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Journal of Virology, December 2007, p. 13835-13844, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00914-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

De Novo Synthesis of N and P Proteins as a Key Step in Sendai Virus Gene Expression{triangledown}

Marian A. Wiegand,1 Sascha Bossow,1 Sabine Schlecht,2 and Wolfgang J. Neubert1*

Department of Molecular Virology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany,1 Bristol-Myers Squibb GmbH & Co. KGaA, Sapporobogen 6-8, 80637 München, Germany2

Received 29 April 2007/ Accepted 30 August 2007


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ABSTRACT
 
Among the members of the paramyxovirus family, the transcription process and the components involved have been studied under in vitro conditions thus far. Here, we reexamined the function of the viral RNA-dependent RNA polymerase through infection studies with Sendai virus (SeV) N and P deletion ({Delta}) mutants. To elucidate solely transcription-specific processes, all virus mutants also were rendered deficient in genome replication. Using mutant SeV {Delta}P, the earlier suspected supplemental role of P protein was clearly demonstrated to be essential during viral gene expression. Moreover, when SeV {Delta}N or {Delta}N P{Delta}2-77 (with the 5' end of the P gene deleted) mutant was used for infections, a completely unexpected new and essential role for N protein was discovered for viral gene expression. In the early phases of an infection and in the absence of de novo viral protein synthesis, primary transcription occurs at hardly detectable levels. In contrast, if newly synthesized N protein is present, primary viral transcription reaches normal levels. From our data, we conclude that de novo synthesis of SeV N and P proteins is a key step for viral gene expression that facilitates the transition from preliminary to normal primary transcriptional activity.


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INTRODUCTION
 
For a long time, Sendai virus (SeV) was used as a model paramyxovirus to study the virus life cycle. Upon infection of a host cell, different stages are completed during replication of the negatively oriented single-stranded RNA genome with its six genes before progeny virus particles are assembled. In the beginning, only one nucleocapsid (NC) is present in the cell. The NC is composed of the RNA genome encapsulated by nucleocapsid (N) proteins (ribonucleoproteins [RNP]) and 30 to 50 associated viral RNA-dependent RNA polymerase molecules (vRdRp) consisting of a P protein tetramer and one L protein. This minimal replication unit is capable of starting viral transcription. During these first transcriptional events, termed primary transcription, the genome is transcribed by the vRdRp and de novo protein synthesis begins. As soon as sufficient amounts of N proteins have been translated, the vRdRp is thought to switch to a replicative mode to synthesize antigenomes. From that template, new genomes are replicated that are concomitantly encapsidated with N proteins (22). Thus, new templates that also can be transcribed before they are assembled into progeny viral particles are present in the cell. This phase of transcription is called secondary transcription and is much more effective than primary transcription.

During in vitro studies, viral transcription can be reproduced by incubating purified RNPs with cell extracts containing SeV P and L proteins (14). For genome replication, additional expression of viral N protein is necessary, which, when bound to a P protein (termed N0-P), serves as a substrate for encapsidation of nascent RNA genomes and antigenomes (20).

However, different puzzling observations have led to an ongoing discussion about other possible functions and tasks for the P protein. During infections, the synthesis of viral P protein is disproportionately high compared to that of the other proteins (23, 26). Furthermore, the P protein is observed in 4 to 10 clusters on the NC in infected cells. In contrast, NCs derived from virions show a uniform distribution of the P protein (27, 28). This difference is proposed to be related to different functions of the P protein. Inside virions, the P protein may stabilize the NC, the half-life of which is estimated to be only 1 to 2 days in a cellular environment (24). On the other hand, the P protein may enhance the processivity of the vRdRp during infections. When RNPs are incubated in the presence of a 5- to 10-fold elevated amount of P protein compared to the level of P as part of vRdRps, only a twofold increase in mRNA synthesis is detected (12). Interestingly, in vitro RNA synthesis studies have shown that mutations in P inhibit viral transcription, even though the interactions between P and L, P and P, and P-L and the template RNA were not impaired (4). These results also may indicate an additional function of the P protein independent of binding to L or to itself.

There have been several observations indicating that the P protein is involved in other undescribed processes during viral replication; no concrete evidence exists for an additional function.

However, these observations all were made under in vitro conditions with about 1011 NCs employed per assay. Under these conditions, it is impossible to estimate the transcriptive power of single NCs inside cells. However, the intent of our studies here was to investigate, for the first time, the influence of SeV P protein on viral gene expression under conditions of a normal infection, i.e., with about one virus particle per cell using specifically designed mutants. We therefore constructed SeV mutants that lacked the P open reading frame (ORF) (SeV {Delta}P) or the N ORF (SeV {Delta}N). Without de novo-synthesized P protein, we expected to elucidate additional functions of this protein, e.g., a possible influence on biphasic mRNA synthesis rates as described by Plumet and colleagues (25); without N protein synthesis, we tried to suppress genome replication. During infection experiments, we could show that de novo synthesis of viral P protein is necessary to bring preliminary primary viral transcription to normal levels. Very surprising and never reported before, we also found that viral N protein not only was necessary for viral replication but also was essential for normal viral primary transcription.


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MATERIALS AND METHODS
 
Cells. Vero cells were obtained from the American Type Culture Collection (ATCC) (CCL-81), and BSR-T7 cells were a gift from K. K. Conzelmann (Munich, Germany). Vero, BSR-T7, and all helper cell lines (HCs) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) in a 5% CO2 atmosphere at 37°C.

