Department of Infectious Diseases and Food Chain Quality, Institute for Animal Science and Health, Lelystad, The Netherlands1
Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Utrecht, The Netherlands2
Author for correspondence: Helene Verheije. Present address: Virology Division, Department of Infectious Diseases and Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, NL-3584 CL Utrecht, The Netherlands. Fax +31 30 253 6723. e-mail h.verheije{at}vet.uu.nl
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Abstract |
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Introduction |
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For vaccine purposes, we aim to generate viable deletion mutants of PRRSV. This raises basic questions about the regions of the viral genome in which deletions are tolerated. In this respect, two considerations are important. Firstly, PRRSV has a concise genome, like other RNA viruses. Since RNA viruses have evolved to optimal fitness, most of the genetic information is expected to be essential. Secondly, the ORFs that encode the structural proteins of the virus are partially overlapping. Deletions in overlapping regions would therefore result in the mutation of two structural proteins, which would almost inevitably lead to the production of a non-viable virus. Earlier studies showed that deletions in many conserved regions were lethal (M. H. Verheije, unpublished results).
In this study, we focused on the 3' end of the PRRSV genome, since this region does not contain sequences that overlap other ORFs. Until now, deletions introduced in the N-terminal and middle parts of the coding region of the N protein have not resulted in viable virus (M. H. Verheije, unpublished results). In the present study, we were more successful by focusing on the region around the N gene stop codon. Alignment of the N protein sequence and the 3' UTR of different PRRSV strains revealed heterogeneity at the C terminus of the N protein and at the 5' end of the 3' UTR. A deletion analysis of this region was therefore performed using the available infectious cDNA clone (Meulenberg et al., 1998a ) of Lelystad virus (LV) in order to determine the limits of the sequences that can be removed while still resulting in the production of a viable virus, but one with reduced pathogenicity. This is the first publication to describe the generation of viable arterivirus mutants that contain a deletion in the viral genome that is maintained stably after multiple passages in vitro.
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Methods |
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Construction of full-length genomic cDNA clones of LV.
PCR mutagenesis was used to introduce sequences into the PacI mutant of the genome-length cDNA clone of LV (pABV437) (Meulenberg et al., 1998a ). The primers used for PCR mutagenesis are listed in Table 1
. PCR fragments generated to introduce deletions into ORF7 were digested with HpaI and PacI and ligated into these sites of pABV437. PCR fragments generated to introduce deletions into the 3' UTR were digested with PacI and XbaI and ligated into these sites of pABV437. Standard cloning procedures were performed essentially as described previously (Sambrook et al., 1989
). Transformation conditions were maintained as described previously (Meulenberg et al., 1998a
). Sequence analysis was performed to confirm the introduced mutations. The constructs are drawn schematically in Fig. 2
.
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In vitro transcription and transfection of BHK-21 cells.
Full-length genomic cDNA clones were transcribed in vitro and the resulting RNA was transfected into BHK-21 cells either using Lipofectin (Gibco BRL) or by electroporation (Meulenberg et al., 1998a ).
Infection of PAMs.
To rescue infectious virus, the culture supernatant of BHK-21 cells was harvested 24 h after transfection and 200 µl of this culture supernatant was used to inoculate PAMs. After 1 h, the inoculum was removed and fresh culture medium was added. Approximately 15 h after infection, the culture supernatant was harvested and PAMs were washed with PBS, dried and stored at -20 °C until the immunoperoxidase monolayer assay was performed.
Immunoperoxidase monolayer assay (IPMA).
Immunostaining of BHK-21 cells and PAMs was performed by methods described previously (Wensvoort et al., 1986 ). MAbs against GP3 (122.14), GP4 (122.1) and the M protein (126.3) (van Nieuwstadt et al., 1996
) and against the different antigenic domains of the N protein [138.22 (domain A), 126.9 (domain B), 126.15 (domain C) and 122.17 (domain D); Meulenberg et al., 1998b
] were used to detect expression of PRRSV proteins.
Genetic analysis of genomic RNA of recombinant viruses.
