Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan1
Author for correspondence: Akihiko Kawai. Fax +81 75 761 2698. e-mail akawai{at}pharm.kyoto-u.ac.jp
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Abstract |
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Introduction |
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The P protein is thought to bind to the nucleoprotein (N) to play its roles in viral RNA synthesis; however, structural requirements seem to be different for its appropriate interactions with the viral N protein, probably depending on the different situations of the N protein (i.e. before, during and after the encapsidation of viral genomic RNA). For instance, Chenik et al. (1994) suggested the existence of at least two independent N protein-binding sites on the P protein, one located in the C-terminal part and the other located between aa 69 and 177. Fu et al. (1994)
reported that the NP interaction of the N-terminal domain of the P protein occurs when the N protein is newly synthesized or during the period of N and P protein synthesis, while the C-terminal-binding site is capable of interacting with the N protein even when both proteins are synthesized separately. Very recently, Jacob et al. (2001)
reported that binding through the C-terminal half of the P protein was much stronger than the interaction through its N-terminal half. In addition to N protein binding, the rabies virus P protein was shown recently to associate with the light chain (LC8) of cellular dynein, which was suggested to be involved in transportation of the viral nucleocapsid (NC) through the neuronal axons (Jacob et al., 2000
; Raux et al., 2000
).
Detailed studies for understanding the function-related conformations and conformational changes of the P protein and its precise roles in the virus replication cycle at the molecular level are, however, lacking. For such kinds of studies to be performed, it would be helpful to use monoclonal antibodies (mAbs) that recognize specific structures or conformations of the protein.
Concerning this problem, we found in our stocks of mAbs, which were selected for their ability to recognize rabies virus-induced cytoplasmic inclusion bodies (Kawai et al., 1999 ), a mAb (#402-13) that recognized the P protein. In this study, this mAb was shown to recognize a linear epitope of the protein that was exposed only when the protein took a specific conformation. Using this mAb, we could further differentiate in the cell at least two forms of the P protein, the 402-13 epitope-positive and -negative forms or conformations. We will discuss a possible role of the 402-13 epitope-positive P protein.
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Methods |
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BHK-21 cells were propagated in Eagle's minimal essential medium (MEM) supplemented with 5% calf serum and 10% tryptose phosphate broth (Difco). Virus-infected cultures were maintained in MEM containing 3% foetal bovine serum.
Expression of P cDNA in Escherichia coli.
For production of the P protein analogues in E. coli, full-length P cDNA was inserted into the NdeI site of an inducible expression vector, pET3a (Novagen), as described previously (Takamatsu et al., 1998 ). Expression of cDNA was induced in E. coli BL21 (DE3) pLysS cells (Novagen) by adding IPTG at a final concentration of 1 mM, according to Studier et al. (1990)
. P gene products were lysed in SDSPAGE sample buffer.
Expression of P and N cDNAs in animal cells.
Reconstructed expression vectors for P and N proteins (pCDM8-P and pCDM8-N) were described in our previous report (Takamatsu et al., 1998 ; Kawai et al., 1997
). Transfections of P and N cDNAs to BHK-21 cells were done using the calcium phosphate method, as described previously, for which RVV-T7 was used to provide the T7 RNA polymerase (Kawai et al., 1999
).
Preparation of C-terminally deleted mutants of the P protein.
The P cDNA of rabies virus (HEP-Flury strain) was cut with one of the following restriction enzymes: HincII, HindIII, BamHI or MunI. It was then ligated with an universal NheI linker [d(CTAGCTAGCTAG)] to introduce stop codons in all translational frames and subsequently transferred into an expression vector, pET3a. By this process, the P cDNA was made to produce C-terminally truncated P protein analogues, termed PC166, P
C89, P
C42 and P
C22, lacking 166, 89, 42 and 22 aa from the C terminus, respectively. In this study, the prokaryotic expression vector pET3a was used for P gene expression in BHK-21 cells with the help of T7 phage RNA polymerase encoded by recombinant vaccinia virus (RVV-T7).