HCs were generated by transfection of Vero cells with an N and/or P and N genes (NNP-HCs and PNP-HCs, respectively) harboring expression plasmids (pRECISE; see below). Two days posttransfection, cells were incubated with selective medium supplemented with G418 (1,500 µg/ml) for N-gene-transfected cells and/or with hygromycin (200 µg/ml) for P-gene-transfected cells. After 2 weeks, positively selected single-cell clones were isolated. The most efficient clone for supporting SeV {Delta}N or SeV {Delta}P replication was identified through test infections with the respective virus deletion mutant. Cell infections were performed as previously described (2).

Construction of mutated genomic SeV and expression plasmids. The wild-type SeV (SeV wt) sequence with an enhanced green fluorescent protein (eGFP) gene just downstream of the P gene has been previously described (3).

The cDNA of the mutant SeV {Delta}P, lacking the P ORF (corresponding to an original position in SeV wt from nucleotide [nt] 1844 to 3550), which was replaced by an XhoI site plus the three additional nucleotides CTC, was created via PCR with an SeV full-length genomic cDNA template containing a modified F gene. The wt monobasic trypsin-dependent cleavage site (VPQSR {downarrow} FF) was mutated to an oligobasic cleavage site (RRQKR {downarrow} FF), called Fmut, allowing F activation in any cell type through a ubiquitous furin-like protease. First, two PCR fragments were produced. The first fragment was produced using the oligonucleotides 5'-CCCTGACACACTCCTTC-3' and 5'-GCGCCGCTCGAGGCGGTAAGTGTAGCCGAAG-3'; the fragment ranged from within the N gene to the 5'-untranslated region (5'UTR) of the P gene followed by an artificial XhoI site (underlined). For the second fragment, the oligonucleotides used were 5'-CCTGCGCTCGAGCTCATCCCGGGTGAGGCATCCC-3' and 5'-GGCGACGCGTCAGTCTCACAGCCTAATTCG-3'; the fragment begins with the 3'UTR of the P gene, is preceded by an artificial XhoI site, and ends within the F gene. The two fragments were ligated together through the XhoI site in an intermediate construct that then was used to replace the respective wt sequence in pRS-ld-eGFP, which contains an eGFP gene downstream of the leader region (1), via SphI (within the N gene) and AfeI (within the F gene), resulting in pRS-ldE-{Delta}P-Fmut.

The cDNA of the mutant SeV {Delta}N was created by eliminating the ORF of the N gene (corresponding to an original position in SeV wt of nt 120 to 1694), which was replaced by a NotI restriction site (underlined in the sequence below) plus the three additional nucleotides, TGA. First, an insert harboring the N ORF deletion was created by a PCR fusion technique employing the following oligonucleotides: M13R (forward; precedes the T7 promoter and SeV leader) and 5'-TCGTATGATCTCAGCGGCCGCTAGGCAGCAAAGCAAAGGGTCTGG-3' for the first fragment, containing a MluI site at its 5' end; and 5'-TGCTTTGCTGCCTAGCGGCCGCTGAGATCATACGAGGCTTCAAGGTAC-3' and 5'-ATCATCGCATGCTTGGCTGGACAATCTGAGACAGAG-3' (reverse) for the second fragment. The final insert was produced in a third PCR in which both fragments annealed with one another through their overlapping sequences. The forward primer of the first PCR and the reverse primer of the second PCR were used for amplification, and the fragment was used to replace the respective sequences of pRS-P-eGFP after SanDI restriction. The resulting plasmid was named pRS-{Delta}N-PE. Finally, the modified F cleavage site was introduced through an insertion of a BstXI fragment into the respective part of pRS-ldE-{Delta}P-Fmut, yielding pRS-{Delta}N-PE-Fmut.

For the cDNA of mutant SeV {Delta}N P{Delta}2-77E Fmut, a PCR fragment harboring the mutation leading to the deletion of amino acids (aa) 2 to 77 of the P protein was created by using the oligonucleotides M13R and 5'-ACCTGATCGATTATCTTGGGTCGACATGCGGTAAGTGTAGCCGAAGC-3' (reverse); the fragment extends from the 5'UTR of the P gene to the 24 nt downstream of the deleted part of the P ORF that includes a ClaI site (underlined). Via MluI and ClaI restrictions, the respective part from pRS-{Delta}N-PE-Fmut was replaced by the new fragment, leading to pRS-{Delta}N-P{Delta}2-77E-Fmut. All recombinant SeV variants were derived from SeV Fushimi strain D52 (murine parainfluenza virus type 1; ATCC VR-105). All modified genomes were created in compliance with the rule of six (7).

The expression plasmids pRECISE-N439, pRECISE-N461, and pRECISE-N471, harboring the C-terminal deletion of the SeV N gene, were created by transferring the respective mutated N genes via XhoI and SalI restriction of the respective pGEM expression plasmids, which were a kind gift from Sue Moyer (Gainesville, FL).

The pRECISE plasmid harbors an expression unit consisting of a cytomegalovirus promoter and enhancer element together with intron A and a woodchuck hepatitis posttranscriptional regulatory element preceding a simian virus 40 poly(A) signal and simian virus 40 enhancer sequence.

