Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Kent Ridge, Singapore 117543
Correspondence
Sek-Man Wong
dbswsm{at}nus.edu.sg
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ABSTRACT |
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INTRODUCTION |
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Hibiscus chlorotic ringspot virus (HCRSV) is a positive-strand RNA virus, a member of the Carmoviruses, and is composed of 3911 nt containing seven open reading frames (ORFs) with two subgenomes (Huang et al., 2000). The ORF encoding p23 is involved in the replication of RNA in kenaf protoplasts (Liang et al., 2002
).
The cis sequence elements involved in the in vitro and in vivo RNA synthesis of HCRSV have not been characterized. Preparation of a soluble, template-dependent RdRp complex of HCRSV would allow us to study which host or virus-encoded proteins, such as p23, p28 and p81, are involved in the replication of HCRSV. Here we have reported the successful purification of a template-specific RdRp complex from HCRSV-infected kenaf (Hibiscus cannabinus L.) and determination of the cis sequence requirements for initiation of minus-strand synthesis of HCRSV. In addition, the requirements for the two putative SLs for the accumulation of HCRSV RNA in vitro and in vivo were investigated. The replication of chimeric constructs containing substitution of the two SLs of HCRSV with the three SLs in the 3'UTR of TCV were carried out in vivo.
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METHODS |
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Purification of RdRp.
RdRp purification was carried out combining the methods of Nagy & Pogany (2000) and Song & Simon (1994)
. Frozen leaves (20 g) were ground with 10 g sterile white quartz sand in 60 ml buffer A [50 mM Tris/HCl, pH 8·2, 15 mM MgCl2, 10 mM KCl, 2 mM EDTA, 20 % (v/v) glycerol, 90 mM 2-mercaptoethanol] with 300 µl protease inhibitor cocktail (Sigma). After centrifugation at 300 g for 10 min, the supernatant was centrifuged at 43 000 g for 20 min. The pellet was resuspended in 8 ml pre-chilled buffer B [50 mM Tris/HCl, pH 8·0, 10 mM MgCl2, 1 mM EDTA, 6 % (v/v) glycerol, 1·2 M NaCl] containing 80 µl protease inhibitor cocktail and 50 µl 2-mercaptoethanol, followed by stirring for 20 min. The pellet was collected by centrifugation at 43 000 g for 20 min. The pellet in was resuspended in 2 ml buffer B containing 2 % taurodeoxycholic acid (TDC) and 1·5 M LiCl and stirred for 1 h. The preparation was centrifuged at 43 000 g for 20 min and the supernatant was centrifuged further at 100 000 g for 1 h. Subsequently, the supernatant was loaded on to a Sephacyl 500 HR column (1x75 cm) (Amersham Pharmacia Biotech), which was pre-equilibrated with buffer B containing 0·5 % Triton X-100. Fractions (1 ml) were collected and stored at 80 °C for up to 6 months without loss of activity. To obtain highly template-dependent RdRp (Miller & Hall, 1983
) and to remove endogenous RNA, micrococcal nuclease (nuclease S7; Roche) was added according to the method of Osman & Buck (1996)
at 30 °C for 30 min. The micrococcal nuclease was inactivated by adjusting the EGTA concentration to a final value of 15 mM. All steps were carried out at 4 °C unless otherwise stated.
Preparation of RNA templates.
For RdRp activity assay, the full-length in vitro transcripts were derived from pHCRSV223 (Huang et al., 2000), which was linearized by SmaI to provide the exact length of the viral sequence and transcribed by T7 RNA polymerase. The RNA products transcribed separately from the full-length cDNA clones of PVX, TMV, Odontoglossum ringspot virus (ORSV) and TCV were used to test template specificity of the extracted RdRp complex. CMV RNAs 1, 2, 3 and subgenomic RNA 4 were used as size markers and as templates for the RdRp assay. The
5'UTR plus-strand RNA template with nt 160 deleted was derived from pHCRSV223 after digestion with BstBI and SmaI, blunt-ending with T4 DNA polymerase, ligation into pBluescript SK() and transcription by T7 RNA polymerase. The
3'UTR was transcribed by T7 RNA polymerase from a cDNA construct
3'UTR 4() (Koh et al., 2002
). After digestion of the plasmid DNA templates with DNase I, the transcribed products were purified using the phenol/chloroform extraction method.