Antibodies.
Hybridoma cells were the same as those described previously (Kawai et al., 1997 , 1999
). mAbs were screened originally according to their recognition of the viral antigens located in the virus-induced cytoplasmic inclusion bodies by fluorescent antibody (FA) staining (Kawai et al., 1997
). Among those, a mAb (#402-13, isotype IgM) was found to recognize the SDS-denatured P protein from rabies virus-infected cells (Fig. 1A
). We also used two anti-N mAbs, #5-2-26 and #1-7-11, which recognize a phosphorylation-dependent linear epitope and a conformational epitope of rabies virus N protein, respectively (Kawai et al., 1997
, 1999
; Anzal et al., 1997
).
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SDSPAGE.
Assay samples were dissolved in SDSPAGE sample lysis buffer (125 mM TrisHCl, 4·6% SDS, 10% 2-mercaptoethanol, 0·005% bromophenol blue and 20% glycerol; pH 6·8) and applied to 10 or 12% polyacrylamide gels prepared using Laemmlis discontinuous buffer system (Laemmli, 1970 ). After electrophoresis, protein bands were subjected to immunoblotting (see below) or simply stained with Coomassie brilliant blue R-250 (CBB) (Merck).
Immunoblotting.
Cell lysates were prepared by lysing mock-infected and virus-infected cells with SDSPAGE sample buffer and applied to SDSPAGE on a 10% polyacrylamide gel. After separation, the proteins were electrically blotted onto a nitrocellulose membrane using a semi-dry-type blotting apparatus. After blocking, the membrane was stained with murine or rabbit anti-P antibody and then incubated with peroxidase-conjugated second antibody. Colour was developed by adding H2O2 and chloronaphtol. To estimate the apparent molecular mass of the proteins, a set of molecular markers (Dr Western, Oriental Yeast) was co-electrophoresed.
Radioactive labelling.
After preincubation of the cultures with methionine-free medium for 30 min, the cultures were metabolically labelled with L-[35S]methionine (final concentration of 10 µCi/ml; 0·37 MBq/ml) for various periods, as described in the text. Then, the radiolabelled cells were washed three times with PBS- and lysed in 100 µl RIPA (I) buffer (140 mM NaCl, 50 mM TrisHCl, 1% Nonidet P-40, 20 nM okadaic acid, 1 mM pefabloc, 25 µg/ml leupeptin; pH 7·4) for immunoprecipitation studies.
Fractionation of rabies virus-infected BHK-21 cells.
Rabies virus-infected BHK-21 cells were lysed in deoxycholate (DOC)-free RIPA (I) buffer. After centrifuging briefly (10 min at 12000 r.p.m.) in a refrigerated microfuge, the lysates were applied to a 1045% sucrose density gradient and centrifuged at 25000 r.p.m for 2 h at 4 °C. Then, the gradient was fractionated into 1012 fractions, each of which was checked for its polypeptide constituents simply by SDSPAGE and staining with CBB before being used for immunoblotting or immunoprecipitation studies. As we described previously (Kawai et al., 1999 ), the top fraction contained free P proteins and free NP complexes as well as other soluble viral proteins and the NC fraction contained the NC and NC-associated proteins (i.e. P and L proteins).
Dissociation and recovery of the P protein from the NC.
The NC fraction was exposed to 1% DOC in the presence of 0·5 M NaCl in a buffer composed of 5 mM TrisHCl (pH 7·4), 1% Nonidet P-40, 1 mM pefabloc and 25 µg/ml leupeptin for 10 min at 4 °C and then placed onto a 20% sucrose cushion, followed by ultracentrifugation for 120 min at 200000 g. The dissociated P proteins were recovered from the top of the tube and subjected to immunoprecipitation with anti-P mAb and pAb and then to SDSPAGE and autoradiography.
Immunoprecipitation and autoradiography.