Virus rescue. BSR-T7 cells (0.5 x 106) were transfected with a plasmid mixture consisting of 0.25 µg pTM-N (for SeV N gene), 0.15 µg pTM-P/C– (for SeV P gene), 0.05 µg pTM-L (for SeV L gene), and 5.0 µg of a plasmid carrying the cDNA of the viral genome flanked by T7 promoter and terminator sequences so that the viral genomic sequence is transcribed by the T7 polymerase. The correct 5' end of the viral genome was produced by a self-catalytic ribozyme sequence (from the hepatitis D virus) located directly downstream of the viral antigenomic sequence. Transfections were performed using FuGENE 6 or FuGENE HD reagents (Roche) at 2.5 µl/µg plasmid DNA. During and after transfection, cells were incubated with DMEM supplemented with 2% FCS. Viral particles generated initially could be harvested from the cell culture supernatant from days 2 to 4 in the case of SeV wt. The rescue procedure for the deletion mutants was slightly modified. Three days posttransfection, the cells were transfected a second time with the following amounts of plasmid DNA: 1.0 µg pRECISE-N for SeV {Delta}N, 1.0 µg pRECISE-P for SeV {Delta}P, and 1.0 µg of each plasmid for SeV {Delta}N P{Delta}2-77. Titers of viral progeny were determined via infection of Vero cells or the respective HC line in a dilution series and counting eGFP-positive cells. Titers are given in cell infectious units (CIU) per milliliter.

Cell transfection. Transfection of BSR-T7 cells, seeded at low density (0.3 x 106 cells in each well of a 6-well plate), was performed with FuGENE 6 or FuGENE HD (Roche) reagents as described by the manufacturer.

Verification of replication deficiency of SeV mutants via RT-PCR. From 1 µg total cellular RNA isolated (RNeasy; Qiagen) from virus-infected and mock-infected cells, viral genomes were selectively reverse transcribed (RT) (Superscript III first-strand synthesis system for RT-PCR; Invitrogen) using a primer that anneals within the leader region from the original SeV genome (nt 25 to 46): 5'-TGGAATATATAATGAAGTCAGA-3' (RT conditions were 50°C for 60 min and 85°C for 5 min). An aliquot of the resulting cDNA (2 µl) was employed in a PCR with the oligonucleotides 5'-GGATACAGCCAAGGAGAGGC-3' (nt 1280 to 1299) and 5'-GGGGCATTGTCGCAGGTC-3' (nt 4749 to 4732) to amplify the sequence from within the N gene to the M gene (36 cycles of the following: denaturing at 94°C for 45 s, annealing at 57°C for 45 s, and synthesis at 68°C for 3 min) (Expand Long Template; Roche). RNA preparations from uninfected cells or SeV wt-infected cells without the addition of enzyme during RT were used as negative controls. The products were analyzed by electrophoresis in 1% agarose gels (in Tris-borate-EDTA buffer).

Flow cytometry. Infected cells were washed with DMEM, trypsinized, and resuspended in medium. After the detached cells were sedimented and washed once with phosphate-buffered saline (PBS), the cell pellet (approximately 0.5 x 106 cells) was resuspended in 200 µl PBS. The number of eGFP-expressing cells was immediately determined by flow cytometry (FACScalibur; Becton Dickinson) using CellQuest software.

Immunofluorescence. After infection and incubation, cells were fixed with methanol (–20°C) for 10 min. Cells then were washed once with PBS and incubated with a primary antibody (antiserum against all SeV proteins, of our own production [16], diluted 1:100 in PBS-3% bovine serum albumin [BSA]) for 1 h at room temperature. After being washed five times with PBS, the cells were incubated with secondary antibody (Alexa Fluor 568 goat anti-rabbit immunoglobulin G [Molecular Probes] diluted 1:200 in PBS-3% BSA) for 1 h. After being washed five times with PBS, the cells were monitored by fluorescence microscopy.

Western blotting. Western blot analysis was performed to compare protein expression levels of SeV wt in non-HCs to those in HCs and transiently transfected BSR-T7 cells. After cell lysis with 300 µl radioimmunoprecipitation assay buffer (10 mM Tris, pH 8, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS]) per 0.5 x 106 cells, protein concentrations were determined (bicinchoninic acid protein assay kit; Novagen). Aliquots of protein were heated for 4 min at 96°C in sample buffer containing 10% ß-mercaptoethanol and were applied to a 10% polyacrylamide gel (the buffer consisted of 25 mM Tris-HCl, 150 mM glycine, 0.1% SDS). After gel electrophoresis, proteins were transferred to polyvinyl difluoride membranes by electroblotting. A 1-h preincubation at room temperature with Roti-Block solution (Carl Roth GmbH, Germany) to block nonspecific binding sites was followed by a 1-h incubation with the primary antibody (antiserum against all SeV proteins, diluted 1:1,000 in Roti-Block solution). After being washed five times with TBST (10 mM Tris [pH 8], 150 mM NaCl, 0.05% Tween 20), the blot was incubated with secondary horseradish peroxidase-labeled antibody (diluted 1:2,000 in TBST; Dako, Germany) for 1 h. The membranes were washed, incubated in ECL reagent (Amersham, Germany) for 1 min, and exposed to X-ray films (Hyperfilm ECL; Amersham).


