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Short Communication |
United States Department of Agriculture, Agricultural Research Service, 1102 S. Goodwin Avenue, University of Illinois at Urbana/Champaign, Urbana, IL 61801, USA
Correspondence
Leslie Domier
l-domier{at}uiuc.edu
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ABSTRACT |
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The viruses in this family possess large messenger-sense, single-stranded RNA genomes and share physico-chemical properties with members of the Picornaviridae. However, rather than expressing single, large polyproteins with structural proteins located proximal to the amino termini, the RhPV genome contains two large open reading frames (ORFs) that express nonstructural and capsid polyproteins from 5'- and 3'-proximal ORFs, respectively (Moon et al., 1998). Both ORFs are preceded by internal ribosome entry sites (IRESs) that are structurally and functionally different (Domier et al., 2000; Kanamori & Nakashima, 2001; Wilson et al., 2000b; Woolaway et al., 2001). The RhPV 5' IRES is located upstream of the initiation codon of the first ORF and directs translation initiation at AUG codons in animal, insect and plant cell extracts (Woolaway et al., 2001). The intergenic (IG) IRESs of RhPV and CrPV contain and direct translation initiation from CCU codons in the second ORF (Domier et al., 2000; Wilson et al., 2000b). Similarly, the IG IRES of PSIV initiates translation at a CUU codon (Sasaki & Nakashima, 1999). Many primary and secondary structural features of the IG IRESs of members of the Dicistroviridae are conserved, including three pseudoknots that are required for activity (Kanamori & Nakashima, 2001). The use of the noncanonical initiation codons is explained by the finding that the CrPV IG IRES initiates translation in a process that does not involve methionyl-tRNA and is independent of translation initiation factor eIF2
(Wilson et al., 2000a). In addition, the activity of the CrPV IG IRES is stimulated by treatments that result in increased phosphorylation of eIF2 (Fernandez et al., 2002), conditions likely to occur late in virus infection due to the physiological stress of virus infection and/or the accumulation of virus-derived, double-stranded RNAs. The goal of these studies was to compare the in vivo activities of the two RhPV IRESs and to examine the effects of perturbations in the structure of the IG IRESs, including the 3'-proximal pseudoknot, on in vivo activity, initiation complex formation and protein binding.
To study the activity of the RhPV IRESs in insect cells, we established a system for the in vivo transcription and translation of DNA templates that used a recombinant baculovirus to express bacteriophage T7 RNA polymerase in Spodoptera frugiperda (Sf9) cells. Similar systems have been developed by others (van Poelwijk et al., 1995; Yap et al., 1997). For these studies, the bacteriophage T7 RNA polymerase gene was amplified from Escherichia coli JM109 DE3 DNA using oligonucleotide primers that added BamHI and EcoRI recognition sites to the 5' and 3' ends of the gene, respectively. The resultant DNA was then digested with BamHI/EcoRI, ligated into pFastBAC (Invitrogen) and recombined with the baculovirus, as recommended by the manufacturer. To test the ability of the baculovirus-expressed T7 RNA polymerase to transcribe a transfected plasmid template, 106 Sf9 cells were infected with the recombinant baculovirus expressing T7 RNA polymerase at an m.o.i. of 100. After 24 h, the cells were transfected using Cellfectin reagent (Invitrogen) with 1 µg plasmid DNA containing either the firefly or Renilla luciferase genes located downstream of the T7 promoter (Promega). Luciferase activities were determined 24 h after transfection and were about 1000- (firefly luciferase) to 5000-fold (Renilla luciferase) higher than the negative controls (data not shown), as assayed using the Dual Luciferase Reporter Assay system (Promega) and a scintillation counter or Turner Designs TD-20/20 luminometer.
For comparison of the in vivo activities of wild-type and mutant RhPV IRESs, we constructed bicistronic plasmids containing the Renilla luciferase gene downstream of a T7 RNA polymerase promoter, followed by an RhPV IRES and the firefly luciferase gene (Fig. 1). The activities of the two enzymes were assayed as above. Each experiment (transfection and luciferase assay) was performed twice. Renilla luciferase was used as a transfection control by normalizing the activity of the linked firefly luciferase to that of Renilla luciferase. The bicistronic plasmid pRL17FL contained the RhPV IG IRES inserted between the Renilla and firefly luciferase genes. In this plasmid, the coding sequence of the firefly luciferase gene was fused in-frame with the ninth codon of RhPV ORF2 (Domier et al., 2000). The firefly luciferase activity produced by pRL17FL was arbitrarily set to a value of 1·0 and all other firefly luciferase activities were expressed as a fraction of the pRL17FL firefly luciferase activity. As a negative control, the wild-type RhPV IG-IRES was replaced with a mutant IG IRES that contained two nucleotide substitutions that destabilized the 3' pseudoknot (mutation T21; Domier et al., 2000) to make plasmid pRL21FL (Fig. 1). Transcripts containing this mutation failed to initiate translation of RhPV ORF2 in the rabbit reticulocyte lysate (RRL) in vitro translation system (Domier et al., 2000). In extracts of Sf9 cells transfected with pRL17FL, the firefly luciferase activity was 36-fold higher than cells transfected with a plasmid containing only a T7 promoter and the Renilla luciferase gene and 4-fold higher than that produced by cells transfected with pRL21FL (Fig. 1). These results showed that the RhPV IG IRES directed internal initiation in Sf9 cells. In addition, it confirmed the in vitro observations with RhPV and PSIV that showed that the 3' pseudoknot was required for IRES activity (Domier et al., 2000; Kanamori & Nakashima, 2001).