To investigate the effects of 3'-terminal nucleotides on minus-strand synthesis, a set of 3'UTR mutant constructs with different deletions or substitutions was generated by PCR using Pfu DNA polymerase (Promega). pHCRSV223 was used as the template and all resultant PCR products contained a T7 RNA polymerase promoter fused directly with the 5' 21 nt of the 3'UTR of HCRSV. Appropriate PCR primers (Table 1) were designed. All mutated regions were confirmed by DNA sequencing. The mutant DNA constructs were transcribed into RNA templates for the RdRp activity assay. To compare the replication efficiency of various mutants in vitro and in vivo, full-length mutants containing corresponding mutations were similarly constructed by PCR. The primer designated 5'Full(+) contained a T7 promoter fused immediately to the extreme 5'-end sequences of HCRSV.
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RdRp assay.
RdRp assays (40 µl) containing 1 mM ATP, 1 mM CTP, 1 mM GTP, 0·65 mM UTP and 0·35 mM DIG-11-UTP were carried out as described previously for TCV (Song & Simon, 1994; Nagy & Pogany, 2000
) with the exception that each reaction contained 3 µg template RNA and was incubated at 25·7 °C for 1·5 h. To verify that the reaction products generated were double-stranded, half of the products were treated with nuclease S1 (Nagy & Pogany, 2000
). The reaction was stopped by adding 40 µl TE buffer (10 mM Tris/HCl, pH 8·0, 1 mM EDTA) containing 0·2 % SDS and 50 mM EGTA, followed by phenol/chloroform extraction twice and ammonium acetate/2-propanol precipitation. The input and nascent RNA were pelleted together and rinsed with 70 % ethanol, dissolved in 6 µl nuclease-free water and subjected to formaldehyde-denaturing 1·5 % agarose gel electrophoresis. RNA was transferred to a nylon membrane (Roche) and detection was carried out as described in the manual of the DIG-labelling detection kit with chemiluminescent detection (Roche). To quantify the relative amounts of RNA synthesis, standard curves were prepared by diluting the labelled positive- and minus-strand HCRSV RNA derived from in vitro transcription in RdRp reaction buffer or hybridization buffer (DIG-Easy Hyb; Roche). Band intensities were quantified by densitometry as described previously (Lewandowski & Dawson, 1998
). All experiments were conducted independently at least three times.
Prediction of RNA structure.
The RNA secondary structure of the 3'UTR of TCV (Carrington et al., 1989) and HCRSV (Huang et al., 2000
) was predicted using MFOLD, version 3.0 (www.bioinfo.rpi.edu/applications/mfold) (Mathews et al., 1999
; Zuker & Jacobson, 1998
). Since the hairpin structure of TCV satellite RNA C has been determined by enzymic digestion and chemical modification and the first SL nearest to the 3' terminus of the TCV 3'UTR has already been predicted (Song & Simon, 1995b
), we compared the 3'UTR sequence of HCRSV with that of TCV and predicted two SL structures, which was in agreement with the MFOLD prediction.
Isolation and transfection of kenaf protoplasts.
Isolation and transfection of kenaf protoplasts were carried out as described previously (Liang et al., 2002). In each of the three experiments, 10 µg of in vitro transcripts of the full-length mutants was transfected into a suspension of 1x106 kenaf protoplasts using a PEG-mediated method. All experiments were carried out independently at least three times.
Northern blot analysis of mutants replicated in kenaf protoplasts.
At 24 h post-inoculation (p.i.), protoplasts were pelleted by centrifugation and 100 µl sterile water and 100 µl RNA extraction buffer [200 mM Tris/HCl, pH 8·5, 1 M NaCl, 1 % SDS (w/v), 2 mM EDTA] were added to homogenize the pellet. After three rounds of phenol/chloroform extraction, total RNA was recovered by adding three volumes of ethanol followed by centrifugation to precipitate the RNA. The pellet was washed with 70 % ethanol. Equal amounts of total RNA from each sample were analysed on a formaldehyde-denaturing 1·2 % agarose gel. After electrophoresis and blotting on to a nylon membrane, DIG-UTP-labelled HCRSV cRNA probes (nt 39113206 or nt 32063911) were used to detect plus- or minus-strand RNA synthesis by the mutants. All experiments were carried out independently at least three times.