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RESULTS
 
Generation of SeV mutants. Using reverse genetic techniques, we constructed different SeV mutants in order to investigate viral transcription, with a particular focus on the role and the function of the viral P protein. Through the deletion of the ORF of the P gene, no new P protein can be synthesized during infections with the SeV {Delta}P mutant. At the same time, this deletion should render the SeV {Delta}P mutant genome replication deficient and thus should prevent an increase in template numbers during infection. Likewise, the mutant SeV {Delta}N with a deletion of the N ORF also is expected to be incapable of genome replication but still encodes intact viral P protein. The mutant SeV {Delta}N P{Delta}2-77 carries two deletions: the N ORF is deleted, and the nucleotides encoding aa 2 to 77 of the P protein that are thought to harbor the domain for the interaction of the N protein with the N0-P complex, which is essential for genome replication (16), is deleted. Each deleted region in SeV {Delta}N P{Delta}2-77 alone should inhibit genome replication. The recombinant wt virus (SeV wt) and all mutants have an eGFP gene inserted in their genomes (Fig. 1). The position of the eGFP gene in the genome, whether just downstream from the leader region as in SeV {Delta}P or behind the P gene as in SeV {Delta}N, has a slight but not significant influence on the expression level of the viral proteins. This was tested with two SeV wt variants, SeV ld-eGFP and SeV P-eGFP, and also was anticipated based on previous studies (18, 29).


Figure 1
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  		FIG. 1. Genomic organization of recombinant wt and mutant SeV. (A) The schematic illustrations show the position of each mutation within the genome of the deletion mutants, starting with the leader (ld) and ending with the trailer (tr). Further, the position of the eGFP transgene within all genomes is indicated, as is the presence of a mutated F gene (Fmut) in the genomes of the deletion mutants. Fmut can be activated by a ubiquitous protease found in most tissues. (B) Illustration of the C-terminal domain of SeV N protein showing the assigned functional sites for the interaction of N protein in RNP (NNC-NNC) with the viral polymerase (NNC-vRdRp) and of unbound N protein with P (N0-P).

The viral mutant genomes were created at the level of a cDNA inserted in expression plasmids. These plasmids were employed in initial virus rescue experiments. The amount of viral particles that could be generated differed among the mutants and was clearly reduced compared to that of SeV wt. These results can be explained by the inability of the deletion mutants to support the production of all necessary viral components for genome replication. While SeV wt particles could be rescued at titers of up to 5 x 105 CIU/ml, titers for SeV {Delta}P were 5 x 104 CIU/ml, for SeV {Delta}N were 2x 104 CIU/ml, and for SeV {Delta}N P{Delta}2-77 were only 5,000 CIU/ml. In order to obtain sufficient viral particles for the subsequent studies, mutant viral particles had to be amplified using different biological systems.

Different amplification systems for SeV {Delta}P and SeV {Delta}N. SeV {Delta}P could be amplified within P HCs (PNP-HCs) that express viral N and P proteins simultaneously up to a titer of 107 CIU/ml. In contrast, SeV {Delta}N could not be amplified efficiently within HCs (NNP-HCs) after screening various cell clones expressing different amounts and ratios of N and P proteins. The amount of SeV proteins provided by PNP-HCs and NNP-HCs represented about 10% of the P protein and 5% of the N protein produced during SeV wt infections, respectively (Fig. 2A).


Figure 2
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  		FIG. 2. Comparison of viral gene expression from viral genomes after transient transfection with plasmids or stable transfection. (A) Twenty-four hours after infection of 0.5 x 106 Vero cells with SeV wt (MOI, 3), protein was isolated and compared to extracts from HCs expressing SeV N (NNP-HC) or SeV N and P (PNP-HC) genes. Ten micrograms of protein was applied per lane; the exposition time of the X-ray film was 15 s. (B) Amount of SeV N and P proteins resulting from transient transfection (0.5 x 106 non-HCs with 0.5 µg pRECISE-N and pRECISE-P each). Cellular extracts were prepared 24 h p.i. or posttransfection (p.t.), respectively. Five micrograms of protein from SeV wt-infected cells showed slightly less SeV protein than 10 µg total protein from the transfected cells (exposition time, 3 s). (C) Transient transfection of non-HCs with 0.5 µg each of pTM or pRECISE plasmid containing cloned SeV N or P gene. Ten micrograms of protein was loaded per lane, and the exposition time of the two blots was 3 and 6 s, respectively.

Thus, conditions for efficient genome replication of SeV {Delta}N through trans-complementation seem to be much more stringent and could be dependent on the necessity of a high level of protein compared to that needed for the replication of SeV {Delta}P. Unfortunately, despite many attempts, it was not possible to elevate N protein expression in NNP-HCs significantly above 5% of the wt level, even with different levels of viral P protein expressed in parallel. Perhaps the constant expression of a foreign protein with no benefit for cellular metabolism and potential cytotoxic side effects (11, 15) restricted the expression of the SeV N protein to such low levels. However, when cells were transiently transfected with the newly created high-level expression plasmid pRECISE (see Materials and Methods), the amount of N protein could be increased to SeV wt levels (Fig. 2B). To be available for the packaging of viral genomes, SeV N proteins have to be bound by SeV P proteins (N0-P). Therefore, both N and P genes had to be transfected in parallel. After double transfection of non-HCs (0 and 4 days postinfection [p.i.]) with both genes, both proteins could be produced at wt levels for at least 8 days. Figure 2C shows constant high levels of SeV N and P expression from pRECISE plasmids, in this case for the first 3 days after transfection. In contrast, the protein expression resulting from transfection of non-HC BSR-T7 cells with T7 promoter-driven pTM plasmids was much lower and less constant. For 8 days, the initially rescued SeV {Delta}N particles could be amplified in transiently transfected cells at a titer of up to 5 x 105 CIU/ml. This titer is the result of extensive optimization but is still lower than that of SeV {Delta}P obtained with P-HCs. This result underlines that requirements for the generation of SeV {Delta}N particles are much more demanding than those for SeV {Delta}P. It can only be speculated whether this higher need of N protein for SeV {Delta}N replication compared to that of P protein for SeV {Delta}P replication is related to an important function during the viral life cycle.

