1 School of Biology and Biochemistry, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK
2 Centre for Cancer Research and Cell Biology, The Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK
3 The Institute for Animal Health, Pirbright, Surrey, UK
Correspondence
B. K. Rima
b.rima{at}qub.ac.uk
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ABSTRACT |
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MAIN TEXT |
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Morbillivirus genomes range in size from 15 690 for CDV to 15 948 nt for PPRV (Bailey et al., 2005). They start with a 52 nt 3'-leader region followed by a conserved 5'-CTT-3' (in the positive sense) intergenic region and six genes encoding the nucleocapsid (N), phospho- (P), matrix, fusion, haemagglutinin and large (L) proteins. Variable-length 5'-trailer regions complete the genome. Two non-structural proteins V and C are encoded within the P gene. V is expressed by co-transcriptional RNA editing (Cattaneo et al., 1987
) and C from an overlapping reading frame (Bellini et al., 1985
). The negative-stranded RNA genomes are tightly encapsidated into a ribonucleoprotein (RNP) complex and in many, but not all, members of the subfamily Paramyxovirinae, 6 nt are likely to be associated with a single N protein as they comply with the rule of six (Calain & Roux, 1993
). The L protein is the RNA-dependent RNA polymerase. Together with the P protein, it is associated with the RNP, which forms the basic infectious unit.
Because RNPs cannot function as mRNA, generation of infectious virus (rescue) from cDNA requires co-expression of the N, P and L proteins in trans. Rescue of infectious virus has been described for MV by Radecke et al. (1995), RPV by Baron & Barrett (1997)
and CDV by Gassen et al. (2000)
. For all three viruses, the functionality of plasmids encoding N, P and L were assessed in rescue of mini-genomic constructs in which authentic morbillivirus genome termini flanked a reporter gene in the antisense. After their transfection into cells infected with the vaccinia vector MVA-T7 (expressing bacteriophage T7 RNA polymerase), together with helper plasmids that express the N, P and L proteins (Baron & Barrett, 1997
; Schneider et al., 1997
; Gassen et al., 2000
), mini-genome RNA becomes encapsidated by N protein, and P and L associate to generate an active replication/transcription complex. The absolute requirement for the N, P and L proteins allows complementation analysis between MV, CDV and RPV proteins.
During the development of our rescue system for CDV, significant difficulties were encountered with endogenous expression of the chloramphenicol acetyltransferase (CAT) enzyme in mini-genomic constructs containing the CDV genome termini. This forced us to use a MV-based mini-genomic construct to assess the functionality of CDV N, P and L proteins (Gassen et al., 2000). Hence, in this study we could only use MV and RPV mini-genomic constructs to assess functional complementation of the N, P and L proteins. The plasmids pEMC-Na, pEMC-Pa and pEMC-La, which encode MV N, P and L proteins, respectively, and p107MV() : CAT, which contains the authentic MV 3' and 5' non-coding genome termini, were kind gifts from Professor M. A. Billeter (Institute for Molecular Biology, Zürich, Switzerland). A similar plasmid to p107MV() : CAT was made for RPV (Fig. 1
). The CDV and RPV plasmids expressing N, P and L proteins were generated in our laboratories (Gassen et al., 2000
; Baron & Barrett, 1997
). In order to assess which combinations of the N, P and L proteins were able to co-operate in the mini-genome rescue assay, expression of the CAT reporter gene of p107MV() : CAT and p()RPVDICAT mini-genomic constructs was measured with all possible combinations of N, P and L proteins derived from RPV, CDV and MV (Fig. 2
). CAT activity was assessed at 20 h post-transfection by measuring the amount of [14C]acetylated chloramphenicol (Gassen et al., 2000
). Experiments were repeated three times and standard deviations were calculated. For negative controls, the L plasmid was omitted. CAT activity was also measured using an ELISA system (Baron & Barrett, 1997
) and, although the assay was less sensitive and had a smaller dynamic range, the results (data not shown) were entirely consistent.