Radiolabelled cell lysates were subjected to immunoprecipitation with mAbs or rabbit antibodies against rabies virus N or P protein. In brief, 12 µl of antibody solution was added to 1020 µl of the lysates and placed on ice for 12 h, followed by precipitation with Pansorbin cells (commercial product of insoluble protein A-containing formalin-fixed Staphylococcus aureus cells) (Calbiochem) in PBS for 2 h at 4 °C. When precipitated with murine mAbs, the precipitates were recovered using the second antibody (rabbit) against the murine immunoglobulin for efficient recovery with Pansorbin cells. The immune complexes recovered with Pansorbin cells were then dissolved in 50 µl of SDSPAGE sample buffer and applied to 810% polyacrylamide gels for SDSPAGE. Broad range molecular mass standards (Bio-Rad) were co-electrophoresed in SDSPAGE. After being stained with CBB, the gels were dried onto 3 mm filter paper (Whatman) and exposed to an imaging plate for autoradiography in a Bio-Imaging Analyser BAS2000 (Fuji Photo Film).
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Results |
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The epitope region was mapped roughly as described. Some C-terminal deletion mutants of the P protein were prepared by cutting the P cDNA at one of the unique restriction sites and ligating it to an universal NheI linker, as described in Methods. Fig. 2 shows that mAb #402-13 did not recognize the mutants that lacked the C-terminal 42 aa (from positions 256 to 297) or more, and recognized very weakly the mutant that lacked 22 aa (from positions 276 to 297), implying that the presumed epitope region is located at around Gln275.
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Identification of the epitope-positive P protein in the cell
(1) Cell fractionation and detection of the NC-associated P protein.
Radiolabelled, infected cell lysates were fractionated into the top soluble and the middle NC-containing fractions by ultracentrifugation in a sucrose density gradient, as noted in Methods. As described in our previous study (Kawai et al., 1999 ), the top fraction contained free P proteins and free NP complexes as well as other soluble viral proteins and the NC fraction contained the NC and NC-associated proteins, including the P and L proteins, which could be co-precipitated by either anti-N or anti-P pAbs.
As shown in Fig. 4 (lanes 2), the top and NC fractions contained the N and P proteins, which were co-precipitated with anti-P pAb. When these fractions were subjected to immunoprecipitation with mAb #402-13, the P protein was recovered mostly from the NC fraction with co-precipitated N and L proteins (Fig. 4B
, lane 3), while little P or N protein was recovered from the top fraction by the mAb (Fig. 4A
, lane 3; about 6% of the total P protein was detected in the band, as estimated by comparing the radioactivity of the bands). From these results, we assume that mAb #402-13 recognized the epitope that is mostly exposed on the P proteins that are associated with NC.
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As noted in our previous report (Kawai et al., 1999 ), P proteins comprising the free NP complex in the top fraction could be dissociated by treatment with sodium DOC. Accordingly, we examined the dissociated P proteins for their reactivity with mAb #402-13. As shown in Fig. 5(A)
, no DOC-dissociated P proteins were precipitated by the mAb, implying that the 402-13 epitope region was not masked by binding to the newly synthesized free N protein.
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As to the third possibility mentioned above, we think that, from the immunoblotting studies shown in Fig. 1 as well as from the results shown in Fig. 5(B)
(if the DOC-mediated dissociation did not cause any change in the P protein modifications), additional modifications (e.g. proteolytic cleavage, phosphorylation, etc.) are not necessary for the P protein to be recognized by mAb #402-13, even if protein modification occurs during or after the encapsidation process.
