Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, LUMC E4-P, PO Box 9600, 2300 RC Leiden, The Netherlands
Correspondence
Willy Spaan
W.J.M.Spaan{at}LUMC.NL
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ABSTRACT |
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INTRODUCTION |
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Two different mechanisms for translation initiation are found within the Flaviviridae. While the polyprotein of flaviviruses is translated in a cap-dependent fashion, translation of the HCV, pestivirus and GBV-B polyprotein is initiated by internal entry of ribosomes, which is promoted by the 5'NTR (Poole et al., 1995; Rijnbrand et al., 1995
, 2000
; Tsukiyama-Kohara et al., 1992
; Wang et al., 1993
). These 5'NTRs contain IRES elements that have similar structures. Four major structural domains can be distinguished in the HCV 5'NTR (Fig. 1a
) in which the 5' border of the IRES element was mapped between nt 38 and 46 (Honda et al., 1999
; Reynolds et al., 1995
; Rijnbrand et al., 1995
) and the 3' border at the AUG start codon was mapped at nt 342 (Rijnbrand et al., 2001
). Domain III comprises the core of the IRES and participates in the formation of a pseudoknot structure essential for IRES activity (Wang et al., 1995
). Although there is some controversy about the importance of domain II in IRES-dependent translation, it is generally considered to be essential for its activity (Honda et al., 1996b
; Reynolds et al., 1996
; Rijnbrand et al., 1995
). Stemloop IV is not essential for IRES activity but the stability of this structure inversely correlates with efficiency of translation (Honda et al., 1996a
). The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved (Fig. 1a, b
), the main differences between their structures being the absence of stemloop IV, an additional stemloop IIId and an additional stemloop I in pestiviruses (Fletcher & Jackson, 2002
; Honda et al., 1999
; Rijnbrand & Lemon, 2000
). Although the structures are similar, their sequences differ significantly from one another. A few short stretches of high sequence identity can be found, mainly in unpaired regions (Fig. 1a
). HCV and pestivirus IRES elements can be distinguished from other viral IRES elements by their mechanism of ribosome entry (reviewed by Hellen & Pestova, 1999
; Hellen & Sarnow, 2001
). The 40S ribosomal subunit is able to bind specifically to the IRES in the absence of any additional translation initiation factors, in such way that the initiation codon is placed directly in the ribosomal P site. Toeprinting of 40S subunits to HCV and CSFV IRES elements showed that ribosomal binding consists of at least two distinct steps: first, the initial attachment, which involves stem 1 of the pseudoknot structure and the loop of subdomain IIId (IIId1 for CSFV); and second, the placement of the AUG start codon in the ribosomal P site. Determinants for this step include stem 2 of the pseudoknot and domains II and IIIa. Besides the small ribosomal subunit, additional translation factors, like eIF3, which interacts specifically with domain IIIb, and noncanonical factors, like PTB, hnRNPL and La, are recruited. These latter proteins are not essential for IRES activity (reviewed by Hellen & Pestova, 1999
; Hellen & Sarnow, 2001
).
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METHODS |
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Construction of dual-luciferase reporter plasmids.
All dual-luciferase reporter constructs were generated by a (fusion) PCR-based strategy from either pDualLuc-HCVwt or pDualLuc-HCVwt/C, which contain consecutively the T7 promoter, the firefly luciferase ORF, HCV genotype 1a (nt 1342 or 1389), the Renilla luciferase ORF, the HCV genotype 1a 3'NTR, the hepatitis
ribozyme and the T7 terminator in a pBluescript backbone (Fig. 2a
). Table 1
gives an overview of the exact composition of the different dual-luciferase reporter constructs with respect to the 5'NTR preceding the Renilla luciferase ORF. All fragments generated by PCR were sequenced completely upon cloning.
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In vivo expression studies.
The vaccinia virus recombinant vTF7-3 (Fuerst et al., 1986), which expresses the T7 RNA polymerase, was used to infect Huh-7 cells. Cells were grown in 12-well plates until
75 % confluency was reached. Cells were then infected at an m.o.i. of 10 in DMEM containing 1 % FCS. After incubating at 37 °C for 1 h, the cells were washed twice with PBS. For each set of two (duplo) wells, 100 µl LipofectACE reagentOptiMEM (1 : 19) mixture was added to 100 ng of plasmid DNA in 100 µl OptiMEM and incubated for 15 min at room temperature. OptiMEM (800 µl) was then added to the mixture and 450 µl was added to each of the two wells. At 7 h post-transfection, cells were rinsed with PBS and lysed using 250 µl passive lysis buffer (Dual-Luciferase Reporter assay, Promega). Cells were stored at -80 °C and thawed just before reporter activities were measured with the dual-luciferase reporter assay using a luminometer. In all experiments, n
4.
