Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC E4-P, Room P4-26, PO Box 9600, 2300 RC Leiden, The Netherlands
Correspondence
Eric J. Snijder
E.J.Snijder{at}LUMC.nl
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ABSTRACT |
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MAIN TEXT |
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Arterivirus particles (Fig. 1a) are 5060 nm in diameter and contain a plus-sense RNA genome of 12·715·7 kb. The RNA is packaged by a small nucleocapsid protein (N) into a putatively icosahedral core structure that is surrounded by an envelope containing six viral membrane proteins (reviewed by Snijder & Meulenberg, 2001
). As in other arteriviruses, the main envelope proteins of EAV are the unglycosylated membrane protein M and the major glycoprotein GP5, which also contains major determinants for the induction of neutralizing antibodies. GP5 and M are present in the virion as a covalently linked heterodimer (de Vries et al., 1995
). In addition, virus particles contain four minor envelope proteins, the small unglycosylated envelope protein (E) (Snijder et al., 1999
) and three minor envelope glycoproteins named GP2b, GP3 and GP4, which were recently demonstrated to exist as a covalently associated heterotrimer in the virion (Wieringa et al., 2003
). The genome (Fig. 1b
; den Boon et al., 1991
) contains a large replicase gene (ORFs 1a and 1b) that is followed by partially overlapping genes (ORFs 2a, 2b, 3, 4, 5, 6 and 7), which encode the seven structural proteins in the order E-GP2b-GP3-GP4-GP5-M-N. As in other nidoviruses, expression of these proteins depends on the synthesis of a nested set of subgenomic mRNAs.
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Arterivirus mutants that are defective in the production of infectious progeny open new possibilities for vaccine development. Their genetic debility will render them capable of only a single cycle of replication, thus resulting in a self-limiting infection and preventing spread to other animals. Disabled virus mutants that produce non-infectious, (sub)viral particles during their abortive cycle of replication may be particularly powerful vaccine candidates, as described above for the EAV minor structural protein knockouts (Fig. 1c and Wieringa et al., 2004
). Upon entry into a susceptible cell, such EAV mutants will replicate their genome, express the full repertoire of non-structural proteins, generate subgenomic mRNAs, express all but one of the structural proteins and release significant amounts of defective particles that contain major antigens of the virus. Such a vaccine would stimulate both the humoral and cellular arms of the immune system and would offer possibilities for development of carrier vaccines, e.g. by incorporation of foreign sequences into one of the major structural proteins.
The large-scale production of such disabled infectious single-cycle (DISC) arteriviruses will require trans-complementing cell lines that can mediate the packaging of the disabled genome into an infectious virus particle by providing the missing structural protein. To investigate the possibility of expressing EAV structural proteins constitutively, we employed SinRep19 (Agapov et al., 1998), an RNA-based expression vector that is based on the genome of the alphavirus Sindbis virus (Sin). Essentially, this vector is an attenuated RNA replicon that expresses a selectable marker from one subgenomic RNA and a gene of choice from its second subgenomic RNA. SinRep19 replicons expressing different EAV structural protein genes were engineered by cloning PCR-amplified genes between the MluI and SphI sites of the vector. In vitro-transcribed vector RNA was transfected into BHK-21 cells, which support both Sin and EAV replication. A vector expressing green fluorescent protein (SinRepGFP; Agapov et al., 1998
) was used as a positive control. After 24 h, puromycin selection was applied to select for cells containing the RNA replicon. Although expression of the GP5, M and N proteins could initially be confirmed by immunofluorescence microscopy, positive cells did not divide and cell lines expressing these proteins could not be established. In contrast, cells transfected with replicons expressing E, GP2b, GP3, GP4 or GFP were found to become puromycin-resistant and cell division, albeit slightly delayed, was readily observed. To assess the stability of this type of RNA replicon-based cell line, the GP4-expressing cell line was passaged for over 3 months. The consensus sequence of the GP4 gene was determined directly from an RT-PCR product and was found to be unchanged, indicating that the majority of cells still expressed the wt GP4 protein.
