Identification of Rhopalosiphum Padi Virus 5′ Untranslated Region Sequences Required for Cryptic Promoter Activity and Internal Ribosome Entry

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.

secondary structure within this region, with only 40%-60% nucleotides being base-paired [16], which may permit access to ribosomes and protein factors for internal translation initiation.

TAAG Motif in L3 Is More Critical for RP110 Promoter Activity in the Baculoviruses Infected Sf21 Cells
Prior studies indicated that IRES elements derived from cellular mRNA, e.g., p27 kip1 [21] and pim-1 [22], possess cryptic promoter activity. Interestingly, a previous study confirmed that promoter activity is present in the cDNA sequence corresponding to the HCV IRES [23]. Our study provides another example of a cDNA sequence of an IRES element derived from RNA viruses acting as a promoter. A striking characteristic of a baculovirus late or very late promoter is its short length, e.g., the respective sizes of the polyhedron promoter and p10 promoter are about 89 and 114 bp. Thus, our finding of RP110 promoter is merely 110 bp in size is consistent with the size of a baculovirus late or very late promoter. Further, it is known that the transcription activity of the baculovirus late or very late promoter depends on baculovirus early gene expression. Thus, our finding that Sf21 cells transfected with pCMV-DRhirE or pCMV-DRhir(L3-4)E did not elicit red or green fluorescence (data not shown), suggests that the RP110 promoter acts as a baculovirus late or very late promoter, and its transcription activity depends on baculovirus infection. To further examine whether L3, L4 tandem repeat of TAAG is necessary for the RP110 promoter activity, the L3L4 deleted transfer vector pCMV-DRhir(L3-4)E(TAAG∆) was constructed ( Figure 3); and used to generate the recombinant baculovirus vCMV-DRhir(L3-4)E (TAAG∆).
The vCMV-DRhir(L3-4)E (TAAG∆) infected Sf21 cells did not emit any green fluorescence ( Figure 4B), which is contrast to the vCMV-DRhir309E infected Sf21 cells ( Figure 4A). This result indicated the L3, L4 tandem repeat TAAG motifs in the cDNA of RhPV IRES is necessary and sufficient enough to be a cryptic promoter in baculovirus infected Sf21 cells. Further investigation was directed to differentiate which of the L3 and L4 TAAG motifs is more important for the cryptic promoter activity. In this regard, two plasmids, in which a single mutation in either L3 or L4 were constructed without changing the size of  Figure 4F). Thus, the L3 TAAG motif was found to be more critical for the promoter activity in the baculoviruses infected Sf21 cells. were separated by 12% SDS-PAGE and then electrotransferred onto PVDF membranes, which were then incubated with rabbit anti-GFP polyclonal antibody followed by an HRP-conjugated goat anti-rabbit secondary antibody. Detected protein bands were visualized on X-ray film using a Western blot chemiluminescence kit. Molecular mass markers (kDa) are indicated at left and the arrow indicated the EGFP protein band.