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RESULTS |
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The ability of RdRp to utilize an endogenous RNA template to produce both single- and double-stranded RNA progeny has been documented previously (Plante et al., 2000; Singh & Dreher, 1997
). To investigate the activity of the crude RdRp preparation, the RdRp complex was assayed with endogenous RNA template purified from HCRSV-infected kenaf leaves and protein extracts from mock-inoculated kenaf leaves using the same RdRp purification procedures. RdRp activity was observed in the preparations from HCRSV-infected kenaf leaves, and both genomic and subgenomic RNAs were generated when endogenous RNA was present. Protein extracts prepared from the mock-inoculated kenaf leaves did not show any RdRp activity (Fig. 1
).
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RdRp preparation is template dependent
To investigate the specificity of the RdRp extract, RNAs from TMV, PVX, ORSV and CMV were extracted and used as templates to test the activity of the solubilized RdRp after pre-treatment with micrococcal nuclease to remove endogenous template. Results showed that no progeny RNA was produced from these templates (Fig. 2A) compared with that of HCRSV RNA [Fig. 2
, lane HCRSV (wt)]. Thus, the extracted HCRSV RdRp complex was highly template dependent in vitro. In addition, the 5'UTR was not required for the RNA synthesis of HCRCV in vitro, but the presence of the 3'UTR was necessary for its minus-strand synthesis (Fig. 2A
). The HCRSV RdRp complex also successfully supported TCV RNA synthesis in vitro (Fig. 2B
).
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Prediction of the secondary structure of the HCRSV 3'UTR
To investigate further why TCV RNA was recognized by HCRSV RdRp, the 3'UTR sequences of HCRSV (accession no. NC003608) and TCV (accession no. NC003821) were aligned. Approximately 50 % of the sequences were identical. The last 122 nt showed 56 % sequence alignment. In addition, comparison of the 3'UTR secondary structures of HCRSV and TCV using the MFOLD software (Mathews et al., 1999) showed that two SLs were predicted in the 87 nt at the 3'-proximal end of HCRSV (
G=33·2) and three SLs in the 122 nt at the 3' end of TCV (
G=52·7) (Fig. 4
). The predicted SL1 structures of HCRSV and TCV matched those of the satellite RNA C of TCV determined using a chemical method (Song & Simon, 1995b
).
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For the in vivo tests using kenaf protoplasts, replication of the corresponding mutants was analysed by Northern blotting. When deletion mutants of SL1 (SL1), SL2 (
SL2) or SL1+SL2 (
SL1+
SL2) were tested, no replication was detected (Fig. 5C
). When the stem structure of SL1 (SL1LL or S1RR) was disrupted, the two mutants were unable to replicate. However, when the SL1 loop was deleted (SL1
loop), replication was still detectable (Fig. 5C
).
For SL2, when the U and I loops were merged (SL2U : I) or deleted (SL2U
I), the mutants could not replicate. When the stem of SL2 was disrupted (SL2LL), the mutant could still replicate. On the other hand, when the U loop of SL2 was deleted (SL2
U), the mutant was unable to replicate. From these results, it was concluded that the U loop of SL2 is more important in replication than the stem, since minus-strand synthesis was not detected among these mutants (Fig. 5C
). The in vitro and in vivo assays thus indicated that both SL1 and SL2 are required for replication.
