Measles Virus Section, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, MS-C22, Atlanta, GA 30333, USA
Correspondence
Paul A. Rota
prota{at}cdc.gov
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ABSTRACT |
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Present address: Australian Animal Health Laboratory, Geelong, Australia.
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INTRODUCTION |
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HeV and NiV have been assigned to a new genus, Henipavirus, within the subfamily Paramyxovirinae of the family Paramyxoviridae (Mayo, 2002). The number and order of genes in HeV and NiV (3'-N-P-M-F-G-L-5') are identical to those found in the respiroviruses and morbilliviruses. HeV and NiV have six transcription units encoding six structural proteins, the nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), glycoprotein (G) and polymerase (L). Like the morbilliviruses and respiroviruses, HeV and NiV are predicted to encode multiple proteins, designated C, V and W, from the P gene (Harcourt et al., 2000
). The genome of NiV is a single-stranded, negative-sense RNA that is 18 246 nt in length and the predicted gene-start, gene-end, RNA editing site and intergenic sequences are conserved compared with the respiroviruses and morbilliviruses (Vidal & Kolakofsky, 1989
; Harcourt et al., 2000
, 2001
; Wang et al., 2000
; Yu et al., 1998a
, b
). The genomic termini of NiV and HeV are highly conserved and complementary, as in other paramyxoviruses (Rima et al., 1995
).
All the viruses in the subfamily Paramyxovirinae have genomes whose lengths are multiples of six (Hausmann et al., 1996). Genomes whose lengths deviate from the rule of six do not replicate efficiently (Calain & Roux, 1993
) and it has been proposed that the templates for transcription and replication are nucleocapsids in which each nucleoprotein subunit is associated with six nucleotides of genomic RNA. Both NiV and HeV have genomes whose lengths are multiples of six, but it was not known whether these viruses conform to the rule of six.
Transcription and RNA replication in NiV are thought to follow the same strategy as in other, related, paramyxoviruses, although to our knowledge no studies have been conducted to address this question directly. Laboratory studies of NiV must be conducted at biosafety level (BSL)-4. Since this biocontainment classification restricts the type and frequency of research projects, alternatives to the use of infectious virus are highly desirable. Reverse genetics systems allow the study of the replication of negative-strand RNA viruses in vitro without the use of infectious NiV. The present study describes the development of an in vitro replication system for NiV that does not require infectious virus and can be used at BSL-2. Here, we use this in vitro replication system to determine the minimum subset of viral proteins required for NiV transcription and RNA replication, and to show that NiV obeys the rule of six and that the N, P and L proteins of NiV recognize cis-acting sequences in the genomic termini of HeV.
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METHODS |
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Construction of plasmids.
NiV RNA was extracted from infected cells as previously described (Harcourt et al., 2001). RT-PCR for the N and P genes was carried out using AMV reverse transcriptase and Taq polymerase (Roche), as described previously (Harcourt et al., 2001
). The L gene was amplified using AMV reverse transcriptase (Roche) and the Elongase enzyme mix (Invitrogen) according to manufacturer's recommendations for long RT-PCR. All primers were based on the published sequence of the NiV genome (Harcourt et al., 2001
; GenBank accession no. AF212302). Restriction sites included in primers are given in parentheses; primer sequences are available on request. The N open reading frame (ORF) was amplified with primers NF (SpeI) and NR (SalI) and ligated between the SpeI and SalI sites of the expression plasmid pTM1. The P ORF was amplified with primers PF (SacI) and PR (XhoI) and cloned into pTM1. In the plasmid expressing NiV P, the C ORF was silenced by mutating the two consecutive start codons to ACG. The NiV L gene was amplified as two fragments. The 5' half of the L gene was amplified with primers LF1 (NcoI) and LR1 (XmaCI). The 3' half of the L gene was amplified with primers LF2 (NheI) and LR2 (XmaCI). The 5' fragment was cloned into pTM1 using the NcoI and XmaCI sites and then the 3' fragment was added, using the internal NheI site at nt 14724 and XmaCI. The sequences of all plasmids were determined and sequence data were analysed with version 10.0 of the sequence analysis software package of the University of Wisconsin Genetics Computer Group (Devereux et al., 1984
). Where necessary, nucleotides that deviated from the consensus sequences of NiV were repaired using the ExSite mutagenesis kit (Stratagene).