The replication deficiency of all mutants was verified by test infections of Vero cells. Over a period of 7 days, neither a spread of green fluorescing cells nor any progeny virus could be detected in the cell culture supernatant. Through semiquantitative RT-PCR analysis, no detectable increase in genome number inside infected cells could be measured from 2 h p.i. to 3 days p.i. (data not shown). These data are in agreement with findings that, in measles virus-infected cells, viral genome replication is completely arrested when de novo synthesis of proteins is blocked through cycloheximide (25).

Viral gene expression with or without de novo synthesis of SeV P protein. The influence of SeV P protein on viral gene expression was investigated during infections of non-HCs and, as a positive control, of HCs providing the missing viral protein component in trans. Keeping in mind earlier work measuring linear and exponential mRNA synthesis rates by molecular biological techniques (25), we decided to introduce biological assays that allowed us to investigate the influences of de novo-synthesized viral capsid proteins on viral gene expression during the infection process. After infection of non-HCs and the respective HCs with SeV {Delta}P (i.e., without de novo P protein synthesis) and SeV {Delta}N (i.e., without de novo N protein synthesis but with de novo P protein synthesis) at a multiplicity of infection (MOI) of 0.05, viral eGFP transgene expression was monitored for 5 days and was evaluated by fluorescence-activated cell sorter (FACS) analysis. We chose an MOI of 0.05 to be able to observe a possible virus spread throughout the culture, which was expected to develop only in HCs.

During SeV {Delta}P infections under these conditions, eGFP expression could be detected in HCs but not in non-HCs over a period of 5 days (Fig. 3A). This result reflects the need for newly synthesized P protein, at least for viral transcription at normal levels. However, when non-HCs were infected with SeV {Delta}N, that is, when newly synthesized P protein can be synthesized from the viral P gene, eGFP expression also was not detected. This result was very surprising, since all viral components so far known to be necessary for SeV transcription, namely, P and L proteins and the RNP template, were present, and the generation of additional vRdRp molecules also was possible due to the presence of P and L genes carried by the virus. Again, analogous to eGFP expression during SeV {Delta}P infections, eGFP expression took place during SeV {Delta}N infections of HCs as shown in Fig. 3 (in this case, 2 days p.i.), which represents averages of the results from days 1 to 5. Obviously, SeV can be transcribed only at a normal level when additional P protein is provided. Although this is the case during SeV {Delta}N infections of non-HCs, gene expression can be detected only during SeV {Delta}N infections of NNP-HCs or during SeV {Delta}P infections of PNP-HCs. These results demonstrate a thus-far-undefined involvement of SeV N protein during viral transcription.


Figure 3
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  		FIG. 3. Comparison of viral transgene expression during infections of non-HCs to that of HCs with SeV {Delta}N, SeV {Delta}P, and SeV wt. Two days after infection with the respective viruses, the cells were analyzed for viral eGFP transgene expression either by fluorescence microscopy (infection at an MOI of 0.05) (A) or via FACS analysis (B); eGFP-positive cells refers to 10,000 cells counted per sample.

In order to document a possible accumulation of viral gene products from SeV {Delta}N, SeV {Delta}P, and SeV wt over a period of 3 days, non-HCs and HCs were infected with the respective viruses. One and 3 days p.i., all viral proteins of the infected cells were detected by immunofluorescence using an antiserum directed against all SeV proteins. This is a very sensitive method for measuring an increase in viral gene expression; a single NC inside a cell can be detected. Very clearly, viral gene products did not accumulate in SeV {Delta}N- and SeV {Delta}P-infected non-HCs. The only signals observed up to 3 days p.i. were very small spots, probably representing proteins of single NCs (Fig. 4). In strong contrast to this, the signal in SeV wt-infected non-HCs already exceeded those from SeV {Delta}N and SeV {Delta}P infections by 2 h p.i. and spread out over the entire cell by 3 days p.i. Again, when NNP-HCs and PNP-HCs were infected with the respective virus mutant, gene expression could be demonstrated early after infection (Fig. 4). Taking these results together, it appears that both proteins SeV N and P have to be synthesized during infections for efficient viral gene expression.


Figure 4
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  		FIG. 4. Development of viral gene expression in non-HCs and HCs infected with SeV {Delta}P, SeV {Delta}N, and SeV wt. Cells were infected with the indicated viruses (MOI, 0.5) or were mock infected. One and 3 days p.i. (except for SeV wt infection of non-HCs, which was evaluated at 2 h p.i. instead of 1 day p.i.), viral gene expression was evaluated via immunofluorescence. The primary antibody was directed against all SeV proteins and was detected with secondary antibodies conjugated with a red fluorescent dye.