In vitro transcription.
Transcripts of HCV replicon cDNA were generated and treated essentially as described by Lohmann et al. (1999).
Electroporation and selection of G418-resistant cell lines.
Electroporation of Huh-7 cells and colony counting was done as described by Lohmann et al. (1999). To correct for differences in transfection efficiency, 500 ng pGL3 control-plasmid (Promega), which carries the firefly luciferase gene under the control of the SV40 promoter/enhancer sequences, was included in every electroporation. For each replicon tested, n
3.
RNA labelling in cell culture.
Of the various replicon cell lines, 0·5x106 cells were incubated for 14 h in the presence of 50 µCi [3H]uridine and 2 µg actinomycin D in 750 µl medium. Total RNA was isolated using Trizol reagent (Invitrogen), according to the manufacturer's instructions, and analysed by denaturing agarose gel electrophoresis. Gels were prepared for autoradiography as described by Bredenbeek et al. (1993).
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RESULTS |
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For domain II, two chimeras were constructed: chimera 1, in which the apical part was replaced; and chimera 2, in which the complete domain II was replaced. For domain III, a set of four chimeric 5'NTRs was generated: chimeras 3 and 4, in which, respectively, the apical part and the complete subdomain IIIb were swopped; chimera 5, in which subdomains IIIa, IIIc, IIId and the basal stem of domain III were swopped; and chimera 6, in which the complete domain III, with the exception of subdomains IIIe and IIIf, was swopped. Furthermore, two chimeras were constructed with a combined substitution of domains II and III: chimera 7, in which domains II and III were exchanged; and chimera 8, in which, in addition to domains II and III, the intermediate region was exchanged, thereby disrupting the pseudoknot structure.
We performed a two-step analysis of these 5'NTRs: (a) in a translation assay, which provided the possibility to study IRES efficiencies independent of possible effects on replication; and (b) in a replication assay, in which we studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated.
Activity of the HCV/CSFV chimeric IRES elements
The translation activity of the HCV/CSFV chimeric 5'NTRs was analysed in a bicistronic context (pDualLuc-HCVwt, Fig. 2a) in which the firefly luciferase is translated by a 5' end-dependent mechanism and the Renilla luciferase is expressed under the control of the IRES element under study. Luciferase expression of the various constructs was analysed in DNA transfected and vTF7-3-infected Huh-7 cells. The relative translation efficiency of the IRES elements was calculated as the ratio of the luciferase activities (Fig. 2b
).
We observed that exchange of the apical part of domain II for its CSFV counterpart (chimera 1) had no effect on translation, while replacing the complete domain (chimera 2) resulted in a 25 % reduction in IRES activity. The exchange of almost the entire domain III (chimera 6) resulted in an increase in translation activity (75 %), whereas the exchange of several stemloop III subdomains in chimeras 3, 4 and 5 showed an inhibitory effect of 50, 75 and 95 %, respectively. When domains II and III were replaced simultaneously (chimera 7), a slight increase of activity was observed, while exchange of both stemloops in combination with the intermediate sequence (chimera 8) rendered the IRES element inactive.
The chimeric 5'NTRs that were translationally active, i.e. chimeras 14, 6 and 7, were selected for further analysis in a replication assay based on the HCV subgenomic replicon system. However, since we observed that an HCV replicon lacking HCV nt 342389, encoding the amino-terminal 16 aa of core protein, was not functional (see below), we had to include this sequence in our chimeric 5'NTR expression plasmids. To analyse whether these extended plasmids had similar translational characteristics as those of the initial plasmids, we analysed them in the translation assay (Fig. 2c). The translation characteristics of the 5'NTRs including the first 48 nt of the HCV ORF were shown to be essentially similar to those obtained with the corresponding plasmids lacking these nucleotides. Subsequently, we analysed the selected chimeras in the replication assay.
