synthesis of minus-strand genome RNA to be packaged into progeny virions (17).

The P proteins of nonsegmented negative-strand RNA viruses appear to be multifunctional and have been found to exist as homooligomers (7, 12, 23, 36). Although no enzymatic activity has been detected in P, it acts as a transactivator of L, which is the catalytic subunit of the RNA polymerase complex. The L protein is also believed to contain posttranscriptional modification activities such as capping, methylation, and polyadenylation of mRNAs (1). The L protein by itself cannot bind to the N-RNA template for initiation of RNA synthesis; it does so efficiently only by forming the L-P complex (29, 34). The P protein also plays an important role in encapsidation of the nascent RNA chains during genome replication. It interacts with the nucleocapsid protein N, thereby preventing nonspecific encapsidation of cellular RNAs by the N protein. The resulting soluble N-P complex participates in the encapsidation process by which nascent plus- and minus-strand genome RNAs form the respective RNPs during replication (11, 18, 28). It appears then that the P protein forms multiple complexes in infected cells, such as L-P, N-P, and P-P; these interactive processes presumably regulate the ability of the RNA polymerase complex to transcribe or to replicate.

The P mRNA of HPIV3, like that of other paramyxoviruses, has the capacity to encode multiple proteins (2, 20, 22). A basic protein, designated C, is synthesized by translation of an alternate +1 open reading frame, and additional proteins, designated P-D and Pt, are synthesized by an RNA-editing mechanism (21, 33); P-D and Pt are N-coterminal with wild-type P but differ in the C-terminal region. The precise functions of these additional P gene products in the virus life cycle remains unknown, as are those of the L-P, N-P, and P-P complexes in the normal life cycle of the virus. To begin to identify various domains of interaction within the P protein of HPIV3, an in vivo two-hybrid system was recently used to study N-P complex formation (44). These studies demonstrated that both the N-terminal 40 amino acids (aa) and the C-terminal 20 aa of P are directly involved in interaction with the N protein. In this respect, HPIV3 is similar to vesicular stomatitis virus (VSV) (41), rabies virus (19), and Sendai virus (10), in which both the N- and C-terminal domains of P are similarly required for interaction with their cognate N proteins. In the Sendai virus system, in addition, the C-terminal domain of P appears to be involved in binding to the N-RNA template, whereas the N-terminal domain functions as a chaperone for N, forming soluble N-P complex, which is presumably required during the replication process (9, 39). Thus, it seems that the terminal domains of P play vital roles in regulating transcription and replication of the genome RNA by their interactions with L and N-RNA template and maintaining N in encapsidation-competent form.

To gain information on the role of P protein in the regulation of transcription and replication in the HPIV3 system, which has remained unexplored, we focused on the role of the terminal domains of P protein in its interaction with the N-RNA template as well as the L and N proteins. The transcription process was studied using a reconstituted in vitro system, whereas replication was studied in vivo using a recently developed HPIV3 minigenome replication system (27). Protein-protein interaction was studied by coimmunoprecipitation of epitope-tagged and untagged interacting proteins. It is evident from these studies that the terminal domains of P play important regulatory roles in viral transcription and replication, several of which appear to be unique to HPIV3.

	MATERIALS AND METHODS

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Cells and viruses. HPIV3 (HA-1, NIH 47885) was propagated in CV-1 cells as described previously (14, 15). Recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3) was grown in HeLa cells. Protein expression and radiolabeling were performed in HeLa cells.

In vitro transcription. HeLa cells in a six-well plate were infected with vTF7-3 at a multiplicity of infection (MOI) of 3. At 1 h postinfection, the culture medium was replaced with a transfection mix containing 20 µl of lipofectin combined with various plasmid DNAs in Opti-MEM in a total volume of 1.5 ml. Unless otherwise indicated, plasmids pHPIV3-P (pP), in which the initiation codon for C protein is mutated, and pHPIV3-L (pL), both under the control of the T7 promoter in vector pGEM, were used at 2 µg of 200 ng per well, respectively. At 5 h postinfection, the medium containing the transfection mix was replaced with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. At 24 h postinfection, the cell monolayers were washed with ice-cold phosphate-buffered saline (PBS) and harvested by scraping in PBS. The cells were pelleted by centrifugation at 800 × g for 10 min. Cytoplasmic extracts were prepared by lysing the cells in three cycles of freezing and thawing in 40 µl of hypotonic buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, and I mM dithiothreitol (DTT). Nuclei and cell debris were removed by centrifugation for 5 min in an Eppendorf centrifuge at 4°C. The soluble cytoplasmic extract was collected for use in the transcription reaction. The protein concentration was estimated as 5 mg/ml.