On the other hand, early in an infection transcription must take place, possibly at a much reduced level, to start the viral replication cycle, even if no newly synthesized P and N proteins are present. Therefore, SeV {Delta}P or SeV {Delta}N also should be able to transcribe in non-HCs, at least very weakly. When the MOI was increased to 5 for SeV {Delta}P infections of Vero cells, 0.01% of the cells displayed a moderate green fluorescence that lasted from 2 to 10 days p.i. (data not shown). This number of fluorescing cells corresponds very well to a statistical (Poisson) distribution of a cellular fraction simultaneously infected with 15 or more NCs. Thus, by increasing the amount of templates, initial viral gene expression can be detected, probably due to the cumulative effect of a few initially produced eGFP proteins. Interestingly, since this viral eGFP expression lasted for 10 days and the half-life of an eGFP protein is approximately 24 h, NCs seemed to remain active for at least 10 days after infection. This differs from results of in vitro replication studies with NCs of defective interfering particle genomes, the half-lives of which are estimated to be 24 to 48 h (24). Thus, by elevating the MOI and independent of de novo P protein synthesis, eGFP expression during SeV {Delta}P infections could be achieved already after 2 days, thereby demonstrating the efficacy of the detection system. In a parallel approach, we infected cells with only one NC each but extended the incubation time to test whether sufficient proteins can be accumulated in this way. However, an eGFP signal was not detected during a period of 10 days. In conclusion, a strong viral gene expression, comparable to that of SeV wt infections, can be detected only during infections when N and P proteins are synthesized at the same time.

Influence of SeV N protein on viral gene expression. The HCs used above provide the missing viral components during infections with SeV {Delta}N and SeV {Delta}P. Thus, in HCs, all viral proteins are present, and the viral mutants regained the ability not only to transcribe but also to amplify their genomes. Using the double deletion mutant SeV {Delta}N P{Delta}2-77, we wanted to test whether the observed gene expression during SeV {Delta}P and SeV {Delta}N infection of P-HCs and N-HCs, that is, in the presence of de-novo-synthesized P and N protein (Fig. 3), respectively, is influenced by propagation of template RNAs. In SeV {Delta}N P{Delta}2-77, each deletion alone renders the virus replication deficient. The partially deleted P ORF encodes a truncated P protein in which aa 2 to 77 are deleted. This single mutant was characterized using in vitro systems and was shown to be incapable of binding SeV N protein in the N0-P complex, which is responsible for allocating N proteins for the packaging of newly synthesized RNA genomes. All other domains of the P protein still should have been functional (16). Unlike infections of HCs with SeV {Delta}N, infections of non-HCs with SeV {Delta}N P{Delta}2-77 in combination with exogenously provided viral proteins offered the possibility of investigating the influence of the SeV N protein on viral transcription while excluding an increase in the size of the template. Thus, we compared eGFP transgene expression under three different conditions during SeV {Delta}N P{Delta}2-77 infections of non-HCs: without any additional viral proteins, with additional viral N protein only, and with additional viral N and P proteins. After transfection of BSR-T7 cells with the respective viral genes, their expression was verified by Western blot analysis (data not shown). When additional viral protein was absent, eGFP expression was not detected (Fig. 5). However, providing additional SeV N protein to SeV {Delta}N P{Delta}2-77 infections resulted in eGFP fluorescence of single cells without a spread to neighboring cells due to the replication deficiency of the mutant (Fig. 5). In contrast, when N and P proteins were expressed from transfected DNA, eGFP fluorescence developed concomitantly with the production and spread of virus progeny, thereby confirming the functionality of the expression plasmid-encoded viral proteins in trans (Fig. 5). These results demonstrate very clearly that the viral N protein is crucially involved in SeV gene expression and, thus, transcription events.


Figure 5
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  		FIG. 5. Viral eGFP expression in SeV {Delta}N P{Delta}2-77-infected non-HCs with and without cellular trans-complementation. BSR-T7 cells (0.25 x 106) were infected with SeV {Delta}N P{Delta}2-77 (MOI, 0.05) and, in parallel, either mock transfected or transfected with pRECISE-N (expression plasmid with the SeV N gene) or with pRECISE-N and pRECISE-P (0.5 µg DNA each), as indicated. The development of eGFP-expressing cells was monitored for 7 days. Here, representative sections from days 2, 4, and 7 are shown. Each section also is shown as a phase-contrast image.

We next wanted to identify and locate the region on the N protein that is responsible for this involvement in viral transcription. According to many functional studies on the N protein (5, 6, 13), the most likely part to be involved in this activity was the C-terminal domain from aa 400 to 524. Based on functional domains identified previously (9) (Fig. 1B), we constructed a series of expression plasmids harboring SeV N genes with C-terminal deletions of a variety of lengths. pRECISE-N471 (with a deletion of aa 472 to 524) encodes an N protein incapable of binding P proteins in the N0-P complex, which is necessary for genome replication. pRECISE-N461 (with a deletion of aa 462 to 524) encodes an N protein that additionally is unable to bind P proteins of the vRdRp when assembled into NCs (NNC-vRdRp binding). The third construct, pRECISE-N439 (with a deletion of aa 440 to 524), encodes an N protein that is unable to interact with other molecules of N when assembled into NCs (NNC-NNC binding) in addition to the two other defects. After demonstrating strong expression of all N protein variants in transfected cells through immunofluorescence, a possible influence on viral gene expression during SeV {Delta}N infections of non-HCs was tested. Again, under these conditions, genome replication was detectable only with the full-length N protein. After a period of 7 days, none of the partially deleted N proteins could substantially support viral gene expression. In the presence of full-length N protein, however, eGFP expression was clearly visible from 2 days p.i. (data not shown). We conclude that the activity of the N protein supporting viral transcription is located within the C-terminal 53 aa or that the deletions cause a loss of function of a nearby region.