A measles virus minigenome, pMV107-CAT, a gift from M. Billeter (Sidhu et al., 1995), was used as a template for the plasmid containing the NiV minigenome. A DNA fragment containing the two T7 terminator sequences and the hepatitis delta virus ribozyme sequence was amplified from pMV107-CAT with primers NGF1 (SacI) and NGR1. NGR1 included nt 141 of the 3' end of the NiV genome. Two additional rounds of PCR, each using the product of the previous amplification as a template, were used to extend the NiV sequence to nt 95. Primer NGR3 included a BamHI site (nt 90 in the NiV genome) allowing this fragment to be inserted into pUC19 using SacI and BamHI (pUC fragment 1). A second fragment was generated by amplification of the chloramphenicol acetyltransferase (CAT) gene from pMV107-CAT with primers NGF2 (BamHI) and NGR4. NGF2 included nt 90115 of the NiV genome, overlapping with pUC fragment 1, and NGR4 included nt 1814718187 of NiV. Two rounds of amplification were used to extend the NiV sequence to nt 18246, and the T7 promoter sequence was added to this fragment by reverse primer NGR6. NGR6 contained a HindIII restriction site, allowing the fragment to be inserted into pUC fragment 1 with BamHI and HindIII to produce pNiV-CAT. Additional nucleotides were added to primer NGR4 to generate plasmids encoding minigenomes with lengths ranging from 873 to 878 nt (see Fig. 3
). To generate the plasmid containing the HeV minigenome, the CAT gene was amplified from pNiV-CAT with primers HGF1 and HGR1. HGF1 included nt 61115 of the HeV genome and HGR1 included nt 1813418184 of the HeV genome. Two additional rounds of PCR, each using the product of the previous amplification as a template, extended the HeV sequence to include nt 1115 from the 3' genomic terminus and nt 1813418234 from the 5' genomic terminus by adding nucleotides to primer pairs HGF2 and HGR2, and HGF3 and HGR3. Primer HGF3 included a BseRI restriction site and primer HGR3 a HindIII site. The DNA fragment was cloned into pNiV-CAT, replacing the NiV termini and the CAT ORF. Additional nucleotides were added to primer HGR3 to generate minigenomes with various lengths as above. The DNA concentrations of the purified plasmid preparations were determined with the fluorochrome Hoechst 33258 (Gallagher, 1996
).
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Northern blot analysis.
Transfections were carried out as described above and 24 h after transfection, 10 µg actinomycin D (actinomycin D-mannitol; Sigma) ml-1 of medium was added to each well. RNA was purified 4245 h after transfection. Poly(A)+ RNA was isolated directly from cell lysates using Oligotex particles and buffers (Qiagen) according to the manufacturer's protocol, which was modified for batch procedure and followed by ethanol precipitation. For the analysis of minigenome replication, cytoplasmic extracts were treated with micrococcal nuclease (S7 nuclease; Roche) as previously described (Bankamp et al., 2002). Samples were additionally treated with RNase-free DNase (Ambion) to remove residual plasmid DNA.
One half of the RNA extracted from a 35 mm dish was separated by electrophoresis on a 3·7 % formaldehyde, 1·5 % agarose gel and transferred to a nylon membrane by vacuum blotting, then fixed by UV cross-linking. In vitro transcription of CAT gene-specific riboprobes (Bankamp et al., 2002) and hybridization and detection of the bands were carried out using the digoxigenin system (Roche) according to the manufacturer's recommended protocol. Hybridization signals were visualized by autoradiography.