In vivo minigenome replication. In vivo minigenome replication was studied following the procedure of Hoffman and Banerjee (27). Briefly, HeLa cell monolayers in 12-well plates, grown to 90% confluency, were infected with recombinant vaccinia virus vTF7-3, which expresses T7 RNA polymerase, at an MOI of 3. After 1 h at 37°C, the minireplicon, pHPIV3-MG(), and support plasmids pHPIV3P (pP), pHPIV3-L (pL), and pHPIV3-N (pN) were transfected using lipofectin (Bethesda Research Laboratories) according to the manufacturer's instructions. The plasmid amounts used were 200 ng of pHPIV3-MG(), 640 ng of pN, 700 ng of pP, and 100 ng of pL. After 4 h, the transfection medium was removed and replaced with 1 ml of DMEM-5% fetal calf serum. At 28 h posttransfection, the monolayers were lysed in 150 µl of lysis buffer, from which 1.5 µl of lysate (equivalent to 2.3 × 10³ cells) was then used to determine luciferase activity in a Dynatech ML2250 luminometer according to the manufacturer's specifications (luciferase assay kit; Roche Biochemicals).

N-RNA binding assay. Binding of wild-type (wt) P and deletion mutants of P to the N-RNA template was performed using in vitro reticulocyte lysate-translated and [³⁵S]methionine-labeled P proteins. The in vitro-synthesized P proteins were clarified by layering onto 40% glycerol (700 µl total volume) in 50 mM HEPES-KOH (pH 8.0)-50 mM NaCl-1 mM DTT and centrifugation at 45,000 rpm for 1 h in microcentrifuge tubes in an SW50.1 rotor. The clarified P from the top of the glycerol cushion was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and quantitated by phosphorimager. Equal amounts of radiolabeled P were incubated with 5 µg of purified N-RNA in 100 mM HEPES-KOH (pH 8.0)-100 mM KCl-5 mM MgCl₂-1 mM DTT at 30°C for 1 h. The reaction mixture was then layered onto a 40% glycerol cushion and centrifuged as described above. The N-RNA and P protein complex was directly dissolved in SDS-polyacrylamide gel sample buffer and analyzed. The recovery of N-RNA was confirmed by staining with Coomassie blue followed by fluorography.

Glycerol gradient analysis of proteins. HeLa cell monolayers in 100-mm plates were infected with vTF7-3 at an MOI of 3, and at 1 h postinfection the cells were transfected with N and wt or mutant P-expressing plasmid DNAs (5 and 10 µg, respectively). At 24 h postinfection, the cells were harvested in PBS and resuspended in 20 ml of hypotonic buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, and 1 mM DTT. The cells were lysed by four cycles of freezing and thawing, and nuclei and cell debris were removed by centrifugation in an Eppendorf centrifuge for 5 min at 4°C. Extracts were layered onto linear 5 to 20% glycerol gradients prepared in buffer containing 100 mM HEPES (pH 8.0), 150 mM NH₄Cl, 5 mM magnesium acetate, and 1 mM DTT and containing a 100-µl cushion of 100% glycerol, as described by Curran et al. (13). Gradients were centrifuged in an SW60 rotor at 40,000 rpm for 20 h at 4°C. Gradient fractions (300 µl) were collected from the top of the tube, and 10-µl aliquots were analyzed in SDS-10% polyacrylamide gels followed by Western blot with anti-RNP antibody and detection of the proteins by enhanced chemiluminescence following the manufacturer's protocol (Amersham).

