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Journal of Virology, July 2000, p. 6223-6226, Vol. 74, No. 13
Department of Molecular Genetics and
Microbiology, State University of New York at Stony Brook, Stony
Brook, New York 11794-5222
Received 19 October 1999/Accepted 23 March 2000
Internal ribosomal entry sites (IRESs) can function in foreign
viral genomes or in artificial dicistronic mRNAs. We describe an
interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion
up to nt 40 in the HCV 5' nontranslated region relieved the replication
block, yielding PV/HCV variants replicating to high titers. Fortuitous
yet crippling interactions between an IRES and surrounding heterologous
RNA must be considered when IRES-based dicistronic expression vectors
are being constructed.
All picornavirus genomes contain a
genetic element named the internal ribosomal entry site (IRES) that
allows translation of viral genomic mRNAs in a 5'- and cap-independent
manner (5, 12, 13, 16, 20, 25). Similar genetic elements
have also been discovered in genomes of Hepatitis C virus
(HCV) (24), a Hepacivirus, and Bovine viral
diarrhea virus (21), a Pestivirus, of the
Flaviviridae family. IRESs are located in the 5'-terminal segment of the genomes, where they control initiation of polyprotein synthesis. Certain insect RNA viruses, e.g., Plautia stali
intestine virus, however, are exceptional in that their cognate IRES
maps several thousand nucleotides downstream of the 5' end of the
genome, separating two cistrons encoding two different polyproteins
(23). It is of interest that we have previously engineered a
dicistronic poliovirus (PV) by inserting the IRES of
encephalomyocarditis virus into the coding region of the PV polyprotein
(16). The genetic organization of this dicistronic PV, which
encodes two polyproteins separated by an IRES element, closely
resembles that of P. stali intestine virus.
IRES elements are defined by function and not by nucleotide sequence.
Indeed, the IRESs of PV, a picornavirus, and HCV, a flavivirus, have
little if any sequence in common, yet both function as promoters of
internal ribosomal entry for initiation of translation. This phenomenon
is most apparent in chimeric PV/HCV viruses in which the cognate PV
IRES has been exchanged with that of HCV (15, 28).
Unexpectedly, the HCV IRES includes a sequence downstream of the AUG
codon initiating its polyprotein (15, 22). In constructing a
viable PV/HCV chimeric virus, a portion of the core protein-encoding sequence was necessary to obtain a viable virus (Fig.
1,
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genetic Analysis of a Poliovirus/Hepatitis C Virus (HCV)
Chimera: Interaction between the Poliovirus Cloverleaf and a
Sequence in the HCV 5' Nontranslated Region Results in a
Replication Phenotype
and
ABSTRACT
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Core) (15). The Core
sequence leads to the formation of a Core/PV fusion polyprotein
which must be processed through cleavage by the PV viral proteinase
2Apro at the Core*1A junction (Fig. 1) (28).
However, the production of core sequence-encoded peptides resulting
from translation of the Core sequence is not necessary for the
function of the HCV IRES in P/H710-d17* (28). (In previous
studies, P/H710-d17 was designated P/H701-2A because the chimeric
genome contained the HCV-specific sequence from nucleotides [nt] 18 to 710 of the HCV genome [28]. The numbering of the
HCV 5' nontranslated region [5'NTR] in this paper conforms to the
numbering of the full-length HCV 5'NTR [see, for example, reference
11].)
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FIG. 1.
Schematic diagram of the genomic organization of
P/H710-d17. The cloverleaf-like RNA structure of PV, an essential
cis-acting replication signal terminated with the
genome-linked protein VPg, is located at the 5' end of the genome.
Noninitiating AUG codons found in the HCV 5'NTR are denoted by stars.
The shaded (HCV) and filled (PV) boxes depict open reading frames
encoding viral polypeptides; the position of the HCV core protein gene
is marked Core. Overall, the HCV-specific sequence in P/H710-d17
spans from nt 18 to 710 (15, 28). PV-encoded polypeptides
within the polyprotein are indicated by 1A, 2B, etc. The PV-encoded
proteinase 2Apro is responsible for the cleavage between
Core and capsid precursor P1.
