1 Institute of Biological Sciences (Genetics), University of Malaya, 50603 Kuala Lumpur, Malaysia
2 Department of Medical Microbiology, University of Malaya, 50603 Kuala Lumpur, Malaysia
3 CSIRO Livestock Industries, Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia
Correspondence
L.-F. Wang
Linfa.Wang{at}csiro.au
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ABSTRACT |
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INTRODUCTION |
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Paramyxoviruses contain a non-segmented negative-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form a helical nucleocapsid (NC) which functions as template for transcription and replication (for review, see Lamb & Kolakofsky, 2001). The large protein (L) interacts with the phosphoprotein (P) to form the polymerase complex (Horikami et al., 1992
; Holmes & Moyer, 2002
), which associates with the nucleocapsid via interaction between P and the assembled N in the NC (Curran et al., 1993
; Buchholz et al., 1994
).
The NP interactions for members of the Paramyxoviridae have been demonstrated both in vivo and in vitro. In Sendai virus (SeV), complexes of NP and PL formed separately and subsequently mixed together are able to support the replication of the genome of defective interfering virus in vitro (Horikami et al., 1992). The SeV P has been found to complex with newly synthesized, unassembled N (N°) during the nascent chain assembly step of genome replication. The PN° complex prevents non-specific aggregation of N and P is consequently being viewed as a chaperone for N° (Curran et al., 1995
; Errington & Emmerson, 1997
; De et al., 2000
). Domains involved in the NP interaction of several paramyxoviruses have been determined. These viruses include SeV (Ryan & Kingsbury, 1988
; Ryan & Portner, 1990
; Homann et al., 1991
; Ryan et al., 1991
; Buchholz et al., 1994
; Tuckis et al., 2002
), Simian virus 5 (SV5) (Randall & Bermingham, 1996
), Human parainfluenza virus-1 (HPIV1) and -3 (HPIV3) (Ryan et al., 1993
; Zhao & Banerjee, 1995
; De et al., 2000
), Porcine rubulavirus (PoRV) (Svenda et al., 2002
), Human parainfluenza virus-2 (HPIV2) (Nishio et al., 1996
, 1999
), Human respiratory syncytial virus (HRSV) (Garcia-Barreno et al., 1996
; Slack & Easton, 1998
; Lu et al., 2002
), Bovine respiratory syncytial virus (BRSV) (Mallipeddi et al., 1996
; Khattar et al., 2000
, 2001a
, b
), Rinderpest virus (RPV) (Shaji & Shaila, 1999
) and Measles virus (MeV) (Huber et al., 1991
; Harty & Palese, 1995
; Bankamp et al., 1996
; Liston et al., 1997
).
Among the many molecular features unique to henipaviruses is the exceptionally large P of both HeV and NiV. With 707 amino acids (aa) for the P of HeV and 709 aa for NiV, the henipavirus Ps are approximately 100 to 400 aa larger than Ps of other known paramyxoviruses (Wang et al., 1998; Harcourt et al., 2000
). Owing to this P size increase and the lack of significant sequence homology between henipavirus P and those of other paramyxoviruses, it was impossible to predict whether the N and P of henipaviruses would interact in a similar fashion as those reported previously. Hence, we conducted this study to explore the function of the henipavirus P in terms of forming the NP interaction and to map the domains required for such interaction.
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METHODS |
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Plasmid construction.
Three bacterial expression systems were used in this study. For expression of His6-tagged proteins, the pRSET vector (Invitrogen) was used, whereas for expression of biotinylated proteins, we used the pDW363 vector (Tsao et al., 1996), kindly provided by Dr David S. Waugh (National Cancer Institute, Frederick, USA) or the PinPoint Xa vector (Promega). A general approach was used in plasmid construction: PCR primers (forward and reverse) with unique restriction sites incorporated in each primer were designed for each gene fragment to be expressed. After amplification using a high-fidelity DNA polymerase, the PCR fragments were gel-purified and cloned in-frame into appropriate expression vectors. The insert sequences were then confirmed by directly sequencing the recombinant plasmids. Owing to the large number of recombinant plasmids constructed in this study, details of the construction of each plasmid are not presented here, but will be provided on request. Instead, we have listed all the plasmids used in this study in Fig. 1
, which summarizes the aa-residue number in each insert.
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Protein-blotting protein-overlay assay (PBPOA).