Conditions required for exposing the 402-13 epitope
We next investigated the conditions required for the P protein to expose the 402-13 epitope region using the P cDNA transfection system in animal cell cultures. The P and N gene products produced in the N and P protein cDNAs co-transfected cells were metabolically radiolabelled with [35S]methionine and lysed in RIPA (I) buffer. Then, the lysates were subjected to immunoprecipitation with anti-P pAb and mAb #402-13 as well as anti-N pAb and mAbs. As shown in Fig. 6 (lane 5), mAb #402-13 precipitated, although not so much, P proteins from the co-transfected cell lysates with the N proteins that compose the NC-like structures. In accordance with this, anti-N mAbs co-precipitated only small amounts of P proteins (data not shown). On the other hand, similar or less amounts of P proteins were precipitated by anti-P mAbs when the P gene alone was expressed in the cells, although large amounts of P proteins were produced in the cells as detected by anti-P pAb (Fig. 6
, compare lanes 3 and 7).
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Interaction of the C-terminally deleted P protein mutants with the NC
Finally, we checked the structural relationship between the NC-binding ability of the P protein and exposure of its 402-13 epitope. The wild-type (wt) P protein and four C-terminally deleted mutants (PC22, P
C42, P
C89 and P
C166) were co-expressed with the N protein in BHK-21 cells and radiolabelled for immunoprecipitation studies with anti-N and anti-P pAbs as well as the anti-N mAb #1-7-11 (this recognizes a conformational epitope that is exposed on the NC-comprising mature form of the N protein but not on the NP complex) (Kawai et al., 1999
). As shown in Fig. 7
, all mutants were detected by anti-P pAbs and were co-precipitated with the anti-N pAb but not with anti-N mAb #1-7-11, except for one mutant P
C22, whose very faint band was detected when co-precipitated with mAb #1-7-11.
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Discussion |
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We demonstrated that mAb #402-13 recognizes a linear epitope that is located at the C-terminal region of the rabies virus P protein, while the epitope region is exposed only when the P protein is associated with the viral NC. The epitope-containing region of newly synthesized P protein would mostly be hidden immediately after synthesis, so, the epitope was undetectable on the free NP complex and on the free P protein; the former case was not due to masking of the epitope region by the N protein binding. We conclude that mAb #402-13 recognizes a linear epitope exposed on the P protein that is intimately associated with the viral NC and that the epitope is concealed again when released from the NC.
Free NP complexes are involved in viral RNA synthesis to encapsidate newly synthesized viral RNA to form the NC. If the P proteins still remain associated with the NC after RNA encapsidation, the amount of NC-associated P proteins would theoretically correspond to that of the P proteins that are co-precipitated by our conformational epitope-specific anti-N mAbs (e.g. #1-7-11) (Kawai et al., 1999 ). In virus-infected cells as well as in N+P cDNA-transfected cells, however, such NC-associated (402-13 epitope positive) P proteins were detected in very small amounts in comparison with those precipitated by anti-P pAbs. These results suggest strongly that, after RNA encapsidation, NP binding was disrupted and most P proteins were released from the newly formed NC. Dissociated P proteins may undergo structural changes to restore the original form and conceal the epitope region and might be pooled again in the cell for recycled uses through the free NP complex formation with newly synthesized N proteins.
Exposure of the epitope region seems to be dependent on structural or conformational change(s) of the P protein, which would occur mostly during its association with the NC under certain conditions, but not due to the post-translational modifications. Concerning this, we think as follows: during or after the encapsidation process, the N proteins undergo conformational changes that could be recognized by anti-N mAbs (e.g. #1-7-11). And, the conformational change(s) of the N protein might also affect the NP association on the newly formed NC; most of the P proteins would be dissociated from the NC. Some such P proteins, however, might undergo (by chance?) certain structural changes to keep their NC-associated state and expose concomitantly the 402-13 epitope.
The P protein may display various types of association with the N protein in relation to its multiple functions, which might be reflected in its different structures or conformations. The P protein may be present in either of three different forms, which are as follows: (i) free P protein; (ii) free NP complexes used for encapsidation of viral RNA; and (iii) the NC-associated P protein thought to be involved in initiation of viral RNA synthesis or elongation. Concerning the latter two situations, two research groups reported previously that both the N- and C-termini of the P protein are involved differently in the formation of free NP complexes and NCP binding, respectively (Chenik et al., 1994 ; Fu et al., 1994
).
