Defence Research Establishment Suffield, Medical Countermeasures Section, PO Box 4000 Station Main, Medicine Hat, Alberta, Canada T1A 8K61
Virology Division, US Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, MD, USA2
Regulatory Division, Pasteur Merieux Connaught, Swiftwater, PA, USA3
Author for correspondence: Les Nagata. Fax +1 403 544 3388. e-mail les.nagata{at}dres.dnd.ca
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Abstract |
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Introduction |
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All alphaviruses share a number of structural, sequence and functional similarities, including a genome with two polyprotein gene clusters (reviewed in Strauss & Strauss, 1994 ; Schlesinger & Schlesinger, 1996
). The genomic organization of these viruses is conserved. The nonstructural proteins are translated directly from the 5' two-thirds of the genomic RNA. A subgenomic, positive-stranded RNA (the 26S RNA), transcribed from a negative-strand RNA, is identical to the 3' one-third of the genomic RNA and serves as the mRNA for the structural proteins (capsid, E3, E2, 6K and E1). The nonstructural proteins (nsP1, nsP2, nsP3 and nsP4) are also synthesized as a polyprotein and processed into the four nsPs by an nsP2 protease. Two versions of the nonstructural polyprotein are synthesized in alphavirus-infected cells, due to frequent readthrough of an opal codon between the nsP3 and nsP4 genes in several alphaviruses (Strauss et al., 1983
). The nsPs function in a complex with host factors to replicate the genome and transcribe the subgenomic mRNA. Alphaviruses have characteristic conserved sequences at the extreme 5' and 3' domains and the intergenic region (Ou et al., 1982
, 1983
; Pfeffer et al., 1998
). These conserved domains are required for virus growth and replication and are believed to be important in promotion of protein synthesis and the initiation of RNA-dependent RNA polymerase activity.
The serological relationship between WEE isolates has been determined by neutralization tests (Calisher et al., 1988 ). Additionally, several strains of WEE were typed by oligonucleotide fingerprinting and found to have greater than 90% nucleotide relatedness (Trent & Grant, 1980
). The N-terminal sequences of the nucleocapsid and the E1 and E2 glycoproteins have been determined, and the E1 and E2 proteins were found to have 82 and 71% identity, respectively, to those of SIN (Bell et al., 1983
). Hahn et al. (1988)
sequenced the 26S region of WEE strain BFS1703 and proposed that WEE originated as a hybrid virus, formed by recombination of an eastern encephalitis virus (EEE) and a SIN-like virus, probably during a co-infection event. They suggested that two crossover events occurred, one within the E3 gene, the other within the 3' nontranslated terminal region (NTR), resulting in a virus the nonstructural domain, intergenic region and capsid protein of which are similar to EEE and with envelope proteins showing similarity to SIN. Comparison of the 3' NTR from a number of alphaviruses revealed that conserved repeated elements are present (Ou et al., 1982
). Two 40 base direct repeats were identified from the 3' NTR of WEE (Hahn et al., 1988
). The sequence motif is found in nearly identical form in a number of other SIN-like viruses, although the position of these motifs and their number (two or three) varies with the individual virus (Ou et al., 1982
; Pfeffer et al., 1998
). Weaver et al. (1993)
sequenced part of the nonstructural domain (nsP2 and nsP3 genes) of WEE strain 5614, demonstrating that this area also shows similarity to EEE. Short regions within the nsP4 gene and the E1 protein/3' NTR have been determined for many WEE strains, allowing a preliminary assessment of the nucleic acid phylogenetic relationships within the WEE antigenic complex (Weaver et al., 1997
). Serological studies (Calisher et al., 1988
) and preliminary sequence determination (Cilnis et al., 1996
; Weaver et al., 1997
) of the HJ genome suggests that this is another closely related virus and is probably a descendant of the same recombinant virus ancestor as modern WEE. A highly conserved region of the alphavirus nsP1 gene has been identified and proved suitable for use in a PCR-based genetic assay for alphaviruses, including WEE (Pfeffer et al., 1997
). Phylogenetic analysis of this PCR fragment yielded results similar to those obtained by Weaver et al. (1997)
for a PCR fragment in the nsP4 gene.
Our interest in WEE covers a number of facets, including the development of subunit vaccines to WEE, the development of a passive immunization approach by using human/humanized antibodies and the diagnosis of WEE by immunological and genetic approaches. As a first step towards these goals, we report the first complete sequence of a WEE virus (strain 71V-1658) and discuss the significance of the data. The structural genes were expressed from a plasmid vector in the initial step towards the development of a subunit vaccine to WEE.
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Methods |
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Nucleic acid preparation.
