1 Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
2 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
3 Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia
Correspondence
Khatijah Yusoff
(at Department of Biochemistry and Microbiology)
khatijah{at}fsas.upm.edu.my
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ABSTRACT |
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MAIN TEXT |
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The NP protein of paramyxoviruses has been the focus of many studies for years due to its crucial functional role during replication of the genomic RNA (Horikami et al., 1992; Curran et al., 1993
; Myers et al., 1999
). Much structural and functional information on this protein has been derived, mainly from other paramyxoviruses such as Sendai, measles and human parainfluenza type 2 (hPIV-2) viruses (Spehner et al., 1991
; Buchholz et al., 1993
, 1994
; Bankamp et al., 1996
; Nishio et al., 1999
). Like many other paramyxoviruses, the nucleocapsid of NDV possesses a herringbone-like structure (Alexander, 1988
). Recombinant NP protein of Newcastle disease virus expressed in E. coli (Kho et al., 2001a
) or the baculovirus expression system (Errington & Emmerson, 1997
) is able to self-assemble to form ring- and herringbone-like structures which morphologically resemble the authentic nucleocapsid structures. Insertion of a 29 residue peptide containing the Myc epitope and His-tag sequences onto the C terminus of the NP protein inhibits assembly of the herringbone-like structures but does not impair formation of the ring-like particles (Kho et al., 2001a
). Furthermore, this foreign peptide was shown to be exposed on the surface of the ring-like particle (Kho et al., 2001a
; Rabu et al., 2002
). It is most likely that the herringbone-like particles are rather flexible and heterogeneous in both shape and size, making it difficult to obtain high-resolution analysis by X-ray crystallography. To date, no high-resolution structure is available for the NP of paramyxoviruses. Current structural models of the nucleocapsid proposed for this group of viruses were derived from the intermediate resolution studies of Sendai virus (Egelman et al., 1989
) and the N-RNA ring structure of rabies virus (Schoehn et al., 2001
), through a combination of electron microscopy and three-dimensional image reconstruction. Although the nucleocapsid-like particles formed by the NP protein of various paramyxoviruses are all helical in structure, significant structural differences have been reported depending on the virus genera (Bhella et al., 2002
).
The existence of an NPNP interaction that gives rise to the organized nucleocapsid herringbone-like features is evident in vesicular stomatitis (Sprague et al., 1983), Sendai (Buchholz et al., 1993
) and measles viruses (Spehner et al., 1991
; Fooks et al., 1993
). The NPNP interacting domains have been mapped to the N-terminal two-thirds of the NP protein for both Sendai and measles viruses (Buchholz et al., 1993
; Liston et al., 1997
) whereas in the case of hPIV-2, the N-terminal 294 amino acids of NP have been shown to be necessary for this interaction (Nishio et al., 1999
). In many cases, these self-assembled nucleocapsid-like particles appeared to enwrap nonspecific RNA of different lengths. However, the detailed in vivo assembly pathway for this group of negative-strand RNA viruses is still far from complete. For NDV, the NPNP self-assembly domain has yet to be identified. Therefore, this study was performed to define the contiguous sequence of the NP protein which is involved in self-assembly by deletion mutagenesis. In addition, the effect of deletions on the NP protein relative to its nucleocapsid-like phenotypes was also examined.
For NP self-assembly study purposes, a total of 11 deletion mutants with successive deletions from either the 5'- or the 3'-end of NP gene were constructed (Fig. 1a). C-terminal deletion constructs NP
C464, NP
C440, NP
C405, NP
C391, NP
C380, NP
C375, NP
C366 and NP
C245 differed from the full-length NP by deletion of 25, 49, 84, 98, 109, 114, 123 and 244 amino acids from the C terminus of the NP protein, respectively. N-terminal deletion mutants NP
N26, NP
N122 and NP
N245 were the result of 25, 121 and 244 amino acid deletions from the N terminus of the NP protein, respectively. Both the N- and C-terminally deleted cDNA fragments were generated by PCR of the plasmid DNA pTrcHis2-NP (Kho et al., 2001a
), which encodes the full-length NP gene, using the respective primer pairs. Each of the deleted cDNA fragments was ligated to the pTricHis2 expression vector (Invitrogen) and then introduced into E. coli strain TOP 10. Recombinant plasmids were isolated from the putative clones and the mutants were confirmed by DNA sequencing as described in Kho et al. (2001a)
.
