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J Virol, May 1998, p. 3571-3577, Vol. 72, No. 5
Department of Molecular Biology and
Biochemistry, University of California
Received 6 October 1997/Accepted 3 February 1998
Hepatitis A virus (HAV), unlike other picornaviruses, has a
slow-growth phenotype in permissive cell lines and in general does not
induce host cell cytopathology. Although there are no published reports
of productive infection of HeLa cells by HAV, HAV RNA appears to be
readily translated in HeLa cells when transcribed by T7 RNA polymerase
provided by a recombinant vaccinia virus. The 5' noncoding region of
HAV was fused to poliovirus (PV) coding sequences to determine the
effect on translation efficiency in HeLa cell extracts in vitro.
Conditions were optimized for utilization of the HAV internal ribosome
entry segment (IRES). Transcripts from chimeric constructs fused
precisely at the initiation codon were translated very poorly. However,
chimeric RNAs which included 114 or more nucleotides from the HAV
capsid coding sequences downstream of the initiation codon were
translated much more efficiently than those lacking these sequences,
making HAV-directed translation efficiency similar to that directed by
the PV IRES. Sixty-six nucleotides were insufficient to confer
increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an
inhibitory effect on translation efficiency. Constructs lacking these
terminal sequences, or those in which the PV 5'-terminal sequences
replaced those from HAV, translated three- to fourfold better than
those with the intact HAV 5'-terminal end.
Hepatitis A virus (HAV) belongs to
the Picornaviridae family and is the sole member of the
Hepatovirus genus. The viral genome is a positive-strand RNA
of about 7.5 kb which functions in infected cells directly as mRNA. It
contains an extremely long, highly structured, uncapped 5' noncoding
region (5'NCR), which constitutes approximately 10% of the viral
genome and includes 10 or more AUG codons that are not used to initiate
translation. The 5'NCR contains an internal ribosome entry segment
(IRES), reported to include sequences from nucleotides (nt) 152 to 735 (8, 15). The IRES is required for cap-independent, internal
ribosome binding and translation. The HAV genome has a single long open
reading frame which encodes a polyprotein that is proteolytically
processed by a virus-encoded proteinase into various structural and
nonstructural proteins. A short (63-nt) 3' noncoding region follows the
coding sequence and precedes a poly(A) tail.
Translation of picornavirus RNAs takes place by a mechanism of internal
ribosome entry utilizing the IRES element (for a review, see reference
11), in contrast to the mechanism of ribosome scanning from the capped 5' end of the mRNA as occurs for the majority
of cellular mRNAs (29). On the basis of primary sequence homology, biochemical probing, and computer-assisted folding
predictions, the IRES elements of members of the picornavirus family
are divided into two groups: type I, present in entero- and
rhinoviruses; and type II, present in cardio- and aphthoviruses
(44). Although the HAV IRES may resemble the type II IRES
(8), it may represent a third type (23). Despite
little similarity in the primary nucleotide sequence of the 5'NCR, and
marked variations in the predicted secondary structures of the IRES
elements among the different genera of picornaviruses, it has been
shown that the intact IRES domains can be readily exchanged en bloc
among different viruses and maintain functional translation properties
(1, 25, 26, 36).
HAV is known to have an extremely slow replication cycle in cultured
cells, often yielding low amounts of virus. It does not induce
detectable cytopathology and does not show any apparent effect on
cellular growth or metabolism, in contrast to other picornaviruses like
poliovirus (PV), in most susceptible cell cultures (for a review, see
reference 42). It has been reported that the HAV
IRES is much less efficient than other picornaviral IRESes for
translation in rabbit reticulocyte lysates in vitro (6, 8,
24) and also in BT7-H cells in vivo (43). These data
led to the suggestion that the relatively poor growth of HAV may be
attributable to the inherently low translation efficiency of its RNA
(31, 43), although there have been some conflicting reports
on the activity of the HAV IRES depending on the reporter gene used to
assess its function (25). In studies reported here, we
determined the efficiency of HAV IRES utilization in a series of
chimeric RNAs between HAV and PV sequences, translated in HeLa cell
extracts. The data show that sequences downstream of the HAV 5'NCR
extending into the capsid coding sequence of HAV have a significant
impact on translation efficiency directed by the HAV IRES in vitro.