The method described by Homann et al. (1991) was followed with minor modifications. Briefly, cells lysed in SDS-PAGE sample buffer were separated on 1013 % polyacrylamide gels and the separated proteins were transferred onto PVDF membranes. The membrane was then blocked by incubating overnight at 4 °C in blotto containing 5 % (w/v) skimmed milk in TBST [10 mM Tris/HCl pH 8·0, 150 mM NaCl and 0·1 % (v/v) Tween 20]. All subsequent incubations were carried out in blotto containing antibody or conjugate solution at pre-determined dilutions, and washings were carried out with TBST. After blocking, the intact membrane or 45 mm membrane strips were incubated for 23 h at room temperature with a chosen cell lysate. After three washings, the membrane was incubated for 1 h at room temperature either with a mAb or with the APstreptavidin conjugate. After washing three times, the membrane incubated with mAb was further processed by 1 h incubation with AP-conjugated anti-mouse antibody, followed by three washings. For colour development, both types of membranes (probed either with APstreptavidin alone or with mAb+AP-conjugated anti-mouse antibody) were incubated with BCIP/NBT substrate mixture (Promega).
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RESULTS |
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Mapping of P domains involved in NP interaction
While the NiV N and HeV N share an overall 92 % sequence identity, the NiV P and HeV P are less conserved, sharing only 68 % sequence identity. However, the two terminal regions of P are more conserved than the middle region (Wang et al., 1998; Harcourt et al., 2000
). The NP interaction studies above indicated that the NiV P and HeV P contained one or more conserved domains, which are involved in binding the Ns of both viruses. To map these putative N-binding domains, various constructs were made to express N-terminal (P4550, P1251, P3220, P1107 and P62251), C-terminal (P253709, P549709, P549637 and P636709) and the less conserved middle region (P200372) of NiV P (see Fig. 1A
for the location of insert in each construct). Various protein fragments were immobilized to a single membrane, and probed with full-length NiV N and HeV N, respectively. The results presented in Fig. 3
showed that both the N- and C-terminal fragments, but not the middle fragment, of the NiV P were able to interact with the Ns of both viruses. There were no significant differences in binding pattern between NiV N and HeV N with the exception of clone P62251, which interacted weakly with NiV N but failed to interact with HeV N (Fig. 3B, C
). P3220 represents the smallest N-terminal fragment which was able to bind N. Neither the 107 aa N-terminal fragment (P1107) nor the 173 aa middle fragment (P200372) was able to bind NiV N or HeV N (Fig. 3B, C
, lanes 5 and 6). This suggests that residues between 107 and 220 are important for binding to P. However, these residues alone are not sufficient to support the binding as demonstrated by clone P62251. On the other hand, further deletion analysis of the C-terminal constructs revealed that an extreme C-terminal 74 aa region of NiV P, in clone P636709, was able to bind both NiV N and HeV N (Fig. 4B
, lane 4). Taking all the results together as summarized in Fig. 1
, it was clear that there were at least two independent N-binding sites present in NiV P, one located at the N terminus (aa 3220) and the other in the C-terminal region (aa 636709).
Mapping of N domains involved in NP interaction
To map the P-binding domains of N, we first divided the NiV N into two overlapping halves and demonstrated that only the C-terminal half was involved in interaction with P (Fig. 5B). Various C-terminal fragments of NiV N were then produced as shown in Fig. 1(B)
. Using the same strategy as that for the mapping of the N-binding domains of P, we immobilized different pRSET-derived NiV N fragments onto a single PVDF membrane, which was subsequently incubated with full-length NiV P or HeV P. Protein binding assay of these truncated N fragments showed that all but the NN1135 and NN378469 fragments were able to bind both NiV P and HeV P (Fig. 5
). The smallest clone still capable of binding to P is NN432532, which suggests that the region between aa 470532 is important for P-binding (Fig. 5
). Amino acid sequence alignment of NiV N and HeV N covering this region showed very high sequence identity (Fig. 7
C). We then produced smaller biotinylated N fragments to further narrow down the P-binding site (Fig. 1B
). Owing to the high sequence identity in this region between the two Ns and the existence of constructs already made for other studies, the HeV N gene was used instead of the NiV N gene. Using the same strategy as above, we immobilized different N fragments onto the same membrane, followed by incubation with NiV P or HeV P. The bound P was detected using the Anti-Xpress mAb. Fig. 6(B)
shows that the 29 aa fragment HN468496 was the smallest fragment that maintained the binding activity to both NiV P and HeV P (Fig. 6B
, lane 9).
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DISCUSSION |
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Our data did not differentiate between the binding of P to N° or NC but provided direct in vitro evidence for NP interaction. The formation of the NP complex by using recombinant N and P produced in bacteria would suggest that the NP interaction could occur in the absence of post-translational modification of either protein and independent of host cellular proteins. We also demonstrated that heterologous N and P proteins of NiV and HeV could form an NP complex, but such interaction was not detected when N, P or V proteins of other paramyxoviruses were used. To the best of our knowledge, this was the first demonstration of heterologous NP complex formation for any two paramyxoviruses by using bacterial recombinant proteins. This observation further supported the close phylogenetic relationship between these two viruses revealed by sequence analysis (Wang et al., 1998; Yu et al., 1998b
; Harcourt et al., 2000
) and is also consistent with our recent findings on the functional heterologous interaction between the fusion and attachment proteins of HeV and NiV (Bossart et al., 2001
, 2002
). It has been shown for several paramyxoviruses that the co-expression of N, P and L proteins was sufficient to support replication of naked virus genome RNA (Horikami et al., 1992
; Grosfeld et al., 1995
; Yu et al., 1995
). Halpin et al. (2004)
have recently demonstrated that the same was true for NiV. Moreover, it was shown that the NiV N, P and L proteins were also able support the replication and transcription of HeV minigenome, but not MeV. We are currently investigating the possibility of functionally mixing the N, P or L proteins in such studies for HeV and NiV.