Coexpression and immunoprecipitation of proteins. HaLa cells in a 12-well plate were infected with vTF7-3 at an MOI of 1. At 1 h postinfection, the cells were transfected with plasmid DNAs expressing L or P proteins tagged with a stretch of eight amino acids (DYKDDDDK) at the C terminus in combination with untagged P or N protein using lipofectin (Gibco-BRL) in Opti-MEM. The quantities of plasmid DNAs used for transfection are indicated in individual experiments. At 12 h postinfection, the medium was replaced with 2 ml of methionine-free DMEM, and incubation was continued at 37°C. At 14 h postinfection, the cells were labeled with 50 µCi of [³⁵S]methionine in 1 ml of methionine-free DMEM for 6 h. Cells were washed with PBS, and cytoplasmic proteins were extracted by treating the cell monolayers with 150 µl of luciferase extraction buffer (Roche Biochemicals) for 12 min. An aliquot (50 µl) was diluted with 750 µl of immunoprecipitation buffer and used for immunoprecipitation with antiflag antibody conjugated to Sepharose beads following the manufacturer's protocol (Sigma). For immunoprecipitation with monoclonal anti-N antibody or anti-HPIV3 antibody (a generous gift from Ranjit Ray), the precipitation reaction was carried out in buffer containing 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM EDTA, and 5% sucrose using protein A-conjugated Sepharose beads. The immunoprecipitated proteins in the beads were boiled in SDS-polyacrylamide gel sample buffer and analyzed in an SDS-10% polyacrylamide gel. The gel was stained with Coomassie blue, dried, and subjected to fluorography.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Development of a reconstitution system for HPIV3 mRNA synthesis in vitro. HPIV3 transcription and replication are thought to follow the general biosynthetic model proposed for the well-studied prototype viruses of the families Rhabdoviridae and Paramyxoviridae, VSV and Sendai virus, respectively, in which the L and P proteins constitute the active RNA polymerase complex (2, 4, 20, 22, 31). In the case of HPIV3, methods for direct demonstration of the role of L and P proteins in transcription and replication has only recently been developed in vivo using a reverse genetic system which relies mostly on the viral RNA polymerase-mediated replicative amplification of N-RNA template to detect mRNA synthesis (17). There was an additional need to develop an in vitro transcription reconstitution system independent of genome replication to study the role of L and P proteins in mRNA synthesis as well as the functions of various domains within these two proteins. Here we describe for the first time the development of such an in vitro transcription reconstitution system using purified N-RNA template and recombinant L and P proteins. The N-RNA template depleted of endogenous RNA polymerase was prepared from intracellular viral RNP by CsCl gradient centrifugation as described in Materials and Methods. The L and P proteins were coexpressed in HeLa cells using a recombinant vaccinia virus expression system and optimized as described in a recently developed minigenome replication system (27). At 24 h postinfection, cytoplasmic extract was prepared and used directly in an in vitro transcription reaction containing purified N-RNA template in the presence of [-³²P]UTP as the labeled precursor, as described in Materials and Methods. The RNA products were analyzed in a 5% polyacrylamide-urea gel followed by autoradiography. As shown in Fig. 1A, the N-RNA template alone had a low level of RNA-synthesizing activity, presumably mediated by the residual template-bound RNA polymerase. Addition of either L or P extract to the template had no significant effect on RNA synthesis, while addition of extract containing coexpressed L and P resulted in efficient synthesis of mRNAs which increased linearly upon addition of increasing amounts of the later extract, with a maximal stimulation of about 20-fold (Fig. 1B). Like Sendai virus (6, 8), separate L and P extracts, when combined in vitro, failed to activate transcription (data not shown) while the extract containing coexpressed proteins did so efficiently where the P protein was required in stoichiometric amounts for efficient RNA synthesis. High-level expression of P under these conditions was confirmed by Western blot using anti-HPIV3 antibody (Fig. 1C), but L could not be detected due to the lack of a suitable antibody, although its expression can be monitored by appropriate tagging of the recombinant protein (see below). Thus, having developed the optimal in vitro transcription reconstitution conditions, we were poised to study the role of the terminal domains (44) as well as other biologically significant domains of P protein in the transcription process in vitro.