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RESULTS |
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When transcribed by T7 polymerase, the plasmid containing the minigenome of NiV, pNiV-CAT, generated an 876 nt RNA containing exact copies of the 3' and 5' non-coding regions of the genomic RNA of NiV (Fig. 1). The 3' terminus of the minigenome RNA was created by self-cleavage mediated by a hepatitis delta virus ribozyme sequence. In the minigenome, all the genes of NiV were replaced with a negative-sense copy of the CAT gene, which was flanked by the predicted N gene transcription start sequence, the 5' non-translated (NTR) region of the N gene mRNA, the 3'NTR of the L gene mRNA and the L gene transcription stop sequence (Fig. 1
). The CAT translation start codon replaced the N start codon and the CAT translational stop codon replaced the L stop codon. pNiV-CAT contained a 4 nt insertion between the CAT stop codon and the start of the 3'NTR of the L gene to make the length of the minigenome RNA evenly divisible by six.
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To determine whether NiV obeys the rule of six, five additional minigenomes were constructed, which each differing in length by a single nucleotide (Fig. 3). One to six nucleotides were inserted at the junction between the CAT gene translational stop signal and the 3'NTR of the L gene mRNA (Fig. 1
), resulting in minigenome RNAs with sizes ranging from 873 to 878 nt (3, 2, 1, +1 and +2 nt in length relative to pNiV-CAT). pNiV-CAT encoded the only minigenome with a size that was evenly divisible by six. Expression of CAT protein was detected only when pNiV-CAT was co-transfected with the plasmids encoding NiV N, P and L proteins (Fig. 3
). No CAT protein was detected when the other minigenome plasmids were used in the replication assays. The results showed that efficient CAT expression was only achieved with pNiV-CAT and strongly suggest that NiV, like other members of the subfamily Paramyxovirinae, obeys the rule of six.
NiV and HeV have been placed in a new genus within the subfamily Paramyxovirinae (Mayo, 2002). Within this subfamily, it has been shown that it is possible to drive replication of a minigenome from one virus with support proteins derived from another virus from the same genus (Pelet et al., 1996
). To test whether the NiV N, P and L proteins could support replication and transcription of a minigenome containing the genetic termini of HeV, two minigenomes for HeV were constructed, pHeV-CAT and pHeV-CAT(-4). The lengths of the 3' leader, 5' trailer, 5'NTR of N and 3'NTR of L are identical in NiV and HeV. For HeV and NiV, the leader and trailer regions share 80 % and 90 % nucleotide identity, respectively, while the NTRs are more heterogeneous (5'NTR of N, 67 %; 3'NTR of L, 58 %). pHeV-CAT contained the same four nucleotides between the CAT protein stop codon and the 3'NTR of the L gene as pNiV-CAT (Fig. 1
) and its length was evenly divisible by six. pHeV-CAT(-4) lacked the four inserted nucleotides.
The replication efficiencies of the HeV mini-genomes were compared with those of pNiV-CAT and pMV107(-)CAT, a minigenome with the termini and non-coding regions of measles virus, a member of the Morbillivirus genus (Sidhu et al., 1995). In assays using the same ratio of support plasmids and minigenome plasmid as the experiments described above, the pHeV-CAT minigenome was efficiently transcribed by the NiV support plasmids (Fig. 4
a) and produced levels of CAT that were slightly higher than those produced with pNiV-CAT. No CAT expression was detected in cells transfected with pHeV-CAT(-4), the minigenome that did not conform to the rule of six. There was no detectable CAT in the cells transfected with pMV107(-)CAT indicating that the N, P and L proteins of NiV did not transcribe this minigenome. Northern blot analysis of poly(A)+-selected mRNA and micrococcal nuclease-resistant RNA demonstrated that the NiV N, P and L proteins supported transcription and replication of the minigenome RNA encoded by pHeV-CAT (Fig. 4b
). These results support the previous observations that the N, P and L proteins of one virus can support replication of other viruses within a genus and lend additional support for inclusion of HeV and NiV as a separate genus within the subfamily Paramyxovirinae.