Our results showed that there are at least two independent N-binding regions in NiV P, located at the N-terminal (aa 3220) and the C-terminal (aa 636709) regions. The protein fragment containing aa 1107 failed to bind N and the truncated fragment containing aa 62251 had weak binding to NiV N, but no binding to HeV N. Together, these mapping data suggested that the first 220 aa and the last 74 aa of P represent two essential and independent binding regions for interaction with N. Although no direct analysis was done to map the N-binding domain for HeV P, it is tempting to say that the homologous regions of the two N-binding points of NiV P in HeV P are the contact points with HeV N because the two N-binding regions of NiV P were able to interact with HeV N as well. In addition, aa sequence alignment of NiV P and HeV P within the two N- and C-terminal N-binding regions showed high similarity between the two viruses (Fig. 7A and B). This is largely consistent with results obtained so far for other paramyxoviruses. There are many reports on the mapping of NP-interacting domains for Ps in the family Paramyxoviridae. One of the best studied viruses is SeV, the type species of the genus Respirovirus. Two non-continuous regions at the SeV P C terminus are required to form the complex with N (Ryan & Kingsbury, 1988
; Ryan & Portner, 1990
; Ryan et al., 1991
). In addition, the N-terminal region of P is able to form a stable complex with N° (Curran et al., 1995
). In the genus Morbillivirus, the MeV P (Harty & Palese, 1995
; Liston et al., 1997
) has also been demonstrated to contain two separate N-binding domains located at N and C termini. For viruses in the genus Rubulavirus, the N-binding domains of HPIV2 P (Nishio et al., 1996
) and SV5 P (Randall & Bermingham, 1996
) have also been mapped to contain both the N- and C-terminal regions. As for pneumoviruses, which are more distantly related to members of the subfamily Paramyxovirinae, two separate N-binding domains also exist in HRSV P (Hengst & Kiefer, 2000
). It can therefore be concluded that the two independent and non-continuous N-binding domain structures are well conserved in all paramyxovirus Ps, including those of henipaviruses. Such conservations have been extended to other negative-stranded RNA viruses including Rabies virus (RABV) and Vesicular stomatitis virus in the family Rhabdoviridae. In both viruses, the P N- and C-terminal regions are involved in binding with N (Takacs et al., 1993
; Chenik et al., 1994
). Like SeV, the N terminus of RABV P is involved in N°P complex binding, whereas the C-terminal domain is required for NCP binding (Fu et al., 1994
). This is consistent with the observation that among different paramyxoviruses the sequences of the N- and C-terminal regions of P are more conserved than those in the middle region and that the significant size increase in the exceptionally large henipavirus Ps seems to be due to the expansion of the middle region. It will be interesting to see whether deletion of this middle region in henipavirus Ps will have any functional effect.
Interestingly, previous mapping studies indicated that most paramyxovirus Ns contain at least one P-interacting domain located at the least conserved C terminus. For SeV N, the P-binding sites are located on the C-terminal and the middle regions (Homann et al., 1991; Buchholz et al., 1994
). For HPIV2 N, two separate C-terminal domains are involved in binding to P (Nishio et al., 1999
). Murray et al. (2001)
reported that the C-terminal region of N is responsible for binding to HRSV P. Liston et al. (1997)
demonstrated that there are two separate independent P-binding sites on the MeV N and they reside at the hypervariable C terminal region and the conserved middle region. In this study, we have demonstrated that the 29 aa C-terminal region (aa 468496) of henipavirus N alone was sufficient to bind both the full-length P and the C-terminal fragment of P (aa 549709). Although we could not directly map any other P-binding sites in this study, we did show that the N-terminal region of P (aa 3220) was able to independently interact with the full-length N, but not the C-terminal region of N. This would suggest that it is highly possible that additional P-interacting site(s) exists in the henipaviruses N, but was not revealed in this study, probably because this site(s) is more conformation dependent, and the current approach of bacterial expression and PBPOA is not suitable for such mapping studies.
In this study, we have successfully demonstrated NP in henipaviruses interaction by using bacterially expressed recombinant proteins. The PBPOA described here provided a simple and rapid method not only allowing the detection of the NP interaction but also mapping of domains in such interaction. The NP interaction detected here was specific and sensitive, as direct analysis of crude bacteria cell lysate was carried out without involving any purification of recombinant proteins.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 24 October 2003;
accepted 15 January 2004.
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