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DISCUSSION
 
Infections with the replication-deficient mutants SeV {Delta}P and SeV {Delta}N did not result in detectable viral gene expression. Viral gene expression took place only when both SeV N and P proteins were present during infections. Initially, this observation seems to disagree with the current opinion that SeV transcription is an interplay of the viral RNA genome encapsidated with N proteins and the vRdRp that is composed of P and L proteins (14) and that additional N protein is not supposed to be needed.

However, this idea is based on experiments performed under in vitro conditions that, in some aspects, substantially deviate from the authentic infection situation. For in vitro studies, RNP and vRdRp complexes are isolated from cell extracts and are purified. Subsequently, large amounts of template (ca. 1011 RNP molecules) are incubated with the protein extracts, thereby amplifying the effect of a single NC (10, 20). The resulting mRNA products are analyzed. However, the detected transcription products could represent the transcriptional activity of vRdRp molecules that are already present at the very beginning of an infection, when no viral protein has been synthesized yet. The extent of initial transcription that is achieved under in vitro conditions with 1011 RNPs clearly differs from that at the beginning of a natural infection, when only one NC is present in the cell. This natural situation is the basis for the results presented here. From infections with SeV {Delta}P, as shown in Results, the minimum amount of genome template required for detection of viral gene expression was estimated to be 15 RNP molecules.

A possible involvement of additional N and P proteins in viral transcription was never evaluated separately from genome replication events in vitro (16). In vivo, this issue has never been addressed due to the lack of the helper systems needed for amplification of the appropriate viral mutants.

The course of transcription during viral infection begins with a single encapsidated genome (RNP) associated with 30 to 50 vRdRp molecules. In order to synthesize enough viral N proteins for encapsidation of only one single progeny genome or antigenome, it can be expected that these vRdRps have to transcribe the N gene very often. Thus, initial transcription alone, that is, without new synthesized viral proteins, most likely is not sufficient to replicate the genome with prior antigenome synthesis. Additionally, the activity of vRdRp complexes is assumed to be limited to ca. 6 h inside cells (25). Therefore, synthesis of new vRdRp molecules seems to be necessary; this could represent at least a part of the present observed need for additional P protein during viral gene expression.

On the other hand, support of viral transcription by additional P proteins during in vitro studies is independent of additional L proteins: a 5- to 10-times-elevated amount of P protein alone only slightly (by a factor of 2) enhanced transcription (12). Thus, the P protein seems to have another, so far undefined, activity during RNA synthesis, but, according to the data presented here, only when N protein also is synthesized de novo. The issue of an N protein function during viral transcription has never really been addressed before and is still unresolved.

With our system, we could monitor viral gene expression during SeV infections, including the phase during which new vRdRp molecules are produced but in the absence of template amplification. One reason why an involvement of N and P proteins in SeV transcription has never been documented previously could simply be due to the experimental conditions. When N protein was added to previously described in vitro or in vivo transcription assay systems for the reconstitution of the complex, genome replication took place, but not much attention was given to the transcription levels. On the other hand, in in vitro studies, the presence of P and L proteins has been shown to be sufficient for supporting viral transcription (20).

With our approach, the separation of transcription and replication events with or without de novo synthesis of N and/or P proteins was possible for the first time during infections. This permits the evaluation of the influence of N and P proteins, combined or separate, exclusively on viral transcription.

In this context, the introduction of a new, additional stage after the preliminary phase of viral transcription, during which transcription is much more pronounced, has to be considered. Along these lines, Plumet et al. (25) reported on a change from linear to exponential mRNA synthesis. This change was confirmed by our gene expression studies. Moreover, using a set of viral gene deletion mutants, viral de novo syntheses of both N and P proteins were identified as essential factors inducing that change. More recently, the strong increase during secondary transcription has been correlated with the synthesis of progeny templates (22). However, during primary transcription another important state appears, during which mRNA synthesis is clearly increased compared to that of initial transcription. This is most likely and at least partially the consequence of early protein synthesis leading to new vRdRp molecules (25). Until then, the N protein production can hardly suffice to package new viral genomes, since for each genome or antigenome replication event more than 2,500 N proteins are necessary. This means that, during this preliminary phase, new vRdRp molecules, together with the initially available ones, transcribe the parental template RNA. Therefore, unlike the vRdRps that already are attached to the RNP, the new polymerases have to find the template first, interact with it, and start transcription. How these steps are efficiently accomplished is still unknown, but the existence of a mechanism that coordinates this interaction would make sense for efficient virus replication. All in all, de novo synthesis of N and P proteins is required for efficient viral gene expression and, thus, viral transcription.

New model of N and P involvement in viral transcription and replication. Based on the results presented here, there must be a previously unknown aspect of transcription that involves both the N and P proteins. Not all questions are convincingly answered in the current proposals on the course of transcription. In the beginning of an infection, vRdRps already are associated with the template. However, how do newly synthesized vRdRp molecules quickly and unerringly find the 3' end of the genome to start transcription? Most important for genome/antigenome replication events (but also demonstrated for transcription) is the precise start of polymerization at nt 1 of the genome/antigenome to fulfill the rule of six (7). Another interesting matter is how multiple vRdRp molecules interact with and transcribe from a single template simultaneously without interfering with each other.

A scanning mechanism has been proposed, in which the viral polymerase traverses the template in both directions in search of the exact transcription start site. This mechanism was deduced from the manner in which the polymerase deals with gene junctions, through which three nucleotides (the intergenic region) are omitted before polymerization is resumed (21).