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For production of purified protein, E. coli cell pellets (from 500 ml culture) were harvested, resuspended in lysis buffer [50 mM Tris (pH 7·9), 0·1 % Triton X-100, 0·2 mg lysozyme ml-1 and 4 mM MgCl2; 25 ml], and then lysed by sonication. The lysate was then treated with RNase (5 µg ml-1) and DNase I (5 µg ml-1) for 1 h at room temperature (25 °C) and finally the cell extract was recovered after centrifugation at 10 000 r.p.m. (JA 14 rotor, Beckman) for 20 min at 4 °C. The mutated proteins were then precipitated by increasing the saturation of ammonium sulphate to 40 %. Results showed that proteins NPC464, NP
C440, NP
C405, NP
C391, NP
C380, NP
C375 and NP
N26 were efficiently precipitated at different percentages of saturation (data not shown). In contrast, proteins NP
C366, NP
C245, NP
N122 and NP
N245 were scarcely precipitated by ammonium sulphate and were almost invisible when analysed by SDS-PAGE after precipitation (data not shown). The difficulty in precipitating these truncated proteins was most probably due to the drastic decrease in protein solubility which resulted in the retention of the insoluble proteins in cell pellets. For subsequent protein purification, all mutated proteins except NP
C366 and NP
N245 were subjected to 10 to 50 % sucrose gradient centrifugation at 38 000 r.p.m. (SW 41 Ti rotor, Sorvall) for 5 h at 4 °C. These two mutated proteins were excluded due to the difficulty in getting sufficient soluble protein for any further studies. After centrifugation, fractions (0·5 ml) were collected by punching a hole at the bottom of the tube, and samples from each fraction were analysed by SDS-12 % PAGE to confirm the presence of the required proteins.
Fig. 2 shows the protein sedimentation profiles of the truncated NP proteins. The profile, which appeared as a major peak located around fractions containing herringbone-like particles, as demonstrated in the full-length NP protein (Fig. 2
) (preparation as described in Kho et al., 2001a
) was observed for mutated proteins NP
C464, NP
C440, NP
C405, NP
C391, NP
C380, NP
C375 and NP
N26, indicating the ability of these mutants to form particles. However, it was obvious that these particles had different sizes or shapes judging from the respective positions of the major peak. In addition, for NP
C464 and NP
C440, there was an additional peak at fractions 2 to 4. Nevertheless, SDS-PAGE analysis showed that the peak consisted mainly of other E. coli proteins. The position of the peak for C-terminal deletion mutants shifted towards the denser fractions, and this was clearly demonstrated in NP
C405 and NP
C391 (Fig. 2
). Furthermore, a small portion of these two mutated proteins was sedimented at the bottom of the centrifuge tubes, indicating the presence of particles with higher density (data not shown). On the other hand, the tendency for peak shifting to denser fractions was not obvious in the N-terminally deleted NP
N26. For NP
N122 and NP
C245 (Fig. 2
) however, no clearly resolvable peak was observed. Thus, it was anticipated that these two mutants did not retain the ring- and herringbone-like structures.
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This study shows that the entire N-terminal region up to residue 375 (77 %) is essential for assembly of the nucleocapsid while the C-terminal 114 amino acids, from positions 376 to 489 (23 %), are dispensable for assembly. Sequence analysis of NP proteins of paramyxoviruses revealed that 77 % of the N-terminal region contains the four strongly conserved segments (Kho et al., 2001b). The biological functions of these segments are still unclear but they have been predicted to be involved in NPNP contacts during assembly (Morgan et al., 1984
).
An interesting observation in this NP self-assembly study was that further amino acid deletions from the C-terminal end of the NP protein (NPC405, NP
C391, NP
C380 and NP
C375) resulted in the formation of longer herringbone-like structures as compared to mutated proteins with shorter deletions (NP
C464 and NP
C440) (Fig. 3
). This suggests that the tendency of these mutated proteins to form a relatively long structure was the result of amino acid deletions covering positions 406 to 489, which somehow enhance the polymerization of NP monomers. This might be achieved via some secondary structural changes of these NP mutant polypeptides in an unknown manner that permits a stronger contact between the truncated NP monomers. Subsequently, upon sucrose gradient centrifugation, these herringbone-like particles which have a higher stability could withstand the centrifugal forces better. Thus, the monomeric ring was not easily shed from the helices of the herringbone-like particles in mutated proteins NP
C405, NP
C391, NP
C380 and NP
C375 compared to NP
C464 and NP
C440, which formed rather short herringbone-like particles, presumably due to stronger NPNP or NPRNA interactions.
The phenomenon of monomeric ring structures being shed continuously from helical structures during sucrose gradient centrifugation was suggested by Bhella et al. (2002) in their study on the nucleocapsid morphology of measles virus, simian virus 5 (SV5) and respiratory syncytial virus. The prediction that the C terminus of NP (amino acid positions 376 to 439) may enhance the NP polymerization activity remains a brave assumption since there is no precedent for this observation in other nonsegmented single-stranded RNA viruses. In addition, it has been reported that nucleocapsids formed by tail-less NP protein of SV5 appeared to be more rigid (Mountcastle et al., 1974
). On the other hand, removal of the C-terminal 23 amino acids of the nucleoprotein of influenza virus, a segmented single-stranded RNA virus, increased the protein oligomerization activity (Elton et al., 1999
). This supports the notion that deletion of the C terminus of NDV NP may allow a stronger NPNP contact, resulting in the formation of long herringbone-like structures. Nevertheless, further investigation along these lines should prove worthwhile.
This NP protein self-assembly study demonstrates that the NPNP binding sites are located in the first 375 amino acids at the N terminus. The C terminus, on the other hand, is not required for nucleocapsid assembly but may play a regulatory role in NP polymerization. The important role of this part, as well as other NP functional domains, remains to be explored further. In this regard, targeting conserved amino acids for site-directed mutagenesis coupled with function analysis could be a fruitful approach to further delineate the multiple important regions of the NP protein.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 20 January 2003;
accepted 7 April 2003.