Department of Microbiology, Mie University School of Medicine, 2-174, Edobashi, Tsu-Shi, Mie-Ken, 514-8507, Japan1
Author for correspondence: Machiko Nishio.Fax +81 59 231 5008. e-mail nishio{at}doc.medic.mie-u.ac.jp
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Abstract |
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Introduction |
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The hPIV-2 P gene encodes the V protein, and the P protein mRNA is produced by the addition of two nontemplated G residues, which results in a frameshift and the expression of the P protein as a fusion protein with the N-terminal 164 aa of the V protein (Ohgimoto et al., 1990 ). We have reported previously that two noncontiguous regions of the hPIV-2 P protein are required for binding to the NP protein; one is located in the N-terminal region, aa 146, which is in the PV common domain, and the other is in the C-terminal part, aa 357395, which is required for granule formation of the NPP complex (Nishio et al., 1996
). We have also demonstrated that the trimerization site of the P protein is located between aa 211 and 248, which is predicted to form a coiled coil (Nishio et al., 1997
). We have shown that the N-terminal domain, aa 146, of the hPIV-2 V protein is involved in binding to the NP protein (Watanabe et al., 1996b
), and that two noncontiguous regions in the V protein, aa 146 and aa 175196, are required for nuclear localization and retention (Watanabe et al., 1996a
).
The NP proteins of paramyxoviruses play a central role in the replication of the viral genomic RNA (Horikami et al., 1992 ; Buchholz et al., 1993
, 1994
; Bankamp et al., 1996
; Myers et al., 1997
; Spehner et al., 1997
). Based on these known functions, the NP protein of paramyxovirus must have at least three domains: one or more binding site(s) for the P protein to form the NPP encapsidation complex, an NPNP interaction site(s) for the assembly of nucleocapsid, and an RNA-binding domain(s) for initiation and elongation during packaging. However, none of these functional sites has been mapped in hPIV-2. To begin the characterization of the functional domains of the hPIV-2 NP protein, we mapped the regions recognized by 41 monoclonal antibodies (MAbs) directed against the NP protein. By Western blot assay using these MAbs, the analysis of deletion mutants of the hPIV-2 NP protein was performed to identify the region essential for NPNP interaction and P-binding sites on NP protein in vitro.
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Methods |
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Constructions of subclones.
A cDNA fragment encoding the hPIV-2 NP, P or deleted P mutants was inserted into the plasmid expression vector pcDL-SR296 to obtain plasmids pDS-NP, pDS-P, pP
c118 and pP
n, respectively, as described previously (Takebe et al., 1988
; Nishio et al., 1996
, 1997
). Most of the NP deletion mutants detailed in Fig. 1
were generated by PCR amplification using the wild-type NP gene as template as described previously (Nishio et al., 1996
, 1997
). The mutant pNP
BglII was derived from pDS-NP by excision of a fragment between the two BglII restriction enzyme recognition sites.
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Transfection.
COS cells were grown in six-well plates at 37 °C in Dulbecco's modification of Eagle's tissue culture medium (DMEM) supplemented with 10% foetal calf serum (FCS). The cells were washed twice with warm DMEM, and then transfected with various plasmids (3 µg each) in lipofectin (Gibco). After incubation for 7 h at 37 °C, DMEM containing 10% FCS was added.
Immunoprecipitation and Western blot assay.
After 2 days of transfection, cell extracts were prepared with lysis buffer (150 mM NaCl, 50 mM TrisHCl, pH 7·4, 0·6% NP40) containing 1 mM DTT. The extracts were incubated overnight with 150 µl MAbs at 22 °C; 50 µl of 50% protein ASepharose in PBS was added and incubation was continued for 4 h at 22 °C. Immune complexes were washed three times with lysis buffer containing 1 mM DTT and once with PBS, then analysed by LDS (lithium dodecyl sulfate)PAGE. Electrophoretic transfer of virus polypeptides from gels onto nitrocellulose membranes was carried out as described previously (Nishio et al., 1996 ).
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Results |
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Discussion |
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In the present study, we mapped the epitopes recognized by 41 MAbs directed against the hPIV-2 NP protein by testing their reactivity with lysates of COS cells expressing various deleted NP proteins. Using these MAbs, we have determined the sites on the hPIV-2 NP protein responsible for the NPNP and NPP interactions by Western blotting.
Many studies on nonsegmented negative-stranded viruses have indicated that the NP protein folds into a globular body with an exposed C terminus and that this exposed region interacts with the P protein (Homann et al., 1991 ; Curran et al., 1993
; Ryan et al., 1993
; Bankamp et al., 1996
). In the two-hybrid system in yeast, the NPNP interaction was directly demonstrated in Sendai virus (SeV) (Horikami et al., 1996
) and in bovine respiratory syncytial virus (Krishnamurthy et al., 1998
). More recently both the central conserved region (CCR, aa 258357) and the N-terminal 255 aa have been found to contain sequences important for NPNP self-assembly by using the fusion proteins between the maltose-binding protein and different regions of the SeV NP protein. In addition, the specific residues in the CCR were proven to be important for binding to RNA (Myers et al., 1997
). We identified the domain on the hPIV-2 NP protein responsible for multimerization to the whole N-terminal region aa 1294. It is conceivable that almost the whole globular domain of the NP protein is required for the NPNP interaction, suggesting that the deletions within the globular body compromise the overall structure of the polypeptide and consequently abolish the NPNP interaction. The sequences of the NP protein required for interaction with the P protein are ill defined in most nonsegmented negative-stranded RNA viruses. Large regions in the NP proteins of vesicular stomatitis virus (Takacs et al., 1993
), human respiratory syncytial virus (Garcia-Barreno et al., 1996
) and SeV (Homann et al., 1991
) have been shown to be essential for interacting with the P protein.
In this report, we have also demonstrated the existence of two independent NPP-binding sites which can interact with different parts of the P protein. Region aa 403494 on the NP protein is essential for the interaction with the N-terminal binding site of P protein, and the C-terminal binding site of P protein interacts with region aa 295402 on the NP protein, which includes part of a domain that is highly conserved among the parainfluenza NP proteins (Miyahara et al., 1992 ). These results suggest that both P-binding sites are located in the C-terminal part of the hPIV-2 NP protein.
The domains on the hPIV-2 NP, P and V proteins identified in this work and our previous works are summarized in Fig. 5. In SeV and measles viruses, the N-terminal two-thirds are important in NPNP interactions and RNA encapsidation, and the C-terminal one-third is important in binding to polymerase complex (PL) (Curran et al., 1993
; Buchholz et al., 1993
, 1994
; Bankamp et al., 1996
). A similar situation exists in the hPIV-2 NP protein. In a previous study, we demonstrated by using immunofluorescence staining that the interaction of the hPIV-2 NP protein with the N-terminal region of the P protein was different from that with the C-terminal region of the P protein. We have also shown that the C-terminal region of the P protein was required for the NPP complex to form granules, which were detected in hPIV-2-infected cells. The C-terminal region of the P protein binds to the NP protein near the NPNP interaction site and part of the CCR which might be a domain important for a common function of the paramyxovirus NP proteins. Thus it might be that the binding between the NP and C terminus of P proteins affects the structure of the NP protein, resulting in granule formation. When NP
n200 and P proteins of hPIV-2 were coexpressed in COS cells, granule formation was not found, although the mutant NP protein did bind to the P protein (data not shown), suggesting that NP protein self-assembly is required for the NPP complex to form granules.
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Acknowledgments |
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References |
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Received 12 February 1999;
accepted .