A preliminary complementation experiment was performed by using the previously described EAV knockout mutants for E, GP2b, GP3 and GP4 expression (2ako, 2bko, 3ko and 4ko, respectively; Table 1). In these constructs, gene expression was prevented by mutagenesis of the translation initiation codon. A disadvantage of these mutations was the fact that they could revert readily to the wt sequence, resulting in the production of wt virus. In particular, the ORF2a- and ORF3-knockout mutations were found to be prone to reversion (data not shown; Table 1
). Thus, following the first indications for successful complementation (Fig. 2
a), improved knockout mutants for ORFs 2b, 3 and 4 were engineered by adding an internal deletion in these genes to the mutation that inactivated translation initiation. Care was taken not to disturb the regulatory RNA sequences that are involved in subgenomic RNA synthesis (Pasternak et al., 2001
). As the 5'- and 3'-proximal parts of ORFs 2b, 3 and 4 each overlap with neighbouring genes (Fig. 1b
), we chose to make these internal deletions in the central part of each gene, which does not overlap with another ORF. The sequences deleted from ORFs 2b, 3 and 4 corresponded to nt 995610088, 1050710562 and 1079810933 of the viral genome, respectively, and a HindIII restriction site was engineered at the position of each of the deletions. At the protein level, these mutations resulted in truncation of GP2b after Asp-45 and in-frame deletions from Val-68 to Asn-86 in GP3 and from Phe-34 to Leu-78 in GP4.
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When transfected into cell lines carrying the appropriate SinRep replicon (SinRepORF3 or SinRepORF4), successful complementation was evident from rapid spread of the infection. At 3648 h p.t., cytopathogenic effect (CPE) affecting the whole cell culture was observed readily with mutants 3ko3 and 4ko
4. Culture supernatants were now anticipated to contain DISC viruses, i.e. infectious virus particles containing the full complement of structural proteins, but carrying a mutant genome with a debilitating deletion in ORF3 or ORF4. Upon infection of non-complementing cells, they should engage in an abortive single cycle of replication. To prove this point, (dilutions of) culture supernatants were used to infect normal BHK-21 cells and, in immunofluorescence assays, they were indeed found to produce single-cell infections without spread of the infection to neighbouring cells (data not shown). However, upon infection of the corresponding complementing cell line with the same inocula, rapid virus spread and CPE were observed, confirming the dependence of the DISC virus on the trans-complementation provided by the cell line. These results were extended and confirmed in plaque assays (Fig. 2b
; Table 1
). As in the case of cells that were transfected with the corresponding mutant full-length RNAs (Fig. 1c
), cells infected with DISC viruses lacking expression of one of the minor structural proteins were found to produce non-infectious, subviral particles containing the GP5, M and N proteins (data not shown; Fig. 1c
).
Theoretically, the internal deletions in the 3ko3 and 4ko
4 viruses could be repaired during replication in the complementing cell line by RNA recombination involving the SinRep replicon RNA carrying the intact copy of the same gene. This might result in a subpopulation of virions containing a wt, instead of mutant, EAV genome. By using undiluted material and low dilutions of culture supernatants (from different time points after transfection) to infect normal BHK-21 cells or SinRepGFP-containing cells (Fig. 2b
), we screened extensively for such potential recombinants in immunofluorescence and plaque assays, but could not find any indications for infectious recombinants.
Finally, to demonstrate the potential for expression of heterologous sequences by DISC EAV, the GFP reporter gene was inserted at the site of the internal ORF4 deletion in mutant 4ko4, which should result in its expression from subgenomic mRNA4. When transfected into SinRepORF4 cells, the 4ko
4GFP construct produced DISC virus titres that were comparable to those of mutant 4ko
4 (data not shown). Cells infected with 4ko
4GFP DISC virus indeed expressed the GFP reporter gene, underlining the potential for the expression and transfer of foreign (marker) sequences by using this approach.
In summary, our SinRep19-based cell lines expressing EAV minor structural proteins constitute the first trans-complementation system for arterivirus gene function. Probably aided by the relatively small amounts of the minor structural proteins that are required for assembly of infectious arterivirus particles, the level of complementation achieved with the alphavirus vectors (virus yields up to approx. 107 p.f.u. ml1, only one log lower than those of the wt virus) can be considered quite efficient. Obviously, the use of other expression systems might further improve this result.
Although arteriviruses are clearly unrelated to the DNA-containing herpesviruses, this work provides an interesting parallel with the prior engineering of DISC herpesviruses that were used in studies on gene therapy (e.g. Rees et al., 2002; Todryk et al., 1999
and references therein) and herpesvirus vaccine development (e.g. Boursnell et al., 1997
; McLean et al., 1994
, 1996
). Likewise, DISC arteriviruses are potentially useful in vaccine development. In addition, the trans-complementing cell lines described here will be useful to study the molecular biology of the arterivirus minor structural proteins. Recent studies on arterivirus assembly have made it clear that this unique set of proteins probably plays a key role in virus infectivity and entry (Wieringa et al., 2003
, 2004
).
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ACKNOWLEDGEMENTS |
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Received 15 July 2004;
accepted 10 September 2004.