Activity of HCV/CSFV chimeric 5'NTRs in HCV subgenome replication
The effect of the chimeric 5'NTRs on HCV RNA replication was analysed using the G418-selectable replicon cell culture system for which it was shown that the number of G418-resistant colonies directly reflects the efficiency with which a replicon multiplies in cells (Bartenschlager & Lohmann, 2001; Lohmann et al., 1999
). In this replicon, the HCV 5'NTR precedes the neomycin ORF, which is expressed as an amino-terminal fusion protein with 16 aa of the core protein. The encephalomyocarditis virus (EMCV) IRES mediates the translation of the HCV NS3NS5 ORF, which is followed by the 3'NTR. The cell culture-adapted HCV genotype 1b-based pFK-I389neo/NS3-5'/5.1 cDNA (Fig. 3a
) was used for our studies. This replicon has a high sensitivity of detection, thereby enabling the detection of mutants with a low efficiency of replication (Krieger et al., 2001
). Since the HCV/CSFV chimeric cassettes, as tested in the translation assay, lack HCV nt 342389 encoding the amino-terminal 16 aa of the core protein, we first analysed whether the pFK-I389neo/NS3-5'/5.1 replicon was still functional without this sequence. We observed that the presence of this region is a prerequisite for replicon activity (data not shown). Therefore, nt 342389 were included in our chimeric cassettes.
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Subsequently, we analysed the relative replication efficiency of replicons containing the chimeric 5'NTRs that were translationally active (Fig. 3b). The number of colonies found for each replicon is normalized to the number of colonies found for the genotype 1b replicon with the 5'NTR from genotype 1a (Fig. 3b, 1a
5'NTR). Mutants containing the apical part of domain II (chimera 1) showed a severe reduction in colony formation to 1 % of the level seen with genotype 1a 5'NTR, while mutants containing the complete CSFV domain II (chimeras 2 and 7) failed to yield G418-resistant colonies at 34 weeks after electroporation. The mutant in which the apical part of subdomain IIIb was replaced by the corresponding part of CSFV (chimera 3) gave a reduction in colony formation to 70 % of the level seen with genotype 1a 5'NTR, whereas mutants containing either CSFV subdomain IIIb (chimera 4) or CSFV domain III (chimera 6) showed a stronger reduction to 20 and 5 %, respectively. In all cases, these colonies were, on average, smaller in size (data not shown).
Characterization of chimeric replicon cell lines
To verify that the selected G418-resistant colonies contained replicating viral RNA and not integrated DNA, we performed a [3H]uridine labelling in the presence of actinomycin D on cell lines generated from isolated clones of chimeras 1, 3 and 4 and analysed the resulting radiolabelled RNAs (Fig. 3c). Synthesis of cellular RNAs was properly blocked, while replication of HCV RNA was shown to be resistant to actinomycin D. The radiolabelled replicon RNAs all had the correct size. It has been shown that the efficiency of colony formation is determined primarily by the initial level of RNA replication upon transfection and that similar levels of viral RNA are obtained in selected cell lines regardless of this initial replication efficiency (Krieger et al., 2001
). Therefore, the RNA synthesis observed for these established colonies cannot be linked to the differences in colony formation (Fig. 3
b).
To verify that the replicating RNA present in the selected colonies contains the chimeric 5'NTR sequence, total RNA was isolated from the cell lines of chimeras 1, 3 and 4 and the corresponding cDNA was sequenced. The replicating RNAs did contain the chimeric 5'NTR sequence (data not shown).
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DISCUSSION |
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The translation activity of the chimeric 5'NTRs in which stemloops II (chimera 2), III (chimera 6) or II and III of HCV (chimera 7) were replaced by corresponding domains of the CSFV 5'NTR demonstrates clearly that IRES activity is regulated by higher order structures rather than primary sequence. Furthermore, the observations with the different stemloop III chimeras in which only subdomains of stemloop III were replaced by CSFV sequences (chimeras 3, 4 and 5) suggest that interactions within stemloop III are important for IRES activity. These substitutions could have resulted in subtle structural changes, thereby destabilizing/stabilizing the RNA tertiary structure or influencing the interaction with essential translation factors like the 40S small ribosome subunit and eIF3 (Hellen & Sarnow, 2001; Rijnbrand & Lemon, 2000
). Chimeras 7 and 8 confirmed the requirement of the pseudoknot structure for a functional IRES (Wang et al., 1995
).
The reduced translation observed with chimeras 24, 6 and 7 is not the only possible explanation for the observed reduction in replication efficiency. The expression of the viral replicase complex is independent of these chimeric 5'NTRs, as it is driven by the EMCV IRES. Furthermore, while the IRES activities of chimeras 2 and 3 are similar, chimera 2 is completely replication incompetent and chimera 3 forms colonies up to 60 % of the wild-type level. Moreover, although the IRES activity of chimera 4 is only one-third of that of chimeras 6 and 7, it forms more colonies than these two chimeras.