In order to analyse the viral RNA in the culture supernatant of PAMs and in the fractions of the sucrose gradient, 200 µl of the culture supernatant or of the fraction was diluted with an equal volume of proteinase K buffer (100 mM TrisHCl, pH 7·2, 25 mM EDTA, 300 mM NaCl, 2% w/v SDS) and 0·08 mg proteinase K was added. After incubation for 30 min at 37 °C, the RNA was extracted with phenolchloroform and precipitated with ethanol. The RNA was reverse-transcribed with primer LV76 and PCR was performed with primers 119R218R and LV20, which flank the region of the viral genome that contained the deletions. Amplified fragments were analysed in 2% agarose gels and the PCR fragments were excised from the gel and purified with SpinX columns (Costar). Sequence analysis of the fragments was performed using the antisense primer of the PCR.
Radioimmunoprecipitation (RIP).
Metabolic labelling and immunoprecipitation of proteins expressed in PAMs were performed essentially as described previously (Meulenberg & Petersen-den Besten, 1996 ). MAb 122.17 was used to immunoprecipitate the N protein. PAMs were infected with passage 5 of the viruses at an m.o.i. of 1 and were labelled for 4 h with Tran[35-S] label (ICN) at 15 h post-infection. Samples were analysed by SDSPAGE using a 14% acrylamide gel.
Virus concentration and purification.
In order to analyse the production of (non-infectious) virus particles, BHK-21 cells were electroporated with RNA transcripts from pABV747 and pABV437 and, 15 h after transfection, the cells were labelled metabolically with 75 µl 10·5 mCi/ml Tran[35-S] label (ICN) for 24 h (Meulenberg & Petersen-den Besten, 1996 ). The particles in the supernatant were concentrated by centrifuging the supernatant through a 0·5 M sucrose cushion at 26000 r.p.m. for 5 h at 4 °C (Meulenberg & Petersen-den Besten, 1996
). The pellet was resuspended in TNE buffer (0·01 M TrisHCl, pH 7·2, 0·1 M NaCl and 1 mM EDTA, pH 8·0) and layered onto a 2050% sucrose gradient (van Berlo et al., 1982
). The sucrose gradient was centrifuged at 32000 r.p.m. for 19 h at 4 °C. Fractions of 0·5 ml were collected from bottom to top and 5 µl aliquots of the fractions were analysed by SDSPAGE using a 14% acrylamide gel.
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Results |
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LV accepts C-terminal truncations of up to six amino acids of the N protein
cDNA clones with deletions in the sequence encoding the two (pABV639), four (pABV694) and nine (pABV695) C-terminal amino acids of the N protein were constructed by PCR mutagenesis and cloning of the PCR fragments into an infectious cDNA clone of LV containing a PacI site at the stop codon of ORF7 (Meulenberg et al., 1998a ) (Fig. 2
). RNA transcripts of these constructs were transfected into BHK-21 cells and tested for their ability to replicate by analysing the expression of the structural proteins in IPMA (Fig. 2
). All transcripts expressed the viral proteins GP3, GP4 and M. In order to analyse the expression of the N protein, and in particular its antigenic domains (Meulenberg et al., 1998b
), we used MAbs 138.22, against antigenic domain A, 126.9, against domain B, 126.15, against domain C, and 122.17, against domain D of the protein in IPMA. For all constructs, we found that the transfected cells could be stained with each of the MAbs. These results indicated that LV genomes containing deletions at the C terminus of the N protein still replicated and that the structural proteins were properly translated. In addition, these deletions did not disturb the antigenic domains of the N protein.
In order to investigate whether the LV mutants with a C-terminally truncated N protein produced infectious virus, we inoculated PAMs with culture supernatants of the transfected BHK-21 cells, as PRRSV cannot infect BHK-21 cells. Twenty-four h later, the cells were fixed and stained with PRRSV-specific MAbs. PAMs inoculated with the supernatant of BHK-21 cells transfected with transcripts of pABV437, pABV639 or pABV694 stained positive. In contrast, no staining of PAMs was observed after inoculation with supernatant from BHK-21 cells transfected with RNA transcripts from pABV695. In conclusion, LV mutants producing an N protein with a C-terminal deletion of up to four amino acids produced infectious virus, whereas mutants producing an N protein with a C-terminal deletion of nine amino acids did not produce infectious virus at all.