Replication of HCRSV containing TCV SLs
To investigate further the template recognition of HCRSV RdRp in kenaf protoplasts, constructs were generated in which HCRSV SLs were substituted with TCV SLs in the 3'UTR. Mutants of HCRSV : TCV SL1, HCRSV : TCV SL2, HCRSV : TCV SL3, HCRSV : TCV (SL1+SL2) and HCRSV : TCV (SL1+SL2+SL3) were constructed (Fig. 6A). At 24 h p.i., RNA blots showed that there was no detectable RNA synthesis generated from the HCRSV mutants substituted with single a TCV SL (Fig. 6B
, lanes 2, 3 and 5). In contrast, the HCRSV mutant substituted with two TCV SLs (Fig. 6B
, lane 4) replicated in protoplasts, but without generating subgenomic RNAs. This could be a result of the corresponding sequences of SL1 and SL2 of TCV in the HCRSV : TCV chimeric minus-strand RNA affecting the accumulation of its plus-strand RNA in protoplasts. While HCRSV : TCV (SL1+SL2+SL3) was able to replicate successfully with both genomic and subgenomic RNAs, the replication efficiency was reduced (Fig. 6B
). This result clearly indicated that the two SLs in the 3'UTR of HCRSV can be replaced by the three TCV SLs located in its 3'UTR. In addition, these data suggested that TCV SL3 might be involved in the initiation of subgenomic RNA synthesis.
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DISCUSSION |
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HCRSV RNA synthesis in vitro versus in vivo
Generally, an in vitro system of RNA synthesis can be used to dissect the mechanism and roles of proteins involved in RNA replication (Ahlquist et al., 2003). However, an in vitro system may lack important properties that can only be found in vivo, since some host proteins are involved in the replication process (Miller & Koev, 2000
). Therefore, in this study, we used an in vivo system to confirm the results obtained from the in vitro system. The requirement of the stemloop C (SLC) of BMV in vitro in minus-strand synthesis has been confirmed in barley protoplasts (Sivakumaran et al., 2003
). While the in vivo results could be predicted from those in vitro, the correlation of the amount of RNA synthesized in vitro was not proportional to that in vivo (Fig. 3C and D
; Fig. 5B and C
). For example, determination of the initiation site as the two 3'-terminal Cs in the 3'UTR of HCRSV was consistent in both in vitro and in vivo systems, but the amount of RNA synthesized in vitro varied compared with that obtained in vivo (Fig. 3C and D
). Also, the addition of one extra A at the 3' terminus reduced the in vitro replication to 13 % of the wt, while this mutant was unable to replicate in vivo (Fig. 3C and D
).
Some differences also existed in the influence of SLs on replication between the in vitro and in vivo systems. Deletion of the U loop of SL2 (SL2U) slightly affected the replication efficiency in vitro but the mutant could not replicate in kenaf protoplasts (Fig. 5
). For mutants SL1
loop and SL2LL, replication was apparently more reduced in protoplasts than in the in vitro assays (Fig. 5B and C
). The requirements of SLs for minus-strand synthesis of TMV carried out in vitro (Osman et al., 2000
) were the same as in tobacco protoplasts. However, differences were noted in the replication efficiency of some TMV mutants in vivo (Chandrika et al., 2000
). Similarly, insertion of 3 nt in the SLC of BMV RNA 3 showed reduced RNA synthesis in vitro and an even greater reduction in vivo. However, a nucleotide change in the SLC led to less RNA synthesis in vitro but not in vivo (Sivakumaran et al., 2003
). It is likely that other RNA elements may modulate RNA replication in vivo resulting in the differences observed in vitro. Other possible reasons for the differences may be that some host factors participate in the interaction between the RdRp replicase and different RNA templates (Osman et al., 2000
) or other host factors, which may contribute to the initiation of minus-strand RNA synthesis by recognizing and utilizing the 3' end of positive-strand RNA (Ahlquist et al., 2003
). Therefore, cis sequence elements and structural requirements for minus-strand RNA synthesis need to be analysed using a combination of in vitro and in vivo approaches.