In general, IRES elements were discovered and studied by constructing artificial dicistronic mRNAs (13), by exchanging IRES elements in viral genomes (1, 6, 8, 15, 28), for example, as in P/H710-d17, or by inserting an extra IRES into a viral genome (1, 16). In all of these cases, the IRESs must function in the context of RNA sequences foreign to them. Since IRESs consist of remarkably large, continuous RNA segments (300 to 400 nt long) that undoubtedly form complex higher-order structures, the possibility exists that elements of the IRES and surrounding sequences may interact by base pairing. Such fortuitous contacts may have consequences either for the function of the heterologous, adjacent RNA elements or for the IRES. Here we describe an interesting observation made with chimera P/H710-d17, in which the poliovirus cloverleaf forms base pairs with nucleotides of the HCV 5'NTR, resulting in a replication phenotype.
We previously noticed that chimera P/H710-d17 yielded plaques of
different sizes when passaged on HeLa cells (28). This observation can be an indicator of the emergence of new genotypes with
different replication properties. We therefore performed a genetic
analysis of the passaged virus, starting with infectious transcripts
derived from a plasmid harboring P/H710-d17 (for experimental details,
see reference 28). When P/H710-d17 was passaged
several times on HeLa cell R19 monolayers, the average plaque size
expanded (Fig. 2A). Similarly, the virus
titer per unit of transfecting RNA increased (Fig. 2A). It was
therefore likely that a variant(s) with improved growth properties,
relative to P/H710-d17, was selected in each passage that eventually
outcompeted the parental virus.
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A mutation(s) resulting in a larger-plaque phenotype and improved
replication properties could reside in the HCV 5'NTR (which in our
constructs begins at nt 18 of the HCV genome) (15), in the
HCV core sequence, or in PV-specific sequences. For further studies, we
selected a large-plaque variant that had emerged after the first
passage of P/H710-d17. The variant (P/H710-d17 G1) was expanded once in
HeLa cells, and its genotype in the 5'NTR was determined. Sequence
analyses revealed a single nucleotide change (A
G) in position 29 of
the HCV-specific sequence (Fig. 2C). To test whether this mutation
alone was responsible for both plaque phenotype and increased virus
yield, we introduced a single AG change into the P/H710-d17 genome
at nt 29. This was done by PCR-based mutagenesis using primers A29G
(5'-CT TAGAATTCGGCGACACTCCgCCATAGATCACTCCCC- 3')
and PVVP4
(5'-CGTTACTAGCTGAATCTCTATAATAATTAATGG-3').
The PCR fragments were gel purified, digested with
EcoRI and SacI, and ligated with cloning vector
P/H710dES (28), lacking HCV IRES and core sequences, to
yield construct P/H710-d17-A29G, which was selected by restriction
mapping and verified by sequencing. As can be seen in Fig. 2D, the
plaque size of the genetically engineered variant P/H710-d17-A29G was
similar to those obtained after multiple passages of P/H710-d17.
Cell-free translations in a HeLa cell extract (17) were carried out with RNA of PV type 1, Mahoney, and with transcript RNAs of P/H710-d17 and P/H710-d17-A29G. Regardless of whether the translations were carried out at 30°C for 16 h or at 34°C for 7 h, the pattern obtained with P/H710-d17-A29G RNA revealed no significant difference from those obtained with P/H710-d17 RNA (data not shown). Although only circumstantial, this result suggests that the increase in plaque size and virus yield is not the result of increased translation efficiency of the IRES in P/H710-d17-A29G.
All PV/HCV chimeric viruses described by us carry a PV-specific
structure, the cloverleaf, at the 5' end of the genome (Fig. 1)
(15, 28). The cloverleaf (Fig.
3A) serves as a cis-acting signal in PV RNA replication (reviewed in reference
27). It forms an RNP of unknown structure with the
PV proteinase 3CDpro (2, 3) and either PV
polypeptide 3AB (10, 26, 27) or cellular RNA binding
poly(rC) binding protein (4, 7, 18). Genetic analysis has
identified stem-loop D as the binding domain of 3CDpro
(3; E. Rieder, W. Xiang, and E. Wimmer, unpublished
data).