Although a requirement for coding sequences for efficient translation
of other picornaviral RNAs has not been described to date, precedence
for coding sequences downstream of the initiation codon contributing to
IRES function was established recently for hepatitis C virus (HCV)
(21, 32, 34) and hepatitis G virus (isolates GBV-A and
GBV-C) (41). Both viruses are members of the
Flaviviridae and possess an IRES element. At least 12 to 30 nt immediately downstream of the HCV translation initiation site were
necessary for efficient IRES activity in a bicistronic construct (34); if the HCV IRES was used to replace the PV IRES in a
full-length construct, inclusion of 369 nt of HCV coding sequence
extending the HCV IRES generated viable virus, in contrast to a
chimeric construct harboring only the HCV IRES sequences in the 5'NCR
(32). The observations presented in this report and the data
from investigations of the translation of HCV RNA and hepatitis G virus
RNA suggest that in some types of IRES elements, coding sequences
downstream of the initiation codon contribute to or are part of the
IRES activity.
Additional findings from these studies extent previous observations of
an effect on IRES activity of sequences upstream of the apparent 5'
boundary of the IRES. Removal of these sequences from the HAV 5'NCR or
replacement with PV 5'-terminal sequences enhanced translational
efficiency in vitro severalfold.
Plasmid constructs.
All HAV/PV chimeras constructed in this
study were derived from plasmid pT7PV1 (17) and plasmid
pT7-HAV1 (19), containing the full-length copy of infectious
PV cDNA (type 1, Mahoney strain) and the cell culture-adapted HAV cDNA
(HM175p35), respectively, under control of the T7 promoter. The
nucleotide numbering used to describe HAV cDNA below corresponds to the
wild-type HAV strain HM175 (9). Fragments generated from
parental plasmids by restriction digestion used to construct chimeric
plasmids were gel purified by using a QIAquick gel purification kit
(Qiagen Inc.) according to the manufacturer's instructions. All
plasmids were propagated by standard methods in Escherichia
coli DH5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Coding Sequences Enhance Internal Initiation of
Translation by Hepatitis A Virus RNA In Vitro
Irvine, Irvine, California
92697
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
or C600, using ampicillin selection. The nucleotide
sequences of any PCR-generated inserts were determined by using a
Sequenase reagent kit (United States Biochemical Corp.) to verify that
no unwanted mutations had been introduced during PCR.
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Transient expression assay. HeLa cell monolayers grown to confluency in six-well plates were simultaneously infected with recombinant vaccinia virus vTF7-3 (14) and transfected with plasmid DNA. The HeLa cells were washed twice with minimal essential medium (MEM). Lipofectin-mediated transfection-infection with the recombinant vaccinia virus was accomplished by adding 500 µl of MEM mixed with 3 to 9 µg of plasmid DNA, 10 µl of Lipofectin reagent (Gibco, BRL), and vTF7-3 (approximately 10 PFU per cell) to each well of the six-well plate. Cells were incubated at 37°C. After 4 h, 2 ml of MEM supplemented with 3% fetal bovine serum was added, and incubation was continued at 37°C. At 24 h posttransfection, the cells were washed twice with phosphate-buffered saline, lysed with 200 µl of lysis buffer (0.1 M potassium phosphate buffer [pH 7.8], 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA) for 15 min, and scraped from the plates. Nuclei were removed by centrifugation, and samples were processed for Western blot analysis.
Western blot analysis. The samples were boiled in Laemmli sample buffer, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on an SDS-11% polyacrylamide gel, and transferred to a nitrocellulose membrane (Schleicher & Schuell). The blot was first incubated for 1 h at room temperature with a rabbit anti-HAV VP1 serum (18) or with a rabbit anti-PV VP2 serum (kindly provided by Bert Semler) to detect the production of HAV or PV antigen, respectively. Alkaline phosphatase-conjugated anti-rabbit antibodies (Promega) were used in a second incubation step for 30 min at room temperature followed by color detection as recommended by the supplier.
Transcription reactions and translation assays. All plasmids were linearized by digestion with the appropriate restriction enzyme, either EcoRI, SnaBI, or BstZ17I, prior to in vitro transcription. Since all constructs are made in the same background vector as the parent pT7PV1 or pT7-HAV1, RNA transcripts were produced from 2 µg of linear cDNA with T7 RNA polymerase in the presence of [3H]UTP, using a MAXIscript in vitro transcription kit (Ambion Inc.) according to the supplier's instructions. After DNase I treatment and phenol-chloroform extraction, the transcripts were analyzed by agarose gel electrophoresis for integrity and quantified by measuring the incorporated tritium.