Essential role of the 3'-terminal CCC nucleotides in minus-strand RNA synthesis
The HCRSV RdRp complex was shown to be template dependent and able to initiate RNA synthesis in vitro. The in vitro assay showed that the 5'UTR was not involved in minus-strand RNA synthesis, but that the 3'UTR was (Fig. 1A). Both in vitro and in vivo experiments showed that the CCC-3' sequence is crucial for minus-strand RNA synthesis, since its initiation began at the 3'-terminal two Cs. When the single 3'-terminal C was removed in vitro or in vivo, the template could still be replicated. However, when the two Cs at the 3' terminus were deleted, no RNA synthesis was detected either in vitro or in vivo. When the 3'-terminal C was substituted with an A, template activity was reduced but detectable, with the RNA synthesis efficiency being reduced to 41 % and 7 % of the wt in vitro and in vivo, respectively (Fig. 3C and D
). Our results are similar to those obtained from BMV (Chapman et al., 1998
), TMV (Osman & Buck, 1996
) and TYMV (Singh & Dreher, 1997
), where initiation of minus-strand RNA synthesis started opposite the two Cs and removal or substitution of the terminal A in the CCA box reduced minus-strand synthesis.
The wt TMV can tolerate up to seven extra nucleotides at the 3' terminus without significant loss of infectivity (Dawson et al., 1986) and an additional C at the 3' terminus of Cistrus tristeza virus (CTV) also did not affect infectivity (Satyanarayana et al., 2002
). For HCRSV, an additional A added to the 3' terminus resulted in a severe reduction in RNA synthesis (Fig. 3C and D
), indicating that CCC-3' is a key element for minus-strand synthesis. The presence of an extra A at the terminus of the 3'UTR might affect the accessibility of HCRSV RdRp to the template.
For TCV, the initiation site of minus-strand synthesis has not yet been identified (Song & Simon, 1995b). In TYMV RNA, the 3' tRNA-like structure presents the CCA-3' in a conformation that is easily accessible to the replicase (Dreher, 1999
). The importance of CCCA-3' was demonstrated in the end-to-end replication by Q
replicase (Tretheway et al., 2001
). From our experiments, we speculate that CCC-3' may also be involved in forming a conformation for replicase access both in vitro and in vivo in HCRSV. However, the presence of a CCC-3' terminal sequence alone is not sufficient for RNA synthesis. The two SLs in the 3'UTR must also be present and they cannot be disrupted or deleted for RNA synthesis to occur (Fig. 3C and D
).
Crucial role of the predicted SLs in minus-strand RNA synthesis
In general, RNA viruses have a specific structure at the 3' end of the genome that is required for initiation of minus-strand RNA synthesis (Ahlquist et al., 2003). In this study, the 3'UTR of HCRSV was predicted to constitute two SLs, with six unpaired nucleotides located at the 3' terminus. The first SL was very similar in structure to the single SL previously described as a minimum promoter for minus-strand synthesis of TCV satellite RNA C (Song & Simon, 1995b
). This suggested that the two predicted SLs in HCRSV might also play a similar role(s) to the TCV satellite RNA C. However, when SL1 or SL2 was deleted, no RNA synthesis was detected in vitro or in vivo, demonstrating that both SLs were indispensable for RNA synthesis of HCRSV (Fig. 5B and C
). This is different from the TCV RdRp interaction with the single SL of satellite RNA C in minus-strand synthesis. Also, disruption of certain SLs in BMV (Dreher & Hall, 1988
; Chapman & Kao, 1999
), TYMV (Deiman et al., 1997
), AMV (Olsthoorn et al., 1999
) and CTV (Satyanarayana et al., 2002
) has resulted in a reduction in minus-strand synthesis, although not inhibiting RNA synthesis completely. In another case, elements located hundreds of nucleotides upstream of the TCV 3'UTR were found to be needed for efficient replication (Carpenter et al., 1995
). However, not all of the 10 SLs of CTV are critical for RNA synthesis (Satyanarayana et al., 2002
). In this study, the two SLs of HCRSV were demonstrated to be absolutely essential for RNA synthesis. In addition, when the U loop located on SL2 was deleted (SL2
U), no RNA synthesis could be detected either in vitro or in vivo. When both the U and I loops were merged (SL2U : I), there was also no RNA synthesis. In contrast, when the left-hand side stem of SL2 was disrupted by replacing the right-hand side stem with the left-hand side stem nucleotides (SL2LL), marginal synthesis was detected (Fig. 5B and C
). Therefore, the U loop is an essential structure for RNA synthesis. The secondary structures of SLs in the 3'UTR of RNA viruses have been shown to be required for protein binding (Lai, 1998
). For example, the hpE loop of AMV is not essential for RNA synthesis, whereas the stem and base pairing of the lower tri-loop are essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site, similar to the process of subgenomic transcription (Olsthoorn & Bol, 2002
). It may be that failure to bind replication factors by HCRSV mutants with disruption or deletion of SL1 or SL2 renders them unable to initiate minus-strand synthesis.