Viral RNA used in construction of a WEE strain 71V-1658 library was prepared by the lysis of virus in 0·5% (w/v) SDS and extracted with caesium chloride/guanidium isothiocyanate (Sambrook et al., 1989 ). RNA was precipitated with sodium acetate and ethanol and stored at -70 °C. Prior to use, RNA was washed with 80% (v/v) ethanol, dried and dissolved in nuclease-free water (Promega). A cDNA library of WEE strain 71V-1658 was made by Invitrogen by the ligation of cDNA into the BstXI site of prepared pcDNAII vector and electroporation into electrocompetent DH1
F' Escherichia coli cells. Manipulation of RNA and DNA followed established procedures (Sambrook et al., 1989
; Ausubel et al., 1995
). Rapid plasmid preparations were made by using the Wizard plasmid purification kit (Promega). Large-scale plasmid preparations were made by the alkaline lysis protocol as modified by Qiagen. For PCR, RTPCR and DNA sequencing, oligonucleotide primer design was guided by information from other partially sequenced WEE strains (Hahn et al., 1988
; Weaver et al., 1993
) and from regions of sequence conservation (Ou et al., 1982
, 1983
). A catalogue of the sequences of primers used in this study can be supplied on request.
Construction of a cDNA clone encoding the structural genes.
The WEE cDNA library was screened by dot-blot hybridization (Sambrook et al., 1989 ) with 32P-labelled, random-primed RTPCR fragments as probes (Amersham). A 3100 bp insert, pcDW-12, was identified and corresponded to the 3' end of the 26S RNA. The missing 5' end of the 26S region was generated by RTPCR by using the primers WEE5'Sst1 (5' TCCAGATACGAGCTCATACT) and WEEP3 (5' CTTCAAGTGATCGTAAACGT). The 1500 bp SstI/NcoI-restricted fragment was inserted into the plasmid phT3T7BM+ (Boehringer Mannheim) to generate an XbaI site on the 5' end. The 1500 bp XbaINcoI fragment was excised, gel-purified and inserted into the XbaI and NcoI restriction sites of pcDW-12. The resulting clone, pcDWXH-7, encoded the complete 26S region of WEE 71V-1658.
Expression of the structural genes.
The 26S region insert from pcDWXH-7 was cloned into the mammalian expression vector pCI (Promega). The pcDWXH-7 plasmid was first linearized with HindIII, followed by a Klenow fragment reaction to fill in the 5' overhang. The insert was then excised with XbaI, gel-purified and ligated into XbaI/SmaI-digested pCI vector. The pCXH-3 expression vector was then transfected into Vero or CHO K1 cells by using the cationic lipid Lipofectamine (Gibco/BRL). Briefly, Vero or CHO K1 cells were grown to 3050% confluence in Costar 6-well plates. The monolayers were transfected with pCXH-3 in accordance with the manufacturers directions for a period of 5 h, followed by a further 29 h incubation after the addition of 5% DMEM. The monolayers were fixed in methanol:acetone (1:1) for 5 min and washed with PBS containing 0·1% (v/v) Tween 20 (PBS-T). The cells were incubated for 45 min at 37 °C with a 1:100 dilution (in PBS-T containing 3% BSA; PBS-TB) of concentrated cell supernatant from hybridoma cell lines expressing monoclonal antibodies to the WEE E1 (clone 11D2) or E2 (clone 3F3) proteins (L. P. Nagata, M. Long, G. V. Ludwig & J. Conley, unpublished data), followed by washing with PBS-TB. Monolayers were incubated with a 1:4000 dilution (in PBS-TB) of goat anti-mouse IgG/IgM (H & L) horseradish peroxidase conjugate (Caltag) for 45 min at 37 °C. After washing with PBS-T, 2 ml TruBlue HRP substrate (Kirkegaard & Perry Laboratories) was added and plates were incubated for a further 30 min at room temperature followed by microscopic examination.
DNA sequencing.
Automated sequencing of the 26S region was performed by using the ABI Prism Dye Terminator or Big-Dye Terminator cycle sequencing kits with plasmid templates according to the manufacturers instructions (PE-Applied Biosystems). Sequencing reactions were purified on Centri-Sep columns (Princeton Separations) and analysed on an ABI 373 or 310 automated sequencer. For the nonstructural region, template cDNAs were generated in a single-step integrated RTPCR procedure by using the Titan RTPCR kit (Boehringer Mannheim), following the manufacturers suggested protocols. RTPCR products were purified by using the QIAquick PCR purification kit (Qiagen) and sequenced (50100 ng DNA per reaction). The extreme 5' end of the genome was not sequenced in WEE 71V-1658. However, a 5' RACE reaction (Frohman et al., 1988 ) was used to obtain a cDNA fragment from the 5' terminus of WEE strain CBA87. Briefly, primer WEE559 (5' GGTAGATTGATGTCGGTGCATGG) was used to prime reverse transcription of the 5' terminus of the viral RNA. After poly(A) tailing of the cDNA with terminal transferase, a plus-sense primer (5' GTACTTGACTGACTGTTTTTTTTTTTTTTT) was used in conjunction with WEE559 to amplify the 5' terminus.
