Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

Changhee Lee and Dongwan Yoo

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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Porcine reproductive and respiratory syndrome virus (PRRSV) open reading frame (ORF) 2a contains a small internal ORF (2b) capable of encoding a protein of 73 aa, termed E protein. The function of E protein is currently unknown. The E protein possesses two cysteines at positions 49 and 54 that are highly conserved among North American isolates. In the present study, it was shown that E protein did not homodimerize with itself nor did it heterodimerize with the nucleocapsid (N) protein. However, E protein was interactive non-covalently with itself or with the N protein as shown by pull-down assays. The significance of the E protein cysteine residues on virus replication was determined using an infectious clone. Each cysteine was substituted by serine and the mutations were introduced into a full-length clone of PRRSV. When transfected into Marc-145 cells, all cysteine mutant clones induced PRRSV-specific cytopathic effects and produced infectious progeny virus. The data indicate that cysteine residues in the E protein are not essential for replication of North American genotype PRRSV.


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Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae in the order Nidovirales, and it causes severe reproductive failure in pregnant sows and respiratory illness in pigs of all ages (Rossow et al., 1999). Other members of the family Arteriviridae include Lactate dehydrogenase-elevating virus (LDV) of mice, Equine arteritis virus (EAV) and Simian hemorrhagic fever virus (SHFV) (Cavanagh, 1997). The PRRSV genome is a single-stranded, positive-sense RNA of approximately 15 kb in size that encodes two large polyproteins, 1a and 1ab, in the 5'-terminal 12 kb region and seven structural proteins in the 3'-terminal 3 kb region: GP2 (glycoprotein 2), small envelope (E) protein, GP3, GP4, GP5, membrane (M) protein and nucleocapsid (N) protein (Meulenberg et al., 1993; Snijder & Meulenberg, 1998; Nelsen et al., 1999). Based on antigenic and genetic differences, PRRSV isolates are divided into two distinct genotypes, European type and North American type. The genetic similarity between the two groups is approximately 63 % (Meng et al., 1995; Nelson et al., 1993; Nelsen et al., 1999; Wootton et al., 2000).

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 N–N interactions (Wootton & Yoo, 2003). Subsequently, using an infectious cDNA clone, the N–N 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. 1a; Snijder et al., 1999). The biological significance of the cysteines in E protein is unknown.



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Fig. 1. (a) Conservation of cysteine residues in E proteins of arteriviruses. Numbers indicate positions of cysteine residues and size of E proteins for each arterivirus. PRRSV-NA, North American-type PRRSV; PRRSV-EU, European-type PRRSV. (b) Co-immunoprecipitation of the N and E proteins of PRRSV-NA. HeLa cells were infected with vTF7-3 vaccinia virus and co-transfected with pCITE-N and pCITE-E for 16 h. The cells were radiolabelled for 5 h with 100 µCi ml–1 [35S]methionine/cysteine, lysed with RIPA buffer and then immunoprecipitated using either a mixture of N-specific mAbs (lanes 2 and 5) or anti-E-specific rabbit antiserum (lanes 3 and 6) followed by 17 % SDS-PAGE under reducing (+{beta}ME) or non-reducing (–{beta}ME) conditions. Lanes 1 and 4, pCITE empty vector; lanes 2, 3, 5 and 6, co-transfection of N and E genes. Arrowheads indicate the E protein; 2N, dimeric N protein. (c) Immunoprecipitation of E proteins expressed in HeLa cells using vTF7-3 vaccinia virus under reducing (+{beta}ME) or non-reducing (–{beta}ME) conditions. N in lanes 1 and 4, N gene transfection; C in lanes 2 and 5, empty vector; E in lanes 3 and 6, E gene transfection; 2N, dimeric N protein. (d) The absence of E protein dimerization in PRRSV-infected cells. Virus-infected cell lysates metabolically labelled with [35S]methionine/cysteine were immunoprecipitated with a mixture of individual antibody specific for E, N or M and resolved by SDS-PAGE under reducing (+{beta}ME; lanes 1 and 2) or non-reducing (–{beta}ME; lanes 3 and 4) conditions. C (lanes 1 and 3) represents mock infection and V (lanes 2 and 4) represents virus infection. GP5–M, heterodimeric form of the GP5 and M proteins. (e) GST pull-down assay. Bacterially expressed GST, GST–VP8, GST–N or GST–E was individually bound to glutathione–Sepharose beads and incubated with lysates from PRRSV-infected Marc-145 cells (upper panel) or from HeLa cells expressing N (middle panel) or E (lower panel) protein. The beads were washed five times, and the bound proteins were eluted in reducing sample buffer followed by SDS-PAGE and autoradiography. Lane 1, radioimmunoprecipitation of input proteins, N (upper and middle panels) or E (lower panel) using N-specific mAb SDOW17 or anti-E-specific rabbit antiserum, respectively. Lanes 2–5, bead-binding assay for N (upper and middle panel) or E (lower panel) by GST alone (lane 2), GST–VP8 (lane 3), GST–N (lane 4) or GST–E (lane 5).