In order to define further the acceptable limits of truncation of the N protein, we made stepwise deletions in the region encoding the five to eight most C-terminal amino acids. The fragments generated by PCR mutagenesis were again introduced into pABV437, resulting in pABV745, 746, 747 and 748, encoding N proteins lacking five, six, seven and eight C-terminal amino acids (Fig. 2). Transfection of their RNA transcripts into BHK-21 cells resulted in the expression of the structural proteins for all constructs, as detected by IPMA. After infection of PAMs with the culture supernatant of the transfected BHK-21 cells, we only detected expression of the structural proteins for vABV745 and vABV746. For mutants lacking the region encoding the C-terminal seven amino acids or more, no staining was observed in IPMA. These results indicate that the maximum region that can be deleted at the 3' end of ORF7 without abolishing the production of infectious virus comprises 18 nucleotides, encoding the six C-terminal residues of the N protein. The virus produced by this deletion mutant (vABV746) was found to express the set of N protein epitopes, as demonstrated by using our panel of MAbs (data not shown).
Deletion of seven but not of 32 nucleotides at the 5' end of the 3' UTR of LV is tolerated
In view of the observed nucleotide sequence variation in the 3' UTR of the PRRSV genome downstream of ORF7 (Fig. 1B), we also investigated how deletion of these nucleotides would affect the infection process. Deletions were again introduced by PCR mutagenesis and the PCR fragments were introduced into pABV437, directly behind the PacI site at the stop codon of ORF7. The first four nucleotides of the 3' UTR were left intact, as they are part of this PacI site. This resulted in the plasmids pABV693, which has a deletion of seven nucleotides, and pABV729, in which a deletion of 32 nucleotides occurs at the 5' end of the 3' UTR. BHK-21 cells transfected with transcripts of pABV693 expressed the structural proteins. However, BHK-21 cells transfected with transcripts of pABV729 did not express these structural proteins to levels detectable by IPMA, suggesting that RNA replication and/or transcription did not occur. Subsequent infection of PAMs with the culture supernatant of the BHK-21 cells that had been transfected with pABV693 showed expression of the structural proteins in IPMA 24 h after infection. These results demonstrated that at least seven nucleotides at the 5' end of the 3' UTR are dispensable for the virus to remain infectious.
Analysis of the stability and growth characteristics of vABV746 and vABV693 in vitro
In order to investigate whether the deletions in the viruses generated from pABV746 and pABV693 were maintained stably in vitro, these viruses were serially passaged on PAMs. After five passages, viral RNA was isolated from the culture supernatant and studied by genetic analysis. The RNA was reverse-transcribed and the region flanking the introduced deletions was amplified by PCR. Sequence analysis of the fragments showed that the introduced deletion was still present in both cases (data not shown) and that no additional mutations had been introduced in the flanking regions. These results indicated that the deletions had been maintained stably during in vitro passaging on PAMs.
The growth characteristics of viruses vABV746 and vABV693 were investigated by determining their growth curves and comparing them with that of wild-type vABV437. PAMs were infected with viruses from passage 5 at an m.o.i. of 0·05 and samples were taken from the culture media at various time-points. Virus titres were determined by end-point dilution on macrophages. As is clear from Fig. 3, no significant differences in growth rates could be observed between recombinant viruses and wild-type virus.
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Discussion |
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The dramatic effect of truncation at the seventh residue of the LV N protein was quite surprising and was not predicted by the sequence. The sequence of the nine C-terminal residues of the LV N protein is very different from that of the VR2332 isolate except for its high content of hydroxy amino acids. In the LV and VR2332 N proteins, six of ten and three of six residues, respectively, at the very C terminus are serines or threonines. The functions of this domain and of these particular residues are unknown. Two other arteriviruses, LDV and SHFV, also contain hydroxy amino acids at the extreme C terminus of their N proteins, namely 3/10 and 4/10 amino acids, respectively. In contrast, hydroxy amino acids are completely absent from the last ten amino acids of the EAV N protein. While coronavirus N proteins do generally have a relatively high serine content (711%; Masters & Sturman, 1990 ), the proportion of serines and threonines at their C termini is quite insignificant; in these viruses, this region is markedly acidic. Obviously, these variable characteristics do not allow predictions about the role of the C terminus of the N protein in the virus life cycle. The truncated N protein had the same antigenic profile as that of the wild-type N protein, since it reacted with all MAbs directed against antigenic domains of the N protein. This is consistent with observations by Meulenberg et al. (1998b
), who identified that domain D, the most C-terminal domain of N, is a conformation-dependent or discontinuous epitope that involves amino acids 5167 and 8090.