Nucleotide sequence analysis and assembly.
Sequence traces were edited manually and assembled by using the Seqman component of the Lasergene DNA analysis software (DNASTAR). Codon preferences and patterns were assessed by using the CodonUse and CodonFrequency programs, while the overall frequencies of mononucleotides and dinucleotides were calculated by using the Composition program of the Wisconsin package, version 9.0 [Genetics Computer Group (GCG), Madison, WI, USA]. Quantitative assessments of sequence similarities (nucleotide and amino acid) were calculated by preliminary alignment with the Pileup program, followed by manual alignment adjustment and analysis with the Distances program (GCG). Amino acid sequences aligned as described were used as the basis for the generation of phylogenetic trees (GCG). The GeneQuest module of the Lasergene program (DNASTAR) was used to predict and calculate RNA secondary structures at the ends of the genomic RNA by using minimum energy calculations. Multiple sequence alignments were accomplished by using the Clustal component of MegAlign (DNASTAR).
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Results |
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Expression of 26S region
Expression of the structural proteins of WEE 71V-1658 was accomplished by placing the structural genes under the control of the cytomegalovirus (CMV) promoter in the pCI vector. Expression was assayed after cationic liposome-mediated transfection of Vero or CHO K1 cells with pCXH-3 by using histochemical staining with E1- or E2-specific monoclonal antibodies (L. P. Nagata, M. Long, G. V. Ludwig & J. Conley, unpublished data), as shown in Fig. 6(a). The control cells transfected with pCI alone showed no staining (Fig. 6b).
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Discussion |
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Comparison of the sequence of WEE 71V-1658 with other partial sequences of WEE (Hahn et al., 1988 ; Weaver et al., 1993
) appears to indicate little variation at the nucleotide level among these viruses (Table 1
), showing an overall nucleotide sequence difference of 1·7% over 8624 bases. Given a calculated rate of divergence of 0·028% per year for the WEE E1 protein (Weaver et al., 1997
), the expected nucleotide divergence for a difference in isolation of 18 years between the strains should be 0·5% (71V-1658 isolated in 1971 and BFS1703 in 1953). The E1 protein itself showed a divergence of 1·5% in nucleotide sequence between 71V-1658 and BFS1703. The lower rate observed by Weaver et al. (1997)
could be due to greater conservation of structure at the C terminus of E1, from where the rates of divergence were calculated. Areas with high rates of divergence between WEE strains 71V-1658 and 5614 were observed at the 3' end of nsP1 and the 5' end of nsP4 (Table 1
). The relatively high interstrain divergence of nsP1 (4·5%) may be due the presence of a small hypervariable region, with 11 of 28 nucleotides changed in strain 5614 (Fig. 2b
). Variation in nsP4 occurred in a stretch of 21 nucleotides at the 3' end of the 5614 sequence; these residues were left out of subsequent comparisons (similarity with the EEE sequence was maintained in this region). Discounting the C-terminal region of nsP3 also gives a more accurate picture of the similarity of the nsP14 nonstructural region (Weaver et al., 1993
). The results of comparisons of nucleotide and amino acid sequences of WEE with other alphaviruses are shown in Table 1
, and are similar to those obtained for nsP2 and nsP3 of strain 5614, when compared with other alphavirus sequences. Phylogenetic analysis of the WEE 71V-1658 deduced amino acid sequences of nsP1, nsP4 and the nsP14 region, as related to other alphaviruses (Fig. 5
), illustrates the close relationship to EEE (HJ sequences were very limited for comparative purposes and were not included).
Assessments of codon-usage frequencies and the frequencies at which certain dinucleotides were found throughout the genome identified a number of statistical anomalies. The slight CpG dinucleotide deficiency described previously within other alphaviruses including WEE was confirmed in this study, at levels comparable to those reported previously (Weaver et al., 1993 ). The CpG under-representation is a typical feature of vertebrate genomes and is not seen in invertebrates. Viruses that infect two hosts, such as the arboviruses, might be expected to utilize an intermediate nucleotide bias, as indicated by the slight CpG under-utilization observed in alphaviruses (Weaver et al., 1993
). Pronounced under-representation of two other dinucleotides, UpA and CpC, was also observed within the WEE genome, a phenomenon noted throughout the genome, although the role of these codon preferences is unclear.