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DISCUSSION |
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In this report, we used in vitro replication assays to demonstrate that, as for the other paramyxoviruses, the NiV N, P and L proteins are necessary and sufficient for both transcription and RNA replication of NiV. Replication was not affected by the C protein because the sequence of the P gene on the expression plasmid was modified to silence the C ORF. Of course, this conclusion that the N, P and L proteins are necessary and sufficient for transcription and replication must be stated with the caveat that other viral proteins, especially C, V and W, might play a role in virus replication. There is a growing body of evidence that the C and V proteins of paramyxoviruses regulate replication (Tapparel et al., 1997; Curran & Kolakofsky, 1999
). For example, the C protein of Sendai virus has been shown to inhibit both transcription and replication by down-regulation of the promoter of genomic RNA (Curran et al., 1992
; Cadd et al., 1996
), while the C and V proteins of measles virus inhibit transcription (Tober et al., 1998
; Reutter et al., 2001
). The V proteins of both Sendai virus and rinderpest virus inhibit replication (Horikami et al., 1996
; Baron & Barrett, 2000
). The minigenome replication assay described in this report will help to define the roles that the V, C and W proteins of NiV and HeV play in regulation of replication. So far, the V proteins of both HeV and NiV have been detected in infected cells (Shiell et al., 2003
; B. H. Harcourt, unpublished results), but the C and W proteins have not.
It was assumed that, like other paramyxoviruses, NiV and HeV adhered to the rule of six (Calain & Roux, 1993) because the lengths of their genomes are multiples of six. The results obtained from the minigenome replication assay support this hypothesis. Also, the hexameric phasing positions of the transcription initiation sites are conserved within a paramyxovirus genus (Kolakofsky et al., 1998
). NiV and HeV have a hexameric phasing pattern that is unique among the paramyxoviruses and the hexameric phasing positions of the RNA start sites for the P, G and L genes and for the P editing site are not found in any other paramyxovirus (Wang et al., 2000
; Harcourt et al., 2001
). The biological implications of these phasing patterns have not been explored.
NiV and HeV are the only members of the genus Henipavirus and these viruses have identical genome structures and a high level of nucleotide and amino acid similarity. The amino acid sequence identity between the N, P and L proteins of NiV and HeV is 92, 71 and 87 %, respectively. Overall, these viruses share a greater level of nucleotide similarity in their protein encoding regions (7080 %) than in the 5'- and 3'NTRs of each gene (4067 %). However, the genomic leader and trailer sequences have 80 % and 90 % similarity, respectively (Harcourt et al., 2001). Given this level of similarity in the region predicted to contain cis-acting regulatory sequences, it is not surprising that the NiV N, P and L proteins supported the replication of the HeV minigenome. The NiV replicase proteins did not support replication of a minigenome from the more distantly related morbillivirus, measles virus. Previous reports have shown that a Sendai virus defective-interfering genome and minigenome could be rescued by the N, P and L proteins from the respiroviruses, human parainfluenza viruses 1 and 3, but not with the corresponding proteins from measles virus or vesicular stomatitis virus (Curran & Kolakofsy, 1991
; Pelet et al., 1996
). The ability of the replicase proteins from one virus to recognize the genomic RNA of another virus appears to be restricted to viruses in the same genus. Therefore, the results of our experiments with the NiV minigenome replication assay lend additional support to the designation of NiV and HeV as a separate genus within the subfamily Paramyxovirinae.
While the results presented in this report suggest that NiV employs a replication strategy that is similar to those of other paramyxoviruses, the minigenome replication assay can now be used to address some of the unique genetic characteristics of the henipaviruses. These features include the large size of the 5'- and 3'NTRs in most genes, hexameric phasing and unique predicted functional motifs in the L protein (Wang et al., 2000; Harcourt et al., 2001
). Also, RNA editing in the P genes of HeV and NiV is unusual in that more P gene mRNAs with two or more G insertions are found in cells infected with HeV and NiV than in cells infected with other paramyxoviruses (B. H. Harcourt, unpublished results). The availability of the NiV minigenome and plasmids encoding functional replicase proteins also paves the way for the development of an infectious clone for NiV, which could be used to study the pathogenesis of this important viral pathogen.
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ACKNOWLEDGEMENTS |
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Received 30 September 2003;
accepted 13 November 2003.