View larger version (66K): [in this window] [in a new window]   FIG. 1.   Requirement for L and P proteins for mRNA synthesis in vitro. The L and P proteins were expressed either separately or in combination in a recombinant vaccinia virus expression system. Cell extracts were prepared at 24 h postinfection, and total protein was estimated as about 2 mg/ml. (A) Equal amounts of protein (2 µg) were used in in vitro transcription reactions containing purified N-RNA template (2 µg) in the presence of [32P]UTP. (B) Increasing amounts of protein (2, 4, 6, and 8 µg) containing coexpressed L and P were used in the reactions. As a negative control, 8 µg of cell extract alone (L+P) was used in the reaction. The in vitro-synthesized radiolabeled mRNAs were analyzed in a 5% polyacrylamide-urea gel followed by autoradiography. (C) Western blot of cell extracts using anti-RNP antibody. Mock, vaccinia virus-infected and mock-transfected cell extract.

Essential role of the terminal domains of P in transcription in vitro and replication in vivo. To investigate the role of the terminal domains of P in transcription, N- and C-terminally deleted P proteins were coexpressed with L at optimal plasmid concentrations, and cytoplasmic extracts were used directly in an in vitro transcription reaction containing purified N-RNA template in the presence of [-³²P]UTP. The RNA products were subsequently analyzed in a 5% polyacrylamide-urea gel followed by autoradiography. As shown in Fig. 2A, the C-terminally deleted P mutants 10C and 20C were totally inactive in mRNA synthesis, whereas the N-terminally deleted P mutants 20N and 40N were highly active; specifically, P40N was as active as wt P. The expression levels of the wt and mutant P proteins were monitored by Western blot with anti-HPIV3 antibody and found to be virtually similar (Fig. 2B). These data indicate that 10 aa in the C-terminal domain of P are required for mRNA synthesis, while a minimum of 40 aa at the N-terminal domain are dispensable.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Activity of mutant P proteins in transcription in vitro. The N- and C-terminally deleted P proteins indicated were coexpressed with L protein in a recombinant vaccinia virus expression system. (A) Cell extracts (total protein, 6 µg) containing the coexpressed L and P proteins were used in transcription reactions containing N-RNA template in the presence of [32P]UTP, and the mRNA products were analyzed in a 5% polyacrylamide-urea gel. (B) Western blot of the cell extracts using anti-RNP antibody raised in rabbit that recognized the HPIV3 N and P proteins. The migration positions of wt and mutant P (Pwt and Pmut) and a nonspecific host protein are shown. The data are representative of three separate experiments with an experimental variability of <10%. Mock, vaccinia virus-infected and mock-transfected cell extract.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Activity of mutant P proteins in minigenome replication in vivo. Terminally deleted P proteins were coexpressed with L and N in the minigenome replication system containing pHPIV3-MG() plasmid as described in Materials and Methods. At 24 h postinfection, cell extracts were prepared and luciferase activity was measured. (Top) Schematic representation of the minigenome replication assay. (A) Expression of luciferase reporter gene by the supporting plasmids expressing N, L, and wt or mutant P proteins. The background level of luciferase activity (Luc. Act.) was determined by omitting the L plasmid DNA, and the value was subtracted from the data. The data represent the averages of three independent experiments, and standard deviations are indicated as error bars. (B) Primer extension analysis (27) of transcriptive (trans) and replicative (rep) RNAs in the presence of various P proteins, as indicated. The primer used anneals to the positive-sense luciferase coding sequence at the initiating AUG codon. Lane ()L, L plasmid DNA was omitted during transfection. Lanes A, C, G, and T represent the sequencing ladder. T7 , T7 RNA polymerase transcription termination signal; Rz, hepatitis delta virus ribozyme; le, leader; GS, gene start signal; GE, gene end signal; tr, trailer (27).