There are several possible explanations for the observed effects on RNA replication. First, the primary sequence of domains II and/or III, which is altered as a consequence of the domain exchanges, could be an important determinant for replication. The sequences of HCV and CSFV 5'NTRs differ significantly from each other. Only a few short stretches of high sequence identity can be found (reviewed by Rijnbrand & Lemon, 2000). At least some flexibility at the nucleotide level is allowed, since the genotype 1b replicon in which the 5'NTR was replaced by the genotype 1a 5'NTR is almost as viable as the original replicon. This is consistent with an earlier observation that transcripts of a chimeric cDNA that encodes a genotype 1b polyprotein flanked by 3' and 5'NTRs of genotype 1a was infectious in chimpanzees (Yanagi et al., 1998
). Interestingly, there are no differences between genotype 1a and 1b sequences within the crucial domain II. Second, the structure of the positive-strand promoter could be disrupted by the domain swops, thereby influencing its activity. Whereas the chimeras were designed to maintain IRES structure, the structure of the positive-strand promoter was not taken into account. Recently, two studies showed by chemical and enzymatic probing in combination with computer predictions that the negative-strand 3'NTR does not fold into a mirror image of the positive-strand 5'NTR (Schuster et al., 2002
; Smith et al., 2002
). Both studies predict an identical structure for the 3'-terminal 220 nt of the negative strand (Fig. 4
), which is folded into five consecutive stemloops (AE) and contains the core of the positive-strand promoter. The predicted structural differences between the genomic 5'NTR and its complement could explain why swops, as in chimeras 1 and 2, which are minor with respect to IRES structure but disrupt a major stemloop in the core promoter structure, have minimal effect on translation but abolish replication. Furthermore, the observation with the domain III exchanges, the efficiency of colony formation decreases with an increase in the presence of CSFV-derived sequences in the HCV 5'NTR, could reflect their various effects on negative-strand structure. This is expected to be minor for chimera 3 and substantial for chimera 6.
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Nevertheless, we realize that it cannot be excluded that the replication determinants reported here are not part of the actual replication signal but rather influence a proper folding of this signal. In addition to this, it has to be kept in mind that in the HCV replicon, the expression of the neomycin selection marker is regulated by the chimeric 5'NTRs.
The involvement of domains II and III in the replication of the HCV replicons was also documented recently in two other studies (Friebe et al., 2001; Kim et al., 2002
). In both studies, cis-acting signals for replication were separated from signals for translation by fusing various parts of the HCV 5'NTR to the poliovirus IRES. It was shown that domains I and II are sufficient for a low level of replication, which is strongly enhanced by the presence of domains III and IV. While in these studies truncated NTRs fused to the poliovirus IRES were analysed, we studied the various domains as part of a functional IRES structure.
Although the 5'NTRs of HCV and pestiviruses are conserved in their higher order structure and use an identical mechanism of IRES-mediated translation, the determinants for replication appear to be more complex for HCV than for the pestiviruses, for which it was shown in studies with bovine viral diarrhoea virus (BVDV), BVDV/HCV and BVDV/EMCV 5'NTR chimeric viruses that only the 5'-terminal 4 nt are required to direct specific replication and only domains Ia and Ib are important for efficient replication (Frolov et al., 1998). When studied in the context of a subgenomic BVDV replicon, mutations in domain Ia were also found to effect replication (Yu et al., 2000
). An overlap between translation and replication signals was also shown for other positive-stranded RNA viruses with IRES-mediated translation, such as poliovirus (Borman et al., 1994
; Ishii et al., 1999
).
In summary, we observed that replication and translation signals overlap in the HCV 5'NTR and that domain II is crucial for replication. Future studies will focus on the effect of the HCV/CSFV sequence exchanges on RNARNA and RNAprotein interactions involved in HCV translation and/or replication. Furthermore, it will be interesting to define the cis-acting replication signals in more detail and to analyse the sequence of chimeras that were impaired, but viable, to look for compensating mutations.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Borman, A. M., Deliat, F. G. & Kean, K. M. (1994). Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis. EMBO J 13, 31493157.[Abstract]
Bredenbeek, P. J., Frolov, I., Rice, C. M. & Schlesinger, S. (1993). Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol 67, 64396446.[Abstract]
Fletcher, S. P. & Jackson, R. J. (2002). Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5' untranslated region important for IRES function. J Virol 76, 50245033.
Friebe, P., Lohmann, V., Krieger, N. & Bartenschlager, R. (2001). Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication. J Virol 75, 1204712057.