The 5' NTR sequence of WEE showed close phylogenetic affiliation to EEE and HJ, although the HJ sequence information was more limited. Ou et al. (1983) had previously predicted (on the basis of minimum free energy calculations) two hairpin structures at the 5' NTR of several alphaviruses, including SIN and EEE. Both structures are present in WEE, the first of which is a 5'-terminal hairpin structure (nt 230) similar to that calculated for EEE (Fig. 3a
, b
). The second is a dual hairpin structure (nt 137162, 165189) that is almost identical to that identified for EEE. The region between the terminal and dual hairpins can itself form a long hairpin structure and includes highly conserved stretches of 92 nucleotides (data not shown). The significance of these structures is currently unknown.
Previous reports (Hahn et al., 1988 ; Pfeffer et al., 1998
) suggested that WEE arose as a result of two recombination events between alphavirus-like ancestral viruses. The first recombination occurred near the junction of the E3 and capsid genes. The second recombination occurred 80 nucleotides from the 3' end of the genome. Evidence for the occurrence of the second recombination event is inferred from sequence similarities of the 3' NTR between WEE, EEE and SIN, in which WEE shows greater similarity to EEE (65%) than to SIN (50%) in the last 100 nucleotides of the 3' end. However, the apparent plasticity of the 3' NTR may simply reflect the selective pressures under which the nascent WEE virus evolved, resulting in rapid selection of 3' sequences that were more similar to EEE, and may not represent an actual recombination event as previously postulated.
The 3' NTRs of alphaviruses are characterized by widespread sequence divergence and yet contain small, strongly conserved motifs (reviewed in Strauss & Strauss, 1994 ; Pfeffer et al., 1998
). Analysis of the 3' NTR indicated the presence of double stemloop structures in SIN and WEE (Fig. 4a
, b
). Interestingly, the 40 base repeat found in SIN and WEE is contained within the double stemloop structure. SIN was found to contain three double stemloop structures and WEE was found to contain two. In SIN, the spacing between the three double stemloop structures was around 30 nucleotides, while in WEE, the distance was zero nucleotides. Additional alphaviruses were assessed and it is interesting to note that double stemloop structures were found in many of the WEE-and SIN-related viruses (SIN, Aura, Babanki, Ockelbo, Kyzylagach, Whataroa, WEE and HJ). The double stemloop structures found in SIN and WEE consisted of the
loop [AUGUA(U/C)UU] and the
loop (GCAUAAU) (Fig. 4b
). Surprisingly, while EEE does not have the 40 base repeat element found in SIN and WEE, it contains the
and
loop structures (Fig. 4c
). The significance of these loop structures conserved between SIN, WEE and EEE has yet to be elucidated, although previous studies have suggested a role in virus replication and/or host specificity (Kuhn et al., 1990
, 1991
). For example, a deletion of 26318 nucleotides from the 3' end of SIN resulted in reduced virus replication in mosquito cells but not in chicken cells (Kuhn et al., 1990
). In contrast, substitution of the SIN 3' NTR with the substantially different RR 3' NTR (which lacks the 40 base repeat and double stemloop structures) had no effect on the growth of the chimeric virus in mosquito cells, prompting the authors to suggest that host proteins interact with the 3' NTRs to cause differential host effects (Kuhn et al., 1991
).
The structural genes of 71V-1658 were placed under the control of the CMV promoter of pCI. Expression of WEE structural proteins from pCXH-3 when transfected into Vero cells indicated that the E1 (Fig. 6a) and E2 proteins (data not shown) were processed properly and were recognized by monoclonal antibodies that were isolated from mice immunized with inactivated whole virus particles. The binding specificities of these monoclonal antibodies have been determined previously by Western blot analysis and immunoprecipitation analysis (L. P. Nagata, M. Long, G. V. Ludwig & J. Conley, unpublished results). The evaluation of this plasmid as a vehicle for DNA immunization is currently under way, as a first step in the development of a potential DNA vaccine to WEE. Preliminary results indicate that WEE-reactive antibodies can be detected by ELISA when the pCXH-3 plasmid is administered intramuscularly to mice (unpublished results).
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Acknowledgments |
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Footnotes |
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b Present address: Dept of Sciences & Technologies, Medicine Hat College, 299 College Dr. SE, Medicine Hat, Alberta, Canada.
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References |
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Received 13 July 1999;
accepted 28 September 1999.