C-terminal domain of P required for N-RNA template binding, and N-terminal domain required for soluble N-P complex formation. Interaction of P with the N-RNA template is an initial step that mediates the binding of RNA polymerase complex L-P to the template to initiate both transcription and replication processes (29, 34). To study the role of terminal domains of P in this interaction, both wt and mutant P proteins were synthesized in vitro in a reticulocyte lysate using coupled transcription-translation conditions and labeled with [³⁵S]methionine. Equal amounts of translated P proteins free of large protein aggregates were then incubated with purified N-RNA under HPIV3 transcription conditions. The radiolabeled proteins forming the complex with N-RNA were pelleted by centrifugation through a 30% glycerol cushion and directly analyzed in an SDS-10% polyacrylamide gel. As shown in Fig. 4, both wt and N-terminally deleted P mutants 20N and 40N bound efficiently to the N-RNA, whereas C-terminally deleted P mutants 10C and 20C bound only 15 to 20% as well as wt P. Based on the fact that P10C and P20C failed to mediate RNA synthesis in vitro and in vivo (Fig. 2 and 3), the low level of binding of the mutants to the template possibly is not sufficient for activating the RNA polymerase for RNA synthesis during transcription and replication. Thus, it seems that the C-terminal 10-aa region is crucial for binding to the N-RNA template and consequently in transcription and replication.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Binding of mutant P proteins to N-RNA template. The terminally deleted P proteins were synthesized in a reticulocyte lysate and labeled with [35S]methionine. Binding of labeled proteins to N-RNA template (5 µg) was studied as described in Materials and Methods. Binding of P proteins was determined by phosphorimager quantitation of the P protein band after analysis in an SDS-polyacrylamide gel, shown as an inset. The data are representative of two separate experiments.

View larger version (60K): [in this window] [in a new window]   FIG. 5.   Glycerol gradient analysis of encapsidation complex. The wt and mutant P proteins were coexpressed with N in HeLa cells using the recombinant vaccinia virus expression system as described in Materials and Methods. Cell extracts were prepared at 24 h postinfection, and the proteins were subjected to 5 to 20% glycerol gradient centrifugation. The proteins in the gradient fractions, as indicated, were resolved in an SDS-10% polyacrylamide gel and detected by Western blot using anti-HPIV3 antibody. (A) Expression of N alone. (B) Expression of P alone (top panel) and coexpression of N and P (bottom panel). (C) Expression of P40N alone (top panel) and coexpression of N and P40N (bottom panel). (D) Expression of P20C alone (top panel) and coexpression of N and P20C (bottom panel). Numbers above each panel indicate glycerol gradient fractions collected from the top of the gradient. The migration positions of N and the wt and mutant P proteins in the SDS-polyacrylamide gel are shown. Host, host protein that nonspecifically reacted with the anti-HPIV3 antibody.

Role of terminal domains in P-P and L-P complex formation. Since the P proteins of some members of the Rhabdoviridae and Paramyxoviridae families have been shown to form dimers, trimers, or tetramers (7, 12, 23, 36, 42), we were interested in studying the oligomeric state of HPIV3 P protein by coimmunoprecipitation of tagged and untagged P proteins. The P protein was flag-tagged at the C terminus and coexpressed with a large excess of untagged wt or mutant P proteins and metabolically labeled with [³⁵S]methionine. Radiolabeled soluble cytoplasmic proteins were coimmunoprecipitated using antiflag antibody conjugated to Sepharose beads, and the proteins were analyzed on an SDS-polyacrylamide gel followed by fluorography. As shown in Fig. 6, coexpression of flag-tagged P with increasing amounts of untagged P resulted in coprecipitation of different amounts of untagged P in the oligomeric complex. The flag-tagged P and untagged P, which were poorly separated and migrated together in the gel as a closely spaced doublet, were quantitated. Routinely, about two untagged P (1.4 to 1.9) per flag-tagged P were seen, suggesting the formation of a P homooligomer, possibly a homotrimer. The flag-tagged P was confirmed to be as active as the untagged P in minigenome replication in vivo (data not shown). The role of terminal domains in the P oligomerization was then studied by coexpressing increasing amounts of P40N and P20C with the flag-tagged P. P40N was coprecipitated with flag-tagged P, and about 2 (1.3 to 1.6) P40N were present per flag-tagged P, indicating that P40N efficiently formed a heterooligomer, possibly a heterotrimer. In contrast, the C-terminally deleted P20C was coprecipitated with flag-tagged P in a significantly lower amount, indicating less efficient incorporation into the complex. Phosphorimager quantitation revealed that about 1 P20C was present per 2 flag-tagged P (0.5 to 0.6 P20C per flag-tagged P), suggesting that 2 flag-tagged P are associated with 1 P20C. The expression levels of P40N and P20C were determined by Western blot using anti-HPIV3 antibody and found to be similar (data not shown). Thus, it seems that the C-terminal domain somehow plays a role in stabilizing a P subunit in the heterooligomer.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Analysis of P-P and L-P complexes in a flag-tagged immunoprecipitation system. The proteins were coexpressed and metabolically labeled with [35S]methionine. The labeled proteins present in the cytosolic fraction were immunoprecipitated with anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS-10% polyacrylamide gel followed by fluorography. (A) Analysis of P-P complex; 200 ng of flag-tagged P (Pf) plasmid DNA was used in the transfection. Numbers above the lanes indicate the amount of untagged mutant (Pmut) and wt P protein-expressing plasmid DNA (in micrograms) used for transfection. The ratios of untagged to flag-tagged P proteins are shown below. (B) Analysis of L-P complex; 200 ng of flag-tagged L-expressing plasmid DNA (Lf) was used in all transfections. The untagged wt and mutant P protein-expressing plasmid DNAs, where indicated, were used at a concentration of 1 µg each. The ratios of untagged P protein to flag-tagged L in each case are shown below. Radiolabeled bands were visualized by fluorography and quantitated by phosphorimager.