At least 30 to 50 vRdRp molecules are found on the NC inside a virion; potentially all could interact with the template at the same time. However, how the action of many vRdRp molecules on one template, some transcribing, some scanning in one or the other direction, can be coordinated is hard to imagine. Furthermore, the issue of how newly synthesized vRdRps get in contact with the template arises. Do they occasionally hit and interact with the 15,384-nt-long template at random and then scan the template until the beginning of the genome is found? Assuming that at least 30 to 50 vRdRps can operate on one template at the same time, this could mean much traffic, which might need regulation.

From our findings we conclude that, for the improvement of transcriptive processivity to normal levels, de novo-synthesized N and P proteins are essential. What could be the role of the proteins during the polymerization process, when no genome replication runs? For P protein, one could imagine some turnover within the polymerase L-P4 complex (24), but not to the extent of de novo synthesis of P that has been reported (23, 26). However, a role for N protein during viral transcription has not been described. One could imagine the existence of another complex consisting of N and P proteins. This complex, instead of a proposed circular arrangement of the RNPs resulting in leader-to-trailer proximity (25), could transport the viral polymerase, like a shuttle, from one end of the genome to the other (Fig. 6) and could position newly synthesized vRdRps on the template.


Figure 6
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  		FIG. 6. Model for supplemental function of de-novo-synthesized SeV N and P proteins during viral RNA synthesis. Proteins could function as a shuttle for the delivery and the uptake of vRdRp molecules at the beginning or end of the genome or the transcriptional termination site inside the genome. (A) vRdRp molecules that enter the cells as part of the NC initially transcribe the viral genome. Once a vRdRp reaches the end of the genome or terminates transcription at an internal gene junction, the polymerase detaches from the template. It can be expected that every vRdRp molecule works in an iterative fashion. (B) The vRdRp molecules interact with a loosely attached shuttle consisting of N and P proteins. (C) After clearing the template for following vRdRps, the shuttle navigates unerringly to the beginning of the template, where the polymerase can start transcription or replication again or for the first time.

Since N proteins cannot remain soluble in the cytoplasm without being bound to P proteins (20), it is likely that these shuttles consist of N and P proteins. The interaction domains between N and P proteins cannot be the same as those for the N0-P complex, since the SeV {Delta}N P{Delta}2-77 mutant defective for building the N0-P complexes still is able to transcribe efficiently in the presence of N protein (Fig. 5). P protein also is known to interact with NC-bound N proteins through a domain residing in its C-terminal end (9, 19).

How do newly synthesized vRdRp molecules interact with the template? Either they make contact with the template randomly, or they are first attached to the proposed shuttle that directs them to the beginning of the genome. In the latter case, vRdRps already could associate with N proteins during the assembly of the four copies of P and one copy of L. Interestingly, during coexpression of N, P, and L proteins of vesicular stomatitis virus, a tripartite complex is observed, the existence of which could not be clearly explained (17).

Even though the proposed functions of N and P proteins described here take place during initiation and termination of both transcription and replication, the frequency with which transcriptional events, especially, occur during infections could clarify the need for a coordinated procedure. As shown through virus release studies and steady-state analysis during infections (8, 18), at least 30,000 mRNA molecules are produced per hour. Therefore, it would not be astonishing if transcription and replication run faster and more efficiently when they are guided and directed.

The model we propose illustrates a possibility that is consistent with our findings that simultaneous de novo synthesis of N and P proteins is necessary for viral gene expression at normal levels. The different amplification conditions for SeV {Delta}N and SeV {Delta}P reflect very clearly the different tasks in which the N and P proteins are involved and the concomitant viral need for these proteins during infections. In N HCs, the amounts of N protein produced (5% of SeV wt infections) suffice only for viral transcription and not for simultaneous genome replication. In an analogous situation, the amount of P protein produced in P HCs (10% of SeV wt infections) effectively supports both SeV transcription and genome replication. During replication, the greater need for N proteins than P proteins, as illustrated by the amplification system of SeV {Delta}N, can be explained by the consumption of N proteins during encapsidation of the newly synthesized viral genome. P proteins, on the other hand, are required only for delivering N proteins to the vRdRp during genome replication and can most probably be reused and, thus, are not needed in the same amounts as N protein.

Even though the data presented here are based on experiments with SeV, a mechanism describing supplemental functions of N and P proteins during RNA synthesis most probably applies to all paramyxoviruses.


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ACKNOWLEDGMENTS
 
We thank Christine Baumann for excellent technical assistance. We also thank Sue Moyer for supplying pGEM expression plasmids with N gene mutants and K. Conzelmann for providing BSR-T7 cells.

This work was supported by grants from the Bundesministerium für Bildung und Forschung (AZ 0312193) and from the European Commission (QLK2-2002-01722).

This work does not necessarily reflect the views of the European Commission and in no way anticipates the Commission's future policy in this area.


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FOOTNOTES
 
* Corresponding author. Mailing address: Max-Planck-Institute of Biochemistry, Department of Molecular Virology, Am Klopferspitz 18, 82152 Martinsried, Germany. Phone: 49 89 8578 2268. Fax: 49 89 8578 2909. E-mail: neubert{at}biochem.mpg.de Back

{triangledown} Published ahead of print on 12 September 2007. Back


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Journal of Virology, December 2007, p. 13835-13844, Vol. 81, No. 24
0022-538X/07/$08.00+0     doi:10.1128/JVI.00914-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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