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The rescue of infectious virus from full-length cDNAs was carried out essentially as described by Baron & Barrett (1997) for RPV and by Gassen et al. (2000)
for MV and CDV using the MVA-T7 system. Complementation analysis was performed for rescue of infectious virus with combinations of N, P and L protein-encoding plasmids. MVA-T7-infected HeLa cells for rescue of MV and CDV, and MVA-T7-infected Vero cells for RPV rescue, were transfected with the full-length anti-genomic plasmids and mixtures of the plasmids encoding the N, P and L proteins. There is no objective quantitative measure available for the rescue of infectious morbilliviruses. The numbers of primary syncytia in each of the six-well plates in which the transfections are carried out are too small and variable between various transfections to use them or titres of recovered p.f.u. as an accurate and reliable measure. Hence, the results were expressed by reporting the number of wells per six-well tray that gave primary syncytia and generated infectious virus after further passage (Table 1
). The results confirmed that homogeneous combinations of plasmids allowed rescue of infectious MV, RPV and CDV. Whether the differences in the efficiency with which various sets of plasmids were able to rescue their homologous virus (e.g. rescue of CDV by C+C+C) compared with rescue of other morbilliviruses (e.g. rescue of RPV by C+C+C) are significant would require further experimentation. Even the heterogeneous sets with the highest activity in mini-genome rescue did not rescue infectious virus in six attempts.
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CDV is very closely related to PDV. MV and RPV are highly related morbilliviruses with the second-highest levels of pair identity after CDV and PDV (Taubenberger et al., 2000). CDV diverges significantly from MV and RPV. However, differences in levels of similarity appear not to be significant in terms of the functional complementation analysis as, with one or possibly two exceptions, none of the heterologous mixtures of plasmids gave rise to significant levels of CAT expression. The failure to generate significant CAT activity in almost all heterogeneous sets involving proteins from two viruses made it unlikely that combinations in which all proteins were derived from different viruses would be functional in rescue.
Mini-genome assays measure the combined outcomes of (i) encapsidation of the T7 RNA polymerase transcript from the mini-genomic plasmid, (ii) subsequent replication of the mini-genomic analogue and (iii) its transcription as well as (iv) the translation of the mRNA into active enzyme. Successful replication is required for the generation of CAT activity in our assays, as it is not possible to rescue CAT activity from MV or CDV plasmids that do not conform to the rule of six (Gassen et al., 2000) or have a mutation in the B box of the anti-genome promoter for replication (L. J. Rennick, W. P. Duprex & B. K. Rima, unpublished results). Thus, the assay reflects the combined replication and transcription efficiency and does not measure the contribution of each process separately. Hence, these data do not formally rule out the possibility that heterologous sets of plasmids may be able to replicate but not transcribe negative-sense mini-genome RNAs.
The lack of a reliable in vitro transcription system for any of the morbilliviruses makes it impossible to assess the activities of viral proteins directly other than in the assays used here. The lack of functional complementation between the N, P and L proteins suggests that they are tightly tuned, functional components in each of the viruses. In contrast, their interaction with genomic and anti-genomic promoters is unrestricted, since the MV, CDV and RPV helper plasmids are all able to rescue mini-genomic as well as full-length constructs of all three viruses. Bankamp et al. (2002) showed that L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from a MV mini-genome than three wild-type L proteins and that this did not depend on whether the promoter sequences in the mini-genomes were derived from a wild-type or a vaccine strain.
Both mini-genomic reporter and full-length genomic constructs were rescued with homogeneous sets of proteins. The M+M+R combination was the exception as it showed low levels of activity in the rescue of the RPV mini-genome but did not rescue infectious virus in six attempts. There is functionally a significant difference between the two types of rescue events. Mini-genomic reporter rescue depends on a continual supply of proteins generated from the helper plasmids, whereas the rescue of genomic constructs only requires an initiation event by the helper plasmids. In the latter, once a single RNA transcript is encapsidated, replicated and transcribed, the system generates a self-sustaining RNP. Its replication may be more sensitive to dominant-negative effects of plasmid-derived homologous viral proteins due to, for example, the generation of non-physiological levels of N protein, which may compete with the N protein generated from the rescued genome for binding to the P, L and M (and V and C) proteins and interfere with the balance of replication and transcription. Hence, rescue of the full-length genome of one morbillivirus may be more efficient using a set of N, P and L plasmids from another morbillivirus than by its own complement. We have limited evidence to support this as in MV rescue primary syncytia were generated earlier and in higher numbers with the R+R+R plasmids than with the M+M+M set. It is difficult to model outcomes since a large number of proteinprotein interactions are involved, including: oligomerization of P to make P3 (or P4); interaction of P3 with No (the soluble form of N not in the RNP) to solubilize NoP3; interaction of NoP3 with RNA to make RNP; interactions between P3 and L to make P3L and for the latter to attach to the RNP (Karlin et al., 2003). At present we do not have an in vitro assay systems for quantitative evaluation of these interactions.
In summary, we have shown that morbilliviruses, either as whole genome or mini-genomic reporter gene constructs, are rescuable by homogeneous sets of plasmid-generated helper proteins from other morbilliviruses, but that heterogeneous sets, with one exception, do not perform well.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 December 2004;
accepted 17 December 2004.
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