Terminally deleted P proteins can function in minigenome replication by forming heterooligomers with wt P. The observation that the N- and C-terminally deleted P proteins form heterooligomers with wt P prompted us to investigate whether the heterooligomeric forms of P can affect minigenome replication in vivo. Accordingly, we cotransfected N- and C-terminally deleted P with wt P in the minigenome replication system containing flag-tagged L. A low concentration of wt P was used so that it would be sufficient for a basal level of luciferase expression but suboptimal for supplemental function to mediate efficient transcription and replication. The roles of N- and C-terminally deleted P proteins were then investigated by cotransfecting increasing amounts of plasmid DNAs expressing these mutants. As shown in Fig. 7A, a basal level of luciferase expression was detected by using an optimal concentration of flag-tagged L and a low concentration of P (0.3 µg of plasmid DNA). The activity was increased linearly to a maximum of about threefold when the P-expressing plasmid DNA was progressively increased to 1.2 µg, indicating that P is required in stoichiometric amounts for efficient minigenome replication. Surprisingly, cotransfection of increasing amounts of plasmid DNA expressing P40N had virtually no effect on the basal level of luciferase expression, suggesting that the heterooligomer containing wt P and P40N is active in basal RNA synthesis but inert in the stoichiometric function for efficient minigenome replication. By contrast, transfection of plasmid DNA expressing P20C, which presumably formed a heterotrimer, showed increased expression of luciferase, which rapidly reached a plateau, unlike that of wt P, indicating that the heterooligomer is active, albeit at a low level, in the stoichiometric function. To ascertain that the observed effect is specific and not due to an alteration of protein expression, we performed Western blot analysis using anti-RNP antibody. As shown in Fig. 7B, high-level expression of N and P proteins (900 ng of plasmid DNA) was observed. Thus, it seems that both P40N and P20C can form a functional heterooligomer with wt P but the former cannot perform the stoichiometric function of P, whereas the latter does. It is important to mention that the increase in luciferase expression correlated with increased mRNA and plus-sense genome RNA synthesis, which was confirmed by primer extension analysis (data not shown).

View larger version (54K): [in this window] [in a new window]   FIG. 7.   Stoichiometric requirement for P for efficient minigenome replication, and role of terminal domains in this process. (A) The complete minigenome replication system contained pHPIV3MG() (200 ng), pN (640 ng), pLf (200 ng), and pP at a suboptimal concentration (300 ng). pP, pP40N, and pP20C (patterned bars from left to right) were expressed, in addition to the components present in the complete system (solid bar), as indicated above, in increasing concentrations (300, 600, and 900 ng), and luciferase activity in the cell extract was determined. (B) Western blot analysis of N and P proteins using anti-RNP antibody that recognized the N and P proteins. The migration positions of N and P are shown. A protein band migrating faster than N may be a degradation product of P40N. The amounts of plasmid DNAs used are equivalent to the amounts indicated for the complete system. In addition, lanes Pwt, P40N, and P20C contained 900 ng of the respective plasmid DNAs for transfection. (C) Immunoprecipitation of radiolabeled proteins from cells that were transfected with plasmid DNAs as described for panel B, except the first lane contained pLf alone. Cells were metabolically labeled with [35S]methionine and immunoprecipitated by using anti-flag antibody conjugated to Sepharose beads. The precipitated proteins were analyzed in an SDS-10% polyacrylamide gel and subjected to fluorography. The data are representative of three separate experiments with an experimental variability of <10%.