Frolov, I., McBride, M. S. & Rice, C. M. (1998). cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras. RNA 4, 14181435.
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 81228126.[Abstract]
Hellen, C. U. & Pestova, T. V. (1999). Translation of hepatitis C virus RNA. J Viral Hepat 6, 7987.[CrossRef][Medline]
Hellen, C. U. & Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev 15, 15931612.
Honda, M., Brown, E. A. & Lemon, S. M. (1996a). Stability of a stemloop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA. RNA 2, 955968.[Abstract]
Honda, M., Ping, L. H., Rijnbrand, R. C., Amphlett, E., Clarke, B., Rowlands, D. & Lemon, S. M. (1996b). Structural requirements for initiation of translation by internal ribosome entry within genome-length hepatitis C virus RNA. Virology 222, 3142.[CrossRef][Medline]
Honda, M., Beard, M. R., Ping, L. H. & Lemon, S. M. (1999). A phylogenetically conserved stemloop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J Virol 73, 11651174.
Ishii, T., Shiroki, K., Iwai, A. & Nomoto, A. (1999). Identification of a new element for RNA replication within the internal ribosome entry site of poliovirus RNA. J Gen Virol 80, 917920.[Abstract]
Kim, Y. K., Kim, C. S., Lee, S. H. & Jang, S. K. (2002). Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication. Biochem Biophys Res Commun 290, 105112.[CrossRef][Medline]
Krieger, N., Lohmann, V. & Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 46144624.
Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110113.
Poole, T. L., Wang, C., Popp, R. A., Potgieter, L. N., Siddiqui, A. & Collett, M. S. (1995). Pestivirus translation initiation occurs by internal ribosome entry. Virology 206, 750754.[Medline]
Reynolds, J. E., Kaminski, A., Kettinen, H. J., Grace, K., Clarke, B. E., Carroll, A. R., Rowlands, D. J. & Jackson, R. J. (1995). Unique features of internal initiation of hepatitis C virus RNA translation. EMBO J 14, 60106020.[Abstract]
Reynolds, J. E., Kaminski, A., Carroll, A. R., Clarke, B. E., Rowlands, D. J. & Jackson, R. J. (1996). Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon. RNA 2, 867878.[Abstract]
Rijnbrand, R. C. & Lemon, S. M. (2000). Internal ribosome entry site-mediated translation in hepatitis C virus replication. Curr Top Microbiol Immunol 242, 85116.[Medline]
Rijnbrand, R., Bredenbeek, P., van der Straaten, T., Whetter, L., Inchauspe, G., Lemon, S. & Spaan, W. (1995). Almost the entire 5' non-translated region of hepatitis C virus is required for cap-independent translation. FEBS Lett 365, 115119.[CrossRef][Medline]
Rijnbrand, R., Abell, G. & Lemon, S. M. (2000). Mutational analysis of the GB virus B internal ribosome entry site. J Virol 74, 773783.
Rijnbrand, R., Bredenbeek, P. J., Haasnoot, P. C., Kieft, J. S., Spaan, W. J. & Lemon, S. M. (2001). The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs. RNA 7, 585597.
Rosenberg, S. (2001). Recent advances in the molecular biology of hepatitis C virus. J Mol Biol 313, 451464.[CrossRef][Medline]
Schuster, C., Isel, C., Imbert, I., Ehresmann, C., Marquet, R. & Kieny, M. P. (2002). Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA. J Virol 76, 80588068.
Smith, R. M., Walton, C. M., Wu, C. H. & Wu, G. Y. (2002). Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences. J Virol 76, 95639574.
Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. J Virol 66, 14761483.[Abstract]
Wang, C., Sarnow, P. & Siddiqui, A. (1993). Translation of human hepatitis C virus RNA in cultured cells is mediated by an internal ribosome-binding mechanism. J Virol 67, 33383344.[Abstract]
Wang, C., Le, S. Y., Ali, N. & Siddiqui, A. (1995). An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region. RNA 1, 526537.[Abstract]
Wengler, G., Bradley, D. W., Collett, M. S., Heinz, F. X., Schlesinger, R. W. & Strauss, J. H. (1995). Flaviviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses, pp. 424426. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo & M. D. Summers. Vienna & New York: Springer-Verlag.
Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. (1998). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161172.[CrossRef][Medline]
Yu, H., Isken, O., Grassmann, C. W. & Behrens, S. E. (2000). A stemloop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA. J Virol 74, 58255835.
Received 23 December 2002;
accepted 6 March 2003.