Micrococcal nuclease-treated HeLa cell extracts were prepared from HeLa S3 cells and programmed with 250 ng to 1 µg of transcripts in a reaction volume of 12.5 µl under the conditions described previously (33) except that the added potassium acetate concentration was reduced to 80 mM, bringing the total potassium concentration to ~115 mM. These conditions were determined to be optimal in our cell-free translation system for the HAV IRES, as opposed to ~150 mM potassium used for the PV IRES. The reaction was stopped with 1 volume of 2× gel sample buffer (30) after 90 min of incubation at 30°C. A 10-µl aliquot was subjected to SDS-PAGE (11% gel) to analyze the [35S]methionine-[35S]cysteine-labeled translation products. Coomassie blue-stained, dried gels were typically exposed for 16 h to Kodak BioMax film (Kodak Corp.). To evaluate the efficiency of translation in vitro, the autoradiographs were analyzed by densitometry using a Scan Jet IIcx/T (Hewlett Packard) and the public domain analysis software package developed by Wayne Rasband, National Institutes of Health, Bethesda, Md. Data were corrected according to the numbers of methionine and cysteine residues in the different translation products.| |
RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Translation of HAV RNA in HeLa cells. Translation of HAV RNA in rabbit reticulocyte lysates has been shown to be both inefficient and inaccurate (24, 27). In the case of PV RNA, which is also translated poorly by rabbit reticulocyte lysate, supplementation with factors from HeLa cells was shown to improve both the fidelity and the efficiency of translation (7, 10), and translation directly in HeLa cell extract generates high yields of accurately translated and processed viral protein (4, 5, 33). In an effort to identify a convenient system that would better support translation of HAV RNA in vitro, we wanted to explore the possible utilization of HeLa cell extracts. However, since there are no published reports of replication of HAV in HeLa cells, and we have failed repeatedly to propagate HAV in HeLa cells (unpublished observations), it was necessary to determine whether translation of HAV RNA occurred in HeLa cells in vivo. To this end, HeLa cells were transfected with supercoiled plasmid pT7-HAV1, encoding the complete HAV cDNA under control of the T7 promoter. Simultaneous infection with recombinant vaccinia virus vTF7-3 generated T7 RNA polymerase to transcribe HAV RNA. Synthesis of viral capsid proteins was detected by Western immunoblotting with antiserum against HAV VP1. Figure 2A, lanes 1 to 6, shows that the transfected HeLa cells produce readily detectable VP1 proteins. The predominant product detected by anti-HAV VP1 was VP1-2A (PX [2]) at ~40 kDa; in addition, a cleavage product of ~34 kDa representing VP1 or an intermediate in VP1 processing accumulated, similar to that seen after transfection of FRhK-4 cells that are permissive for growth of HAV (25). The transfection conditions included saturating amounts of plasmid DNA, and so the production of viral protein was not enhanced by increasing the DNA concentration (Fig. 2A, lanes 4 to 6). Another plasmid, pPsHAV, in which the 5'-terminal 138 nt of HAV were replaced with the first 109 nt of PV sequence (Fig. 1), gave a similar result (Fig. 2A, lanes 1 to 3), as expected, since the 5' boundary of the HAV IRES has been reported to be downstream of nt 139.
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Translation in vitro of HAV RNA in HeLa cell extract. The demonstration that HAV RNA was readily translated in intact HeLa cells prompted us to examine the translation of RNAs that contained the HAV IRES in HeLa cell extract in vitro. The cell-free translation system developed for PV RNA (33) was optimized with respect to mono- and divalent cation concentrations for translation of HAV RNA (see Materials and Methods). The RNAs used to program the HeLa cell translation extract were derived from plasmid DNAs cut so as to encode only capsid protein sequences to simplify the pattern of translation products.
Figure 3, lanes 3 to 5, shows the translation products from HeLa cell extract programmed with decreasing amounts of RNA derived from pT7-HAV1, linearized with BstZ17I at nt 2024 within the HAV VP3 gene. The predicted size of the truncated HAV capsid protein is ~47 kDa, which is clearly detectable on the autoradiograph and distinguishable from the weak background translation seen in the negative control without any added RNA (lane 2). Products derived from aberrant internal initiations were not apparent. HeLa cell extract programmed with 250 or 500 ng of transcripts produced slightly more product than extract programmed with 1 µg of RNA. This poisoning effect of high RNA concentrations in cell-free translation systems has also been noticed with PV RNA.