40N

20C

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this communication we describe the development of an efficient in vitro transcription reconstitution system for HPIV3 using recombinant L and P proteins. In addition, replication in vivo was studied using a recently developed minigenome replication system in our laboratory (27). Using both these systems, we investigated the role of the N- and C-terminal domains of P in viral mRNA synthesis in vitro and replication in vivo. Our data indicate that both the N- and C-terminal domains of P protein play important roles in these RNA synthetic processes (Fig. 2 and 3); the N-terminal domain is primarily involved in replication, whereas the C-terminal domain is involved in both transcription and replication processes. Consistent with these findings, the N terminus of P is required for interaction with N to form soluble N-P complex. The C terminus, on the other hand, is involved in the binding of P to N-RNA template as well as in interaction with L protein to form stable L-P complex, where the P protein appears to form stable homotrimers but was also identified in the higher oligomeric forms when associated with L or N protein.

The in vitro transcription reconstitution system described here confirms that HPIV3 L and P proteins, like Sendai virus (8), must be coexpressed for the formation of active RNA polymerase complex, possibly to protect L from degradation (40). Using flag-tagging, we were able to detect L protein in the absence of P; however, the level of L expression was increased significantly (about fivefold) when coexpressed with the P protein, indicating the protective role for P against L protein degradation. The coexpression studies further demonstrated that for optimal RNA synthesis, stoichiometric amounts of P protein were neededan observation similar to that previously reported with VSV (13) and recently with respiratory syncytial virus (16) and Sendai virus (5). Thus, it is becoming increasingly apparent that the P protein provides two important functions in RNA synthesis (13); (i) formation of a complex with RNA polymerase L-P, for which it is required in catalytic amounts, and (ii) interaction with the N-RNA template to facilitate elongation of RNA chains, for which it is required in stoichiometric amounts. However, it remains unclear whether the stoichiometric function of P protein is manifested while it is complexed with the L protein or independent of the L protein. On the other hand, the minigenome replication system in vivo relies on RNA synthesis as well as concurrent encapsidation of nascent RNA chains during replication and thus provides a means to study the differential role of P protein in RNA synthesis and encapsidation during replication. Our observation that the P protein is also required in stoichiometric amounts for luciferase reporter gene expression in the minigenome replication system (Fig. 7A) raises the possibility that the stoichiometric requirement for P in minigenome replication may be both at the level of RNA synthesis and at the level of encapsidation of nascent RNA chains during replication while complexed with the N protein.

When the terminally deleted P proteins were analyzed in the in vitro transcription and in vivo minigenome replication, the C-terminal 10 aa essential for interaction with N (44) were found to be required for both transcription and replication (Fig. 2 and 3). Since the C-terminal 10 aa are also involved in the binding of P protein to the N-RNA template (Fig. 4), it can be concluded that the observed defect is directly at the RNA synthesis step during both transcription and replication, similar to that observed for VSV (24) and Sendai virus (39). It is important to note, however, that the Sendai virus P protein requires the C-terminal 30 aa for its binding to the N-RNA template, and the N-RNA binding domain is mapped within two noncontiguous boxes including the C-terminal 30 aa (38). At the present time we do not know whether the N-RNA binding domain of HPIV3 P also spans such noncontiguous domains. Additional internal deletions are needed to pinpoint such a domain. It is clear from the in vitro and in vivo results (Fig. 2 and 3) that the N-terminal 40-aa region is required for replication but not transcription, which is consistent with the N-terminal domain's being involved in the formation of the N-P complex (Fig. 5) required for encapsidation (9, 44). This suggests that the observed defect of P40N in minigenome replication in vivo is most likely at the level of encapsidation of nascent RNA chains during the replication process. Further studies are needed to directly confirm this contention. Moreover, it would be interesting to determine whether coexpression of P-D or Pt (normally synthesized by editing of P mRNA), which contain the N-terminal domain but either differ at or lack the C-terminal domain, can complement the P40N defect in the encapsidation process. Their participation in the encapsidation process would suggest an additional regulation by these viral proteins in the switch from transcription to replication by the RNA-editing mechanism.