 
To examine if E protein interacts with N protein, co-immunoprecipitation was first performed in cells co-expressing N and E proteins (Fig. 1b). The construction of pCITE-N expressing the N protein is described elsewhere (Wootton & Yoo, 2003). To construct a plasmid expressing the E protein, ORF2b (E gene) was RT-PCR amplified from PRRSV (PA-8 strain) using a pair of primers (forward, 5'-ggatccGCCACCATGGGGTCCATGCAAAGCC-3' and reverse, 5'-ggatccTCATAAGATCTTCTGTAATTGCTC-3', where lower-case letters indicate BamHI recognition sequences), and subsequently cloned in-frame into pCITE-2a (Novagen) at the BamHI site downstream of the T7 promoter producing pCITE-E. To express N and E proteins, HeLa cells were infected with vTF7-3 vaccinia virus expressing the T7 RNA polymerase and co-transfected with plasmids pCITE-N and/or pCITE-E. The cells were radiolabelled for 5 h with 100 µCi (3·7 MBq) ml–1 EasyTag EXPRESS protein labelling mix ([35S]methionine and [35S]cysteine, specific activity, 407 MBq ml–1) (Perkin-Elmer) and lysed with RIPA buffer (1 % Triton X-100, 1 % sodium deoxycholate, 150 mM NaCl, 50 mM Tris/HCl pH 7·4, 10 mM EDTA, 0·1 % SDS) containing 1 mM PMSF. The cell lysates were then immunoprecipitated using either the N-specific mAb SDOW17 or the E protein-specific anti-peptide anti-rabbit serum followed by SDS-PAGE under reducing and non-reducing conditions. The anti-N antibody and anti-E antibody precipitated N (Fig. 1b, lanes 2 and 5) and E proteins (Fig. 1b, lanes 3 and 6), respectively, indicating the expression of both proteins. Under reducing conditions, the N protein was not co-precipitated with the E protein by the E antibody, and similarly the E protein was not co-precipitated with the N protein by the N-antibody (Fig. 1b, lanes 2 and 3). No band corresponding to a predicted heterodimer of N and E was identified in non-reducing conditions (Fig. 1b, lanes 5 and 6), showing the absence of disulfide-linked heterodimeric association of N and E proteins.