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HAV IRES utilization in HAV/PV chimeras. To determine the contribution of the HAV IRES to the translation efficiency of the PV coding sequences, we constructed a series of HAV/PV chimeras (Fig. 1). In the first set, the HAV IRES sequences were fused to PV coding sequences directly at the initiation codon (HAV nt 741; PV nt 743) to generate pPsH91-740P and pPsH139-740P. Plasmid pPsH91-740P includes the pY1 tract from the HAV 5'NCR, whereas in plasmid pPsH139-740P, the HAV sequences start downstream of pY1, at nt 139. Both chimeric plasmids were linearized with SnaBI at nt 2954 prior to preparation of transcripts for translation. The predicted size of the translation product is ~81 kDa. Figure 4 shows the results of the translation assays in HeLa cell extracts. PV RNA (lane 2), isolated from purified PV virions, was translated for comparison of translation efficiency. The PV polyprotein is mainly uncleaved, since translation was performed for only 90 min, and protein processing requires more extended incubation times. Lanes 4 and 5 (HAV1* and PsHAV*) confirm the three- to fourfold increased translation efficiency conferred by replacement of the HAV 5'-terminal sequences with those from PV. The two HAV/PV chimeric transcripts, PsH91-740P* and PsH139-740P* (Fig. 4, lanes 6 and 7), translated quite poorly compared to transcripts from pPsHAV, which contained the natural HAV coding sequences following the initiation codon (Fig. 4; compare lanes 6 and 7 to lane 5). When corrected for the number of methionine and cysteine residues in the two proteins, the longer chimeric proteins were produced at only 7% (fusion at HAV nt 91) or 16% (fusion at HAV nt 139) of the amount of the smaller HAV protein. All subsequent constructs were therefore generated with the PV cloverleaf structure fused to the HAV IRES at nt 139, lacking pY1 of HAV.
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Effect of HAV sequences downstream of the translation initiation site. To test the possibility that HAV sequences downstream of the translation initiation site comprise part of the HAV IRES or affect the efficiency of IRES utilization, we made additional chimeric constructs that extended downstream of the HAV 5'NCR (Fig. 1). Plasmid pPsH139-806P contained 66 nt extended into the coding region of HAV, coding for VP4; pPsH139-854P* included 114 nt of the HAV coding region, translated into the first 38 amino acids of HAV sequence; and pPsH139-1191P* had 450 nt downstream of the start codon fused to the amino terminus of the PV capsid coding sequences. All of these constructs contain an artificial cleavage site for PV 3C proteinase (AXXQG) between the HAV and PV coding sequences (3). A comparison of translation obtained with transcripts from pPsH1194/1172P*, in which the HAV and PV coding sequences are fused without the introduction of the PV 3C cleavage site, or transcripts from pPsH139-1191P*, containing the cleavage site, showed no significant difference in translation efficiency between these two constructs (data not shown). Transcripts of the constructs pPsH139-740P, pPsH139-806P, pPsH139-854P*, and pPsH139-1191P*, linearized at nt 2954 of the PV coding sequence, were translated over a range of RNA concentrations in HeLa cell extract, and the efficiency of translation was determined by gel electrophoresis and autoradiography of the dried gel. The results of the translation assays are shown in Fig. 5. The predominant product of each transcript migrated according to its predicted size as indicated in Fig. 5 except for the product from pPsH139-854P*, which migrated slightly faster than expected from its predicted mass of ~85 kDa (Fig. 5, lanes 8 and 9).
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The structure of mRNA is an important element in regulation of protein synthesis. Previous studies of translational control of HAV effected by RNA sequences and structure have been focused within the borders of the 5'NCR. The present study of a series of constructs retaining the HAV 5'NCR extending into various lengths of HAV coding sequences, using PV coding sequences as reporter, demonstrates that sequences downstream of the translation initiation AUG codon of the HAV IRES provided a fourfold increase of HAV IRES-driven translation in vitro over a construct without HAV downstream capsid coding sequences. More than 66 nt of the HAV capsid coding sequence are necessary to support this stimulation. This effect has not been described for any other picornavirus. The report of a specific search for effects of downstream coding sequences on IRES activity in EMCV RNA showed only that insertion of additional guanosine residues located immediately after the AUG initiation codon exerted an inhibitory influence on translation initiation (22). However, the requirement for sequences extending into the coding region for optimal IRES function has been reported for both HCV RNA (21, 34) and hepatitis G virus RNA (41).