Analyses of P-P and L-P complexes involved in RNA synthesis during transcription and replication demonstrated that the HPIV3 P protein expressed alone is capable of forming a stable homooligomer (Fig. 6) similar to VSV (12, 23), Sendai virus (7), and HPIV2 (36). Our data suggest that the HPIV3 P protein forms a homotrimer, but this needs to be confirmed by a more sensitive technique similar to that recently used (42) to correctly determine the Sendai virus P oligomeric state as a tetramer rather than the previously reported trimer (7). The oligomerization of the P proteins is predicted to occur via a specific coiled-coil domain present in the P protein (7). Such a domain has been reported to be present in influenza virus HA (3, 43), reovirus cell attachment protein 1 (32), clathrin triskelion (35), and heat shock transcription factor (37). In all of these cases the oligomerization of the protein is thought to be essential for the manifestation of its biological activity. In the case of HPIV3 P protein (603 aa long), the putative oligomerization domain can be found within aa 416 to 457 (7), the role of which in the trimerization process of the protein will be important to study. Interestingly, P40N was found to interact efficiently with wt P, forming a heterooligomer, whereas P20C interacted only poorly (Fig. 6), suggesting that the C-terminal 20 aa provide some regulatory role in the oligomerization process (Fig. 6). In this respect, HPIV3 P protein is similar to the measles virus P protein (507 aa long), in which the C-terminal domain also plays a regulatory role in P-P interaction (26), although the putative oligomerization domain is found within aa residues 309 to 341 (7). Analysis of L-P complex formation, however, revealed that the wt P protein is present in the complex most likely in the trimeric form (Fig. 6), whereas P40N complexes with L in a higher oligomeric form (5 P40N per L) and P20C is weakly complexed with L and is present at about 1 P20C per L. Together, these data strongly suggest that the P protein interacts with L, perhaps in the monomeric form, and subsequently oligomerizes to form trimers or even higher-order oligomers. This conclusion is underscored by the findings that under high P expression conditions, the molar amount of P in the L-P complex was as high as 6 P per L (data not shown). Thus, it seems that the core RNA polymerase complex contains a subunit composition of L and P at 1:1 to stabilize the complex (Fig. 6C); however, P protein composition changes from monomer to multimeric states (L-P_3-6) in the holoenzyme to maximize the RNA-synthesizing activity.

The most interesting findings emerged from these studies when we coexpressed the N- and C-terminally deleted P proteins in the minigenome replication system in the presence of a suboptimal level of wt P. Unlike Sendai virus, in which a heterooligomeric complex containing mutant and wt P proteins inhibited viral transcription in vitro (6), the HPIV3 P mutants, when present in large excess within the heterooligomeric complex, did not inhibit viral transcription or replication in vivo, suggesting that the presence of 1 wt P in a heterooligomer is sufficient for the formation of active RNA polymerase complex. This difference may be due to different systems, e.g., in vitro and in vivo, used in the two studies or may represent a major difference between the P proteins of Sendai virus and HPIV3. Surprisingly, P40N, which is active in RNA synthesis in vitro, failed to provide the stoichiometric function in vivo for efficient minigenome replication in the presence of wt P protein (Fig. 7A). Thus, the stoichiometric requirement for P in in vivo minigenome replication is possibly at a step other than RNA synthesis, most likely at the step of encapsidation. This notion is supported by the previous findings that the efficiency of luciferase expression in the minigenome replication system is mostly dependent on the encapsidation of genome-sense RNA synthesized by viral RNA polymerase and thus amplification of the minigenome template (17). On the other hand, P20C, which is inactive in RNA synthesis in vitro (Fig. 2) but efficiently forms soluble N-P complex (Fig. 5D), can provide the stoichiometric function, albeit at a low level, when coexpressed in stoichiometric amounts with the wt P in the minigenome replication system. Thus, it is tempting to speculate that within the heterooligomeric complex, 1 wt P is necessary and sufficient to form a stable complex with the L protein to form the core enzyme, while additional P subunits present in the complex seem to be required for efficient RNA synthesis during transcription and replication. Similarly, at the level of encapsidation during replication, 1 wt P is sufficient to form the N-P complex, but additional P subunits in the heterooligomer are possibly required to efficiently perform the encapsidation process. Further studies are under way to elucidate the mechanistic role of various complexes of the P protein of HPIV3 during viral transcription and replication.