Substitution of the two HCRSV SLs with three TCV SLs generates both genomic and subgenomic RNAs
In this study, we demonstrated that HCRSV RdRp could support synthesis of TCV genomic RNA in vitro. We then examined whether the replacement of the two HCRSV SLs with the three from the TCV 3'UTR was functional with the HCRSV RdRp complex in vivo. Sequence alignment showed that the 3'UTR of TCV shares 50 % nucleotide identity with the HCRSV 3'UTR. The TCV 3'UTR folds into three predicted SLs. Our results showed that substitution of SL1 or SL2 of HCRSV individually with one of the SLs of TCV resulted in no RNA replication. However, substitution of both SL1 and SL2 with those of TCV resulted in replication of the genomic RNA of HCRSV. Therefore, it seems that the SL1 and SL2 cis-acting sequences are not organized as modules for RNA synthesis. In addition, substitution of both SL1 and SL2 with all three SLs from TCV generated both genomic and subgenomic RNAs of HCRSV (Fig. 6B). This indicated that recognition of heterologous 3' replication elements may be extended to other carmoviruses as a result of their structural conservation. This supports the view that higher-order structure, rather than the primary sequence of the 3'UTR, is an important replication element (Koev et al., 2002
).
When the 3'UTR of BMV RNA3 was replaced by the 3'UTR of CMV-Fny, this resulted in BMV replication in vivo (Rao & Grantham, 1994). Although there was only 56 % sequence identity between the two 3'UTRs, CMV-Fny replicase could synthesize RNA from the BMV tRNA-like structure in vitro (Sivakumaran et al., 2000
). However, substitution of the BMV RNA 3 SLC with that of CMV did not result in replication in vivo, indicating that the requirement of the BMV SLC is highly specific to BMV replicase (Sivakumaran et al., 2003
). Reciprocal experiments of TCV SLs replaced with those of HCRSV could be carried out using TCV RdRp or in protoplasts of a common host.
It was interesting to note that in the absence of TCV SL3, no subgenomic RNA was synthesized (Fig. 6B). Perhaps the subgenomic RNAs of HCRSV were generated but degraded due to lack of encapsidation by the HCRSV capsid protein. However, the presence of genomic RNA indicated that it was not degraded and therefore it is unlikely that encapsidation and RNA degradation of subgenomic RNAs were involved. When the two SLs of HCRSV were replaced by the SL1 and SL2 of TCV to form HCRSV : TCV(SL1+SL2), no subgenomic RNA was detected, compared with HCRSV : TCV(SL1+SL2+SL3), indicating that TCV SL3 may be involved in the synthesis of both subgenomic RNAs. The predicted secondary structures of SL1 and SL2 of TCV and HCRSV were very similar (Fig. 4A and B
). In SL1, both TCV and HCRSV possessed a long stem (10 versus 9 bp) and a small loop (5 versus 7 nt), respectively. The similarities of SL2 included the same number of base pairs (9 nt) in the stem, the exact sequence of the I loop (12 nt), identical nucleotides in the upper stem (3 versus 2 bp) and same size (4 nt) of the U loop (Fig. 4A and B
). Since both SL1 and SL2 of TCV and HCRSV are similar in sequence and/or in structure and HCRSV SL1 and SL2 could support subgenomic RNA synthesis, it is puzzling that HCRSV : TCV (SL1+SL2) could not support replication of subgenomic RNAs of HCRSV in vivo. Additional functions of HCRSV SL1 and SL2 and replication of subgenomic RNA will be delineated in comparative studies with TCV.
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ACKNOWLEDGEMENTS |
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Received 28 November 2003;
accepted 6 February 2004.