Virus particle production appeared to be blocked after truncation of the LV N protein by seven amino acids. This indicates strongly a defect at the level of virus assembly. For a Canadian PRRSV isolate, it has been demonstrated that non-covalent interactions between the C-terminal regions of N proteins are critical for formation of the isometric capsid protein (Wootton & Yoo, 1999 ). In a system expressing only the N protein, they showed that the last 11 amino acids were involved in these interactions. This might indicate that the C terminus of PRRSV is essential for nucleocapsid formation. Our study supports this idea. Other effects of C-terminal truncation of the N protein can, however, not be excluded, as the N protein has been implicated in various other processes, such as interaction with the viral RNA [Dea et al., 2000
; for mouse hepatitis virus (MHV), see Cologna & Hogue, 1998
; Molenkamp & Spaan, 1997
] and interaction with other viral proteins (for MHV, see Narayanan et al., 2000
). Since it has been described for MHV, the best-studied coronavirus, that a 29 amino acid deletion in the putative spacer region preceding the C-terminal domain of the N protein resulted in temperature-sensitive and thermolabile viruses (Peng et al., 1995
), we investigated whether our deletion mutants had similar characteristics; they appeared not to have these characteristics. Moreover, infectious virus was still not produced from the deletion mutants expressing truncated N proteins lacking seven amino acids or more after the incubation temperature was lowered to 30 °C. In an earlier study, we demonstrated that extension of the C terminus of the N protein by a nine amino acid sequence of the influenza virus HA protein significantly impaired virus growth (Groot Bramel-Verheije et al., 2000
). We could not establish whether this was caused by disturbance of virus assembly or disassembly, however. Again, these observations are consistent with the C-terminal region of the LV N protein being involved in NN interactions essential for the production of nucleocapsids during virus assembly.
RNA viruses have at their termini non-coding sequences that play essential roles in RNA replication and sg mRNA transcription. Mutations in these domains are likely to affect the virus life cycle. Consistently, when we introduced deletions in the 5'-terminal region of the LV 3' UTR, we found that removal of a small seven-nucleotide variable sequence was accepted, while removal of a somewhat larger, 32-nucleotide stretch was not. From the inability of the RNA transcripts to express the M and N proteins, we conclude that the defect probably resides in an effect on RNA replication or sg mRNA transcription. This suggests that this region of the 3' UTR contains an essential RNA signal. Our results are in accordance with studies on coronaviruses, which showed that the 5' terminus of the 3' UTR is essential in the initial processes of the virus life cycle (Hsue et al., 2000 ). No host or virus protein was found to bind this region of the viral RNA specifically. However, the exact function of this region remains to be elucidated. Since deletion of the 3'-most 27 nucleotides of the N gene did not abolish RNA replication, this region apparently does not contain a replication signal similar to the one observed within the 3'-terminal region of the EAV N gene (Molenkamp et al., 2000
).
In this study, we aimed to generate viable PRRSV mutants with maximal deletions at the target site. The viruses obtained were characterized in vitro and fulfilled the most important requirements; good growth and genetic stability. Because their growth characteristics in vitro on PAMs were identical to those of wild-type virus, virus production for in vivo studies can be accomplished easily. The growth characteristics in vitro do not necessarily correlate with or predict the behaviour of the virus in vivo. Thus, many vaccines used currently are attenuated in vivo, but show no differences in propagation in vitro (Yang et al., 1998 ). Therefore, only animal experiments will tell how these viruses behave in vivo, whether they are sufficiently attenuated and whether they induce immune responses that will protect against infection with virulent PRRSV.
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Acknowledgments |
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Footnotes |
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References |
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Received 2 May 2001;
accepted 7 August 2001.