Chenik et al. (1994) suggested that there may be at least two independent N-binding sites on the P protein, one located in the C-terminal part, the other in a region spanning from aa 69 to 177. Fu et al. (1994)
reported that the N-terminal domain of the P protein interacts with the N protein only when the N and P proteins were synthesized simultaneously, while the C-terminal site of the P protein is capable of interacting with the N protein even though both proteins are synthesized separately. From these results, they suggested that the N-terminal-binding site is involved in free NP complex binding and that the C-terminal domain is required for NCP binding. Our present observations provide novel evidence that would support the proposal by Fu et al. (1994)
. At present, we can say that the 402-13 epitope site is not the NC-binding site itself because this site is recognized by the mAb. Very recently, Jacob et al. (2001)
described that one of two N-binding domains (probably the NC-binding domain) is mapped to a small region ranging from aa 209 to 215 (Fig. 8
).
When the N and P proteins are co-expressed in animal cells, N and P proteins assemble to form free NP complexes, as seen in infected cells. The NP complexes are used for constructing the NC-like structures that could be recognized by anti-N mAbs (e.g. #5-2-26 and #1-7-11) (Kawai et al., 1999 ). All of the C-terminally deleted mutants showed similar ability of NP complex formation, which is consistent with a suggestion that the N-terminal half of the P protein contains a region required for the formation of free NP complexes with newly synthesized N protein.
On the other hand, since the C-terminally deleted mutant PC22 showed very weak antigenicity to the mAb and weak NC-binding ability, it was suggested that the epitope-containing the C-terminal region is required for the P protein to display NC-binding activity. Structural changes to expose the epitope region would also be important for the P protein to strongly associate with the NC, whereby the P protein would become competent for working in viral RNA synthesis.
In other negative-stranded RNA viruses, similar studies have been done to define two different N protein-binding sites on the N- and C-terminal domains of the P/NS protein (Takacs et al., 1993 ; Harty & Palese, 1995
): the C-terminal of VSV P protein is involved in NCP binding (Gill et al., 1986
). Ryan & Kingsbury (1988)
reported that the C-terminal region of the Sendai virus P protein is required for NC binding, while Curran et al. (1995)
described that both the N- and C-termini of the P protein are required for PNPo complex formation (which may correspond to rabies virus NP complex formation). It has also been reported that the C-terminal region of the respiratory syncytial virus P protein is involved in the NP interaction (Garcia-Barreno et al., 1996
). All of these descriptions concerning the C-terminal domain implicate its possible involvement in a certain important function common to P proteins, although one which has not yet being defined precisely.
As to the possible role(s) of the 402-13 epitope-positive P protein, we have to remember the fact that the C-terminal epitope-containing region is one of the highly conserved regions of the P protein when compared with some rabies virus strains (Tordo et al., 1986 ; Larson & Wunner, 1990
; Conzelmann et al., 1990
; Takamatsu et al., 1998
). A similar situation has also been described for the VSV P protein, which has a highly conserved region composed of 21 aa at the C terminus (Gill & Banerjee, 1985
). Consistent with these considerations, we observed that the C-terminal sequence mimicked in synthetic oligopeptides specifically inhibited VSV transcriptase activity in vitro by reducing the frequency of RNA synthesis initiation (Yamashita & Kawai, 1990
). Based on these considerations, we think that exposing the C-terminal epitope-containing region on the NC-associated P protein allows the P protein to be ready for accepting the L protein, as implicated from the results shown in Fig. 4(B)
. Possible roles of the C-terminal region of the P protein are now under investigation from the viewpoint of NC binding of the P and L proteins as well as the possible involvement of other viral and cellular components.
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Acknowledgments |
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Footnotes |
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c Present address: Department of Virology I, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo, 1628460, Japan.
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References |
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Received 29 April 2002;
accepted 22 July 2002.
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