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). It is possible that the relative activities of the two RhPV IRESs determined in this in vivo system may not reflect the activities of the IRESs in RhPV-infected aphids cells, since baculoviruses express antiapoptotic proteins (Clem et al., 1991; Crook et al., 1993) and, being DNA viruses, do not accumulate double-stranded RNA late in infection that could stimulate the activity of the IG IRES (Fernandez et al., 2002). In addition, Woolaway et al. (2001) reported that the activity of the RhPV 5' IRES was increased when 100 nt, including the AUG initiation codon at position 580, were deleted from the 3' end of the 5' IRES. Hence, it is possible that a truncated 5' IRES might have a greater activity than the sequence used here.
To test for interactions between the two RhPV IRESs, the 5' IRES was inserted upstream of the Renilla luciferase in pRL17FL to make p5'RLIGFL, which mimicked the structure of the RhPV genome. In extracts of Sf9 cells transfected with p5'RLIGFL, the activity of firefly luciferase was about 70 % of the activity of pRL17FL. The reduced activity of the IG IRES when in cis with the 5' IRES could have resulted from competition between the two elements for ribosomal subunits or other translation initiation components. Similar inter-IRES competition was observed in bicistronic plasmids that contained the 5' IRESs of Equine rhinitis B virus and Encephalomyocarditis virus (Hinton & Crabb, 2001). Because of the stimulation of IG IRESs activities of members of the Dicistroviridae by stresses that would probably inhibit translation from the 5' IRESs (Fernandez et al., 2002), the competition, if it exists in RhPV-infected aphid cells, is likely to be relieved late in infection.
The interactions of the RhPV 5' and IG IRESs with RRL proteins were compared using in vitro transcripts labelled with [-33P]UTP. Plasmids containing the RhPV 5' (nt 1–790) and IG (nt 6806–7283) IRESs were constructed, transcribed in the presence of [-33P]UTP (Sambrook & Russell, 2000) and cross-linked to proteins by UV light (Niepmann et al., 1997). Transcripts of the coding region of a modified green fluorescent protein (mGFP) (Haseloff et al., 1997) were used as negative controls. The labelled RNAs were incubated for 10 min at 30 °C in 25 µl RRL (Promega) supplemented with 110 mM KCl, 1·0 mM Mg2+ and 4·0 mM guanylyl-imidodiphosphate (GMP–PNP), exposed to 254 nm UV light for 60 min on ice and degraded by the addition of RNase A (1 µg ml-1). Proteins were precipitated with trichloroacetic acid, solubilized in 6 M urea, run on SDS-polyacrylamide gels and visualized with a Molecular Dynamics Phosphorimager. In these experiments, the RhPV IG IRES preferentially labelled a protein of approximately 68 kDa (Fig. 2). The 5' IRES preferentially labelled protein bands of approximately 30 and 36 kDa more intensely than the other two RNAs. In addition, protein bands of 36, 44, 48 and 120 kDa were labelled by all three RNAs. The difference in labelling by the two RhPV IRESs likely reflects differences in the mechanisms by which they interact with host proteins in translation initiation. The identities of the proteins labelled by the RhPV IRESs remain to be determined. Since translation initiation from the CrPV IG IRES can occur in the absence of initiation factors (Wilson et al., 2000a), the protein labelled by the RhPV IG IRES probably was not one of the initiation factors. The protein could represent double-stranded RNA-dependent protein kinase, PKR, which has an apparent mass of 68 kDa on SDS-polyacrylamide gels and is bound by RNAs of other viruses. For example, Epstein–Barr virus small RNA EBER-1 and adenovirus VA RNA1 specifically bind and inactivate PKR, thereby preventing the shutdown of translation in response to virus infection (Katze, 1995). It is also possible that the labelled proteins represent ribosomal proteins that come into close contact with the IRESs during translation initiation.
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-33P]UTP as above, incubated with 25 µl RRL for 10 min in the presence of 110 mM KCl, 1·0 mM Mg2+ and 4·0 mM GMP–PNP, as described by Niepmann et al. (1997), and sedimented through 10–30 % sucrose gradients. The resulting gradient fractions were either counted with a scintillation counter or separated either on denaturing urea polyacrylamide or on 10 % SDS-polyacrylamide gels. Sucrose gradients of RRL incubated with wild-type RhPV IG IRES contained two radioactive peaks (Fig. 3). The radioactive peak at the top of the gradient contained short RNA species and unincorporated nucleotides (Fig. 3B). The peak that sedimented the fastest corresponded to 80S ribosomal subunits. Polyacrylamide gel analysis of sucrose gradient fractions showed that the peak that cosedimented with the 80S subunits contained intact RhPV IG IRES. The effects of deletions in stem-and-loop (SL) structures 1 and 2 (D6, D7 and T8, Fig. 3A; Domier et al., 2000) on 80S complex formation were evaluated also. Like intact RhPV IG IRES, deletion mutant D6, which lacked the first SL, formed normal levels of 80S complexes. However, mutants D7 and T8, which lacked all of SL1 and large portions of SL2, failed to form 80S initiation complexes (Fig. 3C). SL2 contains sequences conserved among members of the Dicistroviridae and a portion of one of the three predicted RhPV pseudoknots. Hence, deletions D7 and T8 could dramatically change the structure of the RhPV IG IRES. Hence, these studies also indicated that SL1 is not required for 80S preinitiation complex formation but that intact SL2 is required. These results are similar to those reported from in vitro studies with RhPV and PSIV (Domier et al., 2000; Kanamori & Nakashima, 2001).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Crook, N. E., Clem, R. J. & Miller, L. K. (1993). An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif. J Virol 67, 2168–2174.
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Received 16 August 2002;
accepted 30 October 2002.
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