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 GST–E fusion protein was coupled to glutathione–Sepharose 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 GST–N 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 GST–N, the GST–E 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 GST–rotavirus 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 GST–N (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), GST–N 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 12206–12208 and 12221–12223 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 12191–12218) and 2b-C49S-Rev (5'-CCAATCTGATGCtAAAGACCACCAGCC-3'; nucleotide positions 12191–12218); for C54S and C49/54S mutations, 2b-C54S-Fwd (5'-GCATCAGATTGGTTaGCTCCGCGGTATTCCG-3'; nucleotide positions 12207–12238) and 2b-C54S-Rev (5'-CGGAATACCGCGGAGCtAACCAATCTGATGC-3'; nucleotide positions 12207–12238), 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 Eco47III–BsrGI 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. 2c, 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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Fig. 2. Infection of E-cysteine mutant genomic clones P129-E-C49S, P129-E-C54S and P129-E-C49/54S. Cells were transfected with corresponding full-length genomic cDNA clones using the reagent Lipofectin (Invitrogen) and CPE was observed daily (a–e). The photographs were taken at 5 days post-transfection using an inverted microscope attached to a digital camera (Nikon Coolpix 950). For immunofluorescence (f–j), cells were fixed with cold methanol at 4 days post-transfection, stained with SDOW17 and Alexa green-conjugated goat anti-mouse antibody and were examined using a fluorescent microscope (x20; Olympus mode AX70). Plaque morphology of cysteine mutants (k–o). Standard plaques assays were performed with a slight modification. Cells were transfected with individual DNA clones and at 24 h post-transfection overlaid with 0·8 % agarose in Dulbecco's modified Eagle's medium. Plaques were stained with 0·01 % neutral red at 5 days post-transfection and photographed 5 h later. The P129-E-C54S and P129-E-C49/54S mutant virus plaques were smaller in size (n and o) compared with plaques of P129-WT (l) and P129-E-C49S (m).

 
Culture supernatants were harvested from cells at 5 days post-transfection and designated passage 1 (P1). P2 and P3 stocks were prepared from P1 for each mutant, and virus titres were determined to be 1·5x103, 1x104 and 7x105 p.f.u. ml–1 for P1, P2 and P3 of P129-WT, respectively, and 8x102, 1x104 and 5x105 p.f.u. ml–1 for P1, P2 and P3 of P129-E-C49S, respectively (Fig. 3a). P129-E-C54S and P129-E-C49/54S were titrated to be 1·5x104 and 1x104 p.f.u. ml–1 for P1, and 5x105 and 7x105 p.f.u. ml–1 for P2, respectively. The P3 titres of P129-E-C54S and P129-E-C49/54S decreased to 1x104 p.f.u. ml–1 for both (Fig. 3a).



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Fig. 3. Growth kinetics of cysteine mutant viruses. (a) Virus titres at different passages determined by standard plaque assay. The culture supernatant from DNA transfected cells was designated ‘passage 1’ (P1) and sub-sequence passages were designated P2 and P3. Error bars represent standard deviations of the arithmetic mean values from three independent experiments. (b) One-step growth curves for cysteine mutants. The titres are presented as mean values of two independent experiments, each in duplicate.

 
The stability of cysteine mutations was confirmed by RT-PCR and sequencing from RNA extracted from cells and culture supernatants infected with P3. The RNA preparations were treated with RNase-free DNaseI to eliminate any possible carry-over of transfected DNA, and the E gene was amplified. The sequencing results confirmed the stable incorporation of cysteine mutations in the viral genome up to at least three passages in cell culture. The growth kinetics was determined for each mutant and compared to that of P129-WT using P3. P129-WT and P129-E-C49S both reached a titre of 1x105 p.f.u. ml–1 by 2 days post-infection and the titres increased to maximum 7x105 p.f.u. ml–1 by 5 days post-infection (Fig. 3b). P129-E-C54S and C49/54S reached a titre of less than 1x103 p.f.u. ml–1 at 2 days post-infection, but by 3 days post-infection their titres exceeded 1x105 p.f.u. ml–1. This observation was consistent with a slow onset of the CPE appearance showing gradual cytopathology by 4 days post-transfection followed by an abruptly progressive development of CPE.

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 GP2b–GP3–GP4 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 GP2–GP3–GP4, 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 N–RNA 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 N–E 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 N–E non-covalent interaction may be a requirement for PRRSV replication.


   ACKNOWLEDGEMENTS
 
This study was supported by funding to D. Y. from NSERC Canada, Ontario Pork, OMAF, and the USDA NRI Integrated Programme for PRRS of the USA. The authors are grateful to Jay Calvert and Jenny Welch at Pfizer Animal Health USA for providing the infectious cDNA clone for this study and discussion of the results.


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Received 2 May 2005; accepted 27 July 2005.



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