RP110 Acts as an IRES in CHO Cells
A previous study indicated that a small fragment in the RhPV 5′UTR IRES, corresponding to nts 425-579, retains significant IRES activity in mammalian, plant and insect translation systems [24]. Furthermore, Groppelli et al. demonstrated that a region within the RhPV 5′UTR IRES can direct internal translation initiation [24]. The M300/429 fragment that spans the RP110 promoter region also exhibited IRES activity in all of the tested translation systems [24]. Our previous studies also demonstrated that the 579 nts RhPV 5′UTR function as an IRES, but not as a promoter, in mammalian cells [19]. Thus, it would be interesting to explore whether the 110 nt RP110 promoter or any other constructs listed in Figure 1A can function as an IRES element in mammalian cells, given that no promoter activity was found in vCMV-DRhir(L1-2)E, vCMV-DRhir(L5-6)E or vCMV-DRhir(L6)E infected Sf21 cells (Figure 2). Transient transfection of CHO cells with any of the truncated constructs listed in Figure 1 emitted red fluorescence and green fluorescence ( Figure 5A-F). These transient transfection results support previous findings obtained using a rabbit reticulocyte lysate (RRL) in vitro translation system, which indicated that a good part of the RhPV 5′UTR may be deleted without significantly affecting its IRES activity. Interestingly, the 110 nt RP110 promoter can mediate IRES activity in CHO cells ( Figure 5D). This result implies that the novel baculovirus RP110 promoter identified in the RhPv 5′UTR cDNA can function as an IRES in mammalian cells and may thus facilitate the development of a "shuttle" bi-cistronic baculovirus gene expression or delivery vector. Pictures were taken in the same field using a conventional rhodamine channel (Red) with a 510/560-nm filter set and a FITC channel (Green) with a 450/490-nm filter set. All pictures were taken with the same exposure time (900 and 400 ms for EGFP and DsRed, respectively). Scale bar is 50 μm.
Identification of this novel RP110 promoter in the RhPV 5′UTR cDNA will impact baculovirus biology basic research and applications using baculovirus-based gene expression and gene delivery vectors. Among the six TAAG motifs in the DNA sequence of RhPV 5′UTR IRES, only the L3, L4 tandem repeat TAAG possess promoter activity in baculovirus-infected Sf21 cells. It has been proposed that if TAAG sequences in an appropriate context are present within a gene, transcription can be initiated within that gene. Linker-scan mutational analyses indicated that the TAAG motif is absolutely essential for transcriptional initiation to occur and that it appears to be the primary determinant of transcriptional initiation from baculovirus late promoters [25]. For example, the vp39 late promoter has three transcriptional initiation sites, each starting within a TAAG site. The identified 110 bp fragment may serve as a model for the artificial design of enhanced baculovirus promoters. Furthermore, baculoviruses are not only used in recombinant protein production, but are also recognized as potential mammalian gene delivery vectors. Expression of a gene of interest in a baculoviruses-based gene delivery vector is generally controlled by insect-inactive mammalian promoters, which complicates isolation and quantification of the recombinant virus. Use of this short functional RP110 promoter to drive recombinant gene expression in insect cells will facilitate virus selection and titer determination. By virtue of its IRES activity in mammalian cells, the transfected cells can be easily identified by the evaluation of downstream selection markers.

Recombinant Virus Production and Titer Determination
Using Cellfectin (1 μL), the Sf21 cells (2 × 10 5  , these recombinant viruses were identified by X-gal staining according to the manufacturer's protocol. The recombinant viruses were selected and purified by a series of end-point dilutions. Sf21 monolayers were used for virus propagation, and all viral stocks were prepared and titers determined according to the end-point dilution as described before [26].

Western Blot Analysis
Proteins were separated by SDS-PAGE on a mini Protein III system (Bio-Rad, Hercules, CA, USA). After SDS-PAGE fractionation, proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The resulting membranes were blocked with Tris-buffered saline (TTBS; 100 mmol/L Tris (pH 7.4), 100 mmol/L NaCl, and 0.1% Tween 20) containing 5% (v/v) non-fat, dry milk at room temperature for 1 h with gentle shaking. Subsequently, the membrane was incubated with 1:3500-diluted anti-EGFP antibody (BD Biosciences, San Jose, CA, USA) for the cryptic promoter activity in Sf21 cells. The antibody was diluted in TBS with 0.5% (v/v) non-fat, dry milk and incubated shaking at 4 °C overnight. Unbound antibodies were removed by 3 washes each of 10 min in TTBS buffer at room temperature with shaking. Then the membrane was incubated with 1:2500-diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (BD) for 1 h at room temperature. The HRP on the membrane was detected by an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) following the protocol provided by the manufacturer (Fusion-SOLO, Newberg, OR, USA).

Transfection of CHO-K1 Cells
Transfections of CHO-K1 cells were performed using Lipofectine reagent (Invitrogen). The cells (at 5 × 10 4 /well) were plated onto 24-well plates. Before transfection, the cells were repeatedly washed with serum free media to remove all traces of sera. 1 μg of plasmid was diluted in 100 μL serