Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Correspondence
Dongwan Yoo
dyoo{at}uoguelph.ca
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ABSTRACT |
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MAIN TEXT |
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The viral genome is enclosed in the isomeric capsid structure composed of N proteins. The N protein, as the sole protein component of the viral capsid, interacts with itself through covalent and non-covalent interactions (Wootton & Yoo, 2003). North American PRRSV N proteins contain three highly conserved cysteine residues at amino acid positions 23, 75 and 90. By mutational analysis using the expressed protein, the cysteine at position 23 has been shown to be responsible for disulfide linkages for NN interactions (Wootton & Yoo, 2003
). Subsequently, using an infectious cDNA clone, the NN interaction has been shown to be essential for virus infectivity (Lee et al., 2005
). Lee et al. (2005)
also showed that the cysteine at position 90 appeared to be essential for virus infectivity, while the cysteine at position 75 was not. Unlike North American PRRSV, cysteine 90 is not found in the European genotype, suggesting a possible linkage of the N protein by the cysteine at position 90 to another structural protein in the case of North American PRRSV, and this possible linkage is essential for North American PRRSV replication.
Besides N, there are six other M-associated proteins that constitute the virion: GP2, E, GP3, GP4, GP5 and M proteins (Mardassi et al., 1996; Meulenberg et al., 1995
; Wu et al., 2001
). Among these, GP5 and M proteins form a disulfide-linked heterodimer, which is an essential requirement for infectivity in LDV and EAV (Faaberg et al., 1995
; Snijder et al., 2003
). A recent study shows that GP2, GP3 and GP4 proteins form a disulfide-linked heterotrimer in EAV and that this heterotrimerization is essential for EAV infectivity (Wieringa et al., 2003a
, b
). Although the possible structural or non-structural nature of GP3 is currently under debate for European and North American types, GP4 has been shown to co-precipitate GP3 for North American PRRSV, implicating a possible association of GP3 with GP4 in North American PRRSV as well (Mardassi et al., 1998
). Therefore, since the E protein has not been shown to be associated with any other M proteins, it has led us to postulate that the E protein may have a possible interaction with the N protein.
The E protein is a newly identified structural component for arteriviruses. For PRRSV, the E protein is encoded in the small internal open reading frame (ORF) within ORF2a, and is composed of 73 and 70 aa for the North American and European types, respectively (Wootton et al., 2000; Wu et al., 2001
). The E protein is non-glycosylated and M-associated and, in EAV, is essential for virus replication (Snijder et al., 1999
). E proteins of North American PPRSV contain two cysteine residues at amino acid positions 49 and 54. These cysteines, however, are not well conserved among arteriviruses. European genotype PRRSV isolates contain only a single cysteine at position 51, while E proteins of LDV, EAV and SHFV contain a single cysteine residue at different positions (Fig. 1
a; Snijder et al., 1999
). The biological significance of the cysteines in E protein is unknown.
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A further study was conducted to examine if the E protein forms a homodimer. The 35S-labelled E protein was expressed in HeLa cells using vTF7-3 vaccinia virus, and the cell lysates were immunoprecipitated by E-specific antiserum followed by SDS-PAGE under non-reducing conditions. While the majority of N proteins was shifted from a monomeric form of 15 kDa to a dimeric form of 30 kDa under non-reducing conditions (Fig. 1c, lane 4; Wootton & Yoo, 2003
), the E protein remained in its monomeric form of 10 kDa (Fig. 1c
, lane 6). Similar experiments were conducted in PRRSV-infected cells, but it was not possible to identify dimeric E protein under non-reducing conditions (Fig. 1d
, lane 4). It was concluded that the PRRSV E protein does not undergo cysteine-linked homodimerization.
An X-ray crystallographic study has suggested a structural model where the N protein may interact with a cytoplasmic portion of an M protein residing in the virion envelope (Doan & Dokland, 2003). To examine if the E protein interacted with N non-covalently, glutathione S-transferase (GST) pull-down assays were carried out. The RT-PCR amplified E gene fragment was cloned in-frame into pGEX-2T (Amersham Pharmacia) at the BamHI site, constructing the plasmid pGEX-E, which was then used in an inducible Escherichia coli expression system. The E protein expression as a GST-fusion product was confirmed by 12 % SDS-PAGE followed by Coomassie blue staining (data not shown), and the GSTE fusion protein was coupled to glutathioneSepharose beads (Amersham) followed by incubation with the radiolabelled N protein expressed either in PRRSV-infected Marc-145 cells (Fig. 1e
, upper panel) or HeLa cells by vTF7-3 vaccinia virus (Fig. 1e
, middle panel). After extensive washing of beads, bead-bound proteins were dissociated and resolved by SDS-PAGE under reducing conditions (Fig. 1e
, upper and middle panels). The GSTN fusion protein precipitated the N protein (Fig. 1e
, upper and middle panels, lane 4), confirming the specific interaction of N with N as described previously (Wootton & Yoo, 2003
). As with GSTN, the GSTE fusion protein also precipitated both authentic and recombinant N proteins efficiently (Fig. 1e
, upper and middle panels, lane 5), indicating a specific interaction between N and E proteins. Either GST alone or GSTrotavirus VP8 fusion protein did not precipitate the N or E proteins (Fig. 1e
, lanes 2 and 3 in all panels). To confirm the non-covalent interaction between N and E proteins, a reverse experiment was performed to precipitate E using GSTN (Fig. 1e
, lower panel, lane 4). The radiolabelled E protein was prepared in HeLa cells by vTF7-3 vaccinia virus and used as an input protein. As shown in Fig. 1(e)
(lane 4, lower panel), GSTN precipitated the E protein specifically. Together, our data show that the N and E proteins of PRRSV do not form cysteine-linked covalent linkages but that they do form cysteine-independent non-covalent associations.
To determine the role of the cysteines in the E protein for virus infection, a reverse genetics system was applied. To substitute cysteine (C) codons for serine (S) codons at amino acid positions 49 and 54 (nucleotide positions 1220612208 and 1222112223 of the viral genome), PCR-based site-directed mutagenesis was conducted using the shuttle plasmid pTB-shuttle-PRRSV-3997 (Lee et al., 2005) and the following primer pairs: for C49S mutation, 2b-C49S-Fwd (5'-GGCTGGTGGTCCTTTaGCATCAGATTGG-3'; nucleotide positions 1219112218) and 2b-C49S-Rev (5'-CCAATCTGATGCtAAAGACCACCAGCC-3'; nucleotide positions 1219112218); for C54S and C49/54S mutations, 2b-C54S-Fwd (5'-GCATCAGATTGGTTaGCTCCGCGGTATTCCG-3'; nucleotide positions 1220712238) and 2b-C54S-Rev (5'-CGGAATACCGCGGAGCtAACCAATCTGATGC-3'; nucleotide positions 1220712238), where the lower-case letters represent mutated nucleotides in a codon and underlining indicates codon changes for amino acid substitutions from cysteine to serine. The shuttle plasmids carrying respective cysteine mutations were digested with Eco47III and BsrGI, and the 908 bp Eco47IIIBsrGI fragment was purified and subcloned into the full-length wild-type genomic clone pCMV-S-P129 to replace the corresponding fragment. The newly generated full-length cDNA clones were screened by XmaI digestion, and specific mutations in the E gene were confirmed by nucleotide sequencing. The full-length mutated genomic clones for C49S, C54S and C49/54S in the E protein were designated P129-E-C49S, P29-E-C54S and P129-E-C49/54S, respectively.
Marc-145 (a subclone of MA 104 cells) is an established cell line permissive for PRRSV infection (Kim et al., 1993), and therefore infectivity of the P129-E-C49S, P29-E-C54S and P129-E-C49/54S mutant clones was individually examined in Marc-145 cells by DNA transfection. Cells were transfected for 24 h and the appearance of PRRSV-specific cytopathic effects (CPE) was monitored daily. All dishes transfected with either P129-E-C49S, P129-E-C54S or P129-E-C49/54S produced specific CPE by 3 days post-transfection, and the CPE became extensive by 5 days post-transfection (Fig. 2
c, d and e). The specificity of CPE was confirmed by immunofluorescence cell staining using the N-specific mAb SDOW17 (Fig. 2h, i and j
). P129-E-C54S and P129-E-C49/54S mutants induced a distinct CPE development compared with P129-WT and P129-E-C49S. CPE mediated by P129-E-C54S and P129-E-C49/54S was similar in shape but slower in appearance to that of wild-type CPE until 4 days post-transfection. Thereafter, further development of CPE by these mutants was sudden and explosive, resulting in a rapid cell death. The plaque morphology was also distinct for P129-E-C54S and P129-E-C49/54S mutants. The plaques were smaller in size and their shapes were different from those of wild-type and P129-E-C49S mutant virus (Fig. 2n and o
). Cells in the centre of the plaques died readily and detached early, resulting in the clear and transparent plaques for P129-E-C54S and P129-E-C49/54S mutants. The cysteine at position 54 may represent a distinctive role for this cytopathology. The data demonstrate that all cysteine mutants were viable and produced infectious progeny viruses, indicating that both cysteines of the E protein are not essential for PRRSV replication.
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In the present study, a possible association of E protein with N protein and the role of cysteines of the E protein for virus multiplication were examined. It appears that the E protein does not form a disulfide-linked heterodimer with the N protein or a homodimer with itself, rejecting our initial hypothesis. The role of cysteine 90 of the North American PRRSV N protein still remains to be determined. Using an European PRRSV isolate, Wissink et al. (2004) have described a heterotrimeric complex of GP2, GP3 and GP4 and, without evidence, speculated a possible incorporation of E protein with this trimeric complex. Wieringa et al. (2004)
have recently shown a covalent association of the E protein with the GP2bGP3GP4 heterotrimers in EAV, suggesting a possible role of the heteromultimeric complex in the virus entry process. It is noteworthy that E proteins of European PRRSV and EAV contain a single cysteine at positions 51 and 58, respectively, which corresponds to position 54 in the North American PRRSV genotype (Fig. 1a
). Therefore, if the E protein associates with GP2GP3GP4, cysteine 51 would be the residue utilized for the linkage of the E protein with the complex. Alternatively, cysteines at positions 49 and 54 may form an intramolecular disulfide-bond in North American E proteins, rather than associating with other minor proteins. Nevertheless, the current study using full-length genomic clones shows that the cysteine residues in the E protein or any possible multimeric association of E via cysteine-linkages is not essential for virus replication in North American PRRSV.
In enveloped viruses, specific interaction between viral capsid protein and M proteins leads to the incorporation of capsid into virions (Simons & Garoff, 1980). Although the E protein appeared not to form a disulfide-linked heterodimer with N protein, E and N proteins were interactive non-covalently as shown by the GST-pull-down assay (Fig. 1e
), and this interaction may be a basis for virion assembly. The E protein contains a cluster of basic amino acid residues in the hydrophilic C-terminal domain (Snijder et al., 1999
). Therefore, it is postulated that the interaction between N and E may be initiated by the binding of RNA to the N protein (Yoo et al., 2003
). Subsequently, the NRNA interaction may promote the N protein association with E, providing stable assembly of the core structure in the virion. Further studies are needed to determine whether highly basic amino acid residues on the E protein contribute to NE interactions through the RNA bridging. A preliminary study indicates that a single mutation at arginine 51 in the cluster of basic residues on E is lethal for PRRSV (C. Lee & D. Yoo, unpublished results), supporting a premise that the NE non-covalent interaction may be a requirement for PRRSV replication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Doan, D. N. & Dokland, T. (2003). Structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus. Structure 11, 14451451.[CrossRef][Medline]
Faaberg, K. S., Even, C., Palmer, G. A. & Plagemann, P. G. (1995). Disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity. J Virol 69, 613617.[Abstract]
Kim, H. S., Kwang, J., Yoon, I. J., Joo, H. S. & Frey, M. L. (1993). Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch Virol 133, 477483.[CrossRef][Medline]
Lee, C., Calvert, J. G., Welch, S. W. & Yoo, D. (2005). A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity. Virology 331, 4762.[CrossRef][Medline]
Mardassi, H., Massie, B. & Dea, S. (1996). Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus. Virology 221, 98112.[CrossRef][Medline]
Mardassi, H., Gonin, P., Gagnon, C. A., Massie, B. & Dea, S. (1998). A subset of porcine reproductive and respiratory syndrome virus (PRRSV) GP3 is released into the culture medium of cells as a non-virion-associated membrane-free (soluble) form. J Virol 72, 62986306.
Meng, X. J., Paul, P. S., Halbur, P. G. & Lum, M. A. (1995). Phylogenetic analysis of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe. Arch Virol 140, 745755.[CrossRef][Medline]
Meulenberg, J. J., Hulst, M. M., de Meijer, E. J., Moonen, P. J. M., den Besten, A., de Kluyver, E. P., Wensvoort, G. & Moormann, R. J. (1993). Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV. Virology 192, 6272.[CrossRef][Medline]
Meulenberg, J. J., Petersen-den Besten, A., De Kluyver, E. P., Moormann, R. J., Schaaper, W. M. & Wensvoort, G. (1995). Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155163.[CrossRef][Medline]
Nelsen, C. J., Murtaugh, M. P. & Faaberg, K. S. (1999). Porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents. J Virol 73, 270280.
Nelson, E. A., Christopher-Hennings, J., Drew, T., Wensvoort, G., Collins, J. E. & Benfield, D. A. (1993). Differentiation of U.S. and European isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. J Clin Microbiol 31, 31843189.[Medline]
Rossow, K. D., Shivers, J. L., Yeske, P. E., Polson, D. D., Rowland, R. R., Lawson, S. R., Murtaugh, M. P., Nelson, E. A. & Collins, J. E. (1999). Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterized by marked neurovirulence. Vet Rec 144, 444448.
Simons, K. & Garoff, H. (1980). The budding mechanisms of enveloped animal viruses. J Gen Virol 50, 121.[Medline]
Snijder, E. J. & Meulenberg, J. J. (1998). The molecular biology of arteriviruses. J Gen Virol 79, 961979.
Snijder, E. J., van Tol, H., Pedersen, K. W., Raamsman, M. J. & de Vries, A. A. (1999). Identification of a novel structural protein of arteriviruses. J Virol 73, 63356345.
Snijder, E. J., Dobbe, J. C. & Spaan, W. J. M. (2003). Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 77, 97104.
Wieringa, R., de Vries, A. A. F. & Rottier, P. J. M. (2003a). Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus. J Virol 77, 62166226.
Wieringa, R., de Vries, A. A. F., Post, S. M. & Rottier, P. J. M. (2003b). Intra- and intermolecular disulfide bonds of the GP2b glycoprotein of equine arteritis virus: relevance for virus assembly and infectivity. J Virol 77, 1299613004.
Wieringa, R., de Vries, A. A., van der Meulen, J. & 7 other authors (2004). Structural protein requirements in equine arteritis virus assembly. J Virol 78, 1301913027.
Wissink, E. H., Kroese, M. V., Maneschijn-Bonsing, J. G., Meulenberg, J. J., van Rijn, P. A., Rijsewijk, F. A. & Rottier, P. J. (2004). Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production. J Gen Virol 85, 37153723.
Wootton, S. K. & Yoo, D. (2003). Homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages. J Virol 77, 45464557.
Wootton, S. K., Yoo, D. & Rogan, D. (2000). Full-length sequence of a Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate. Arch Virol 145, 22972323.[CrossRef][Medline]
Wu, W. H., Fang, Y., Farwell, R., Steffen-Bien, M., Rowland, R. R. R., Christopher-Hennings, J. & Nelson, E. A. (2001). A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b. Virology 287, 183191.[CrossRef][Medline]
Yoo, D., Wootton, S. K., Li, G., Song, C. & Rowland, R. R. (2003). Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin. J Virol 77, 1217312183.
Received 2 May 2005;
accepted 27 July 2005.
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