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Inspection of stem-loop D and the HCV-specific 5'-terminal sequences
revealed the possibility of an interaction between these regions by
base pairing (Fig. 3B). If so, the single mutation (AG) in
P/H710-d17 G1 at nt 29 would destabilize this interaction. It seemed
unlikely to us, however, that the change from an A-U to a G-U would be
sufficient to relieve P/H710-d17 G1 from its growth restriction. We
favor the hypothesis that the AG change at nt 29 leads to the
formation of a new stem-loop structure in the HCV 5'NTR (Fig. 3C). This
new stem-loop structure would resist base pairing with the cloverleaf
and thus explain the replication phenotype of P/H710-d17-A29G.
The possibility of base pairing between the PV cloverleaf and HCV sequence (Fig. 3B) was eliminated altogether by changing the HCV sequence between nt 17 and 31 from GGCGACACUCCACC to CCGCTGTCUCCTGG (nucleotides changed are in boldface), thereby generating variant P/H710-d17-IM. This was done by PCR-based mutagenesis, using primers IM (5'-ACTTAGGAATTCCCGCTGTCTCCTGGATAGATCACTCCCCTGTGAGGAACTACTGTC-3') and PVVP4 (5'-CGTTACTAGCTGAATCTCTATAATAATTAATGG-3'). The cloning strategy was the same as for P/H710-d17-A29G. This variant expressed plaque and replication phenotypes matching, but not surpassing, that of P/H710-d17-A29G (data not shown).
If a single A29G mutation at nt 29 or the drastic change of the sequence between nt 17 and 31 relieves virus P/H710-d17 from replication restriction, deletions within this region of the HCV 5'NTR are likely to yield a virus with a phenotype matching that of P/H710-d17-A29G. Accordingly, we have deleted the HCV-specific sequence up to nt 40 (W. D. Zhao and E. Wimmer, unpublished data). As can be seen in Fig. 2B, construct P/H710-d40 produced large plaques upon transfection, yielding 105 PFU of virus per ml. On further passage, the plaque phenotype produced by this virus did not change. The growth properties of variant P/H710-d40 will be described elsewhere (Zhao and Wimmer, unpublished data).
IRES elements have been used to construct artificial dicistronic mRNAs (12, 13, 20) in a variety of different eukaryotic expression vectors, either for the study of IRES function per se or for the expression of reporter genes or delivery of gene products for therapeutic or commercial reasons. IRESs have been exchanged between different virus genomes for the studies of viral replication (1, 6, 16, 19), host range (8, 9), or IRES function (15, 28). By necessity, the IRES elements in these constructs are surrounded by foreign RNA sequences. A fortuitous interaction between sequences of surrounding heterologous RNA segments with the IRES RNA could influence the function of either RNA, resulting in either reduced reporter gene expression or genome replication. The evidence presented in this report can be interpreted to mean that a fortuitous interaction between an essential cis-acting element of the PV genome and a sequence in the HCV 5'NTR resulted in impaired viral proliferation. No differences were detected in the translation of the chimeric RNAs, regardless of whether the predicted interaction between cloverleaf and HCV 5'NTR was interrupted. We suggest, therefore, that the chimera P/H710-d17 is impaired in the ability to replicate its genome.
Interactions between the IRES and surrounding heterologous sequences, resulting in an expression phenotype, may be common. They may escape detection because the engineered hybrid RNAs in most expression vectors are not responsive to genetic analysis.
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ACKNOWLEDGMENTS |
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We are indebted to A. Nomoto, Tokyo University, for his generous gift of HCV cDNA subclones of HCV. Critical reading of the manuscript by A. Paul is highly appreciated. Special thanks go to R. Duggal, S. Mueller, X. Z. Peng, T. Pfister, and E. Rieder for suggestions and to F. Maggiore for excellent technical assistance.
This work was supported in part by NIH grants NIAID 2R01AI15122-25 and 5R01AI32100-07. F.C.L. was supported in part by a fellowship from the Schering-Plough Research Institute.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (631) 632-8787. Fax: (631) 632-8891. E-mail: ewimmer{at}ms.cc.sunysb.edu.
Present address: Schering-Plough Research Institute, Kenilworth, NJ 07033.
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Journal of Virology, July 2000, p. 6223-6226, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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