HAV shows many features different from the other members of the picornavirus family, including an extremely slow replication cycle in cultured cells. It was suggested that the slow growth of HAV was attributable to inefficient IRES-mediated translation of HAV RNA (31, 43). However, all constructs used for previous investigations of HAV IRES-driven translation efficiency used fusions between the HAV 5'NCR and reporter genes directly at the translation initiation codon of the HAV 5'NCR. In contrast, the present study shows that inclusion of HAV capsid coding sequences with the HAV 5'NCR stimulated efficient utilization of the HAV IRES.
The coding sequences of HAV may assist in formation of RNA structures required for trans-acting factors to bind within the IRES, or they may cause the ribosome complex to pause at the initiating AUG codon to allow a functional translation initiation complex to form. Studies using Sindbis virus mRNA have demonstrated that structures downstream of the translation initiation codon enhance translation efficiency (12, 13). In any event, translation is but one of many processes in virus replication, and the efficiency of IRES utilization may not be the sole or major determinant of virus growth rate. Indeed, replacement of the HAV IRES by the encephalomyocarditis virus IRES in the context of the HAV genome did not contribute to improved growth properties of the chimeric virus in cultured cells (25).
Analysis of the 5'-terminal end of the HAV 5'NCR showed that sequences upstream of the predicted 5' border of the HAV IRES are inhibitory for translation of HAV RNA in vitro. Transcripts of HAV RNA from which the first 138 nt of the HAV 5'NCR were deleted showed greater translation efficiency than constructs containing the entire 5'NCR of HAV. This effect has been described in translation assays before, using a construct containing the HAV 5'NCR fused to the chloramphenicol acetyltransferase coding region (43). Deletions of the first 161 or 354 nt showed increased translation efficiency in BT7-H cells. In the study presented here, replacement of the 5'-terminal 138 nt of the HAV 5'NCR by the 5'-terminal cloverleaf structure of PV eliminated the inhibitory effect of the 5'-proximal HAV sequences on translation in vitro as well. The deletion of pY1 in the region between nt 99 and 138 from HAV RNA also showed no apparent effect on virus replication in vivo (38). A similar inhibitory effect of 5'-terminal sequences in the 5'NCR has been reported to occur with HCV (21, 28, 35, 45). For HAV, and likely for HCV as well, the 5'-terminal sequences are essential for RNA replication (25). Thus, a balance between RNA replication and translation activity may be necessary to support the optimal growth pattern during virus infection. The stimulatory effect seen by replacing the 5'-proximal 138 nt of the HAV 5'NCR with the PV 5'-terminal cloverleaf structure could be due to a functional impact of the PV cloverleaf structure on translation, as suggested for the PV 5'NCR (40). It is more likely, however, that the PV sequences provide stabilization of an important secondary structure of the HAV IRES.
It is noteworthy that relative IRES activity may vary in different cell extracts in vitro and in different cell types used for RNA transfection assays or laboratory infection. Different cell types may vary in the composition of factors that mediate IRES utilization. This effect was seen clearly for the various strains of HCV, where IRESes were utilized with different relative efficiencies in different cells (28), and is well documented for Sabin strains of PV, where translation is restricted in neuronal cells. Our purpose in using HeLa cell extracts for translation of HAV RNA in vitro was to find a convenient system that would support translation of HAV RNA.
In the translation assays described here, HAV RNA was translated efficiently in HeLa cell extracts, derived from cells in our laboratory which are not permissive for growth of HAV. Translation of HAV RNA occurred readily in HeLa cells in vivo as well. Transient transfection of HeLa cells with DNA containing the HAV genome under control of the T7 promoter and coinfection with recombinant vaccinia virus providing T7 RNA polymerase provided ample HAV RNA for efficient translation. These experiments demonstrated that the block for viral growth in HeLa cells is not due to restriction of translation but might be due to failure to support receptor-mediated virus adsorption or failure to support HAV RNA replication.
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ACKNOWLEDGMENTS |
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We thank B. L. Semler for providing the anti-PV VP2 antibody, and we are grateful to Oliver Richards, Xi-Yu Jia, and Larry Blyn for helpful discussions.
This work was supported by Public Health Service grants AI26350 and AI12387 from the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Present address: National Institutes of Health, NIAID, LID, Hepatitis Virus Section, Building 7, Room 200, 7 Center Dr., MSC 0740, Bethesda, MD 20892-0740. Phone: (301) 496-6227. Fax: (301) 402-0524. E-mail: jgraff{at}atlas.niaid.nih.gov.
Present address: Center for Scientific Review, National Institutes
of Health, Bethesda, MD 20892.
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Discussion
References
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J Virol, May 1998, p. 3571-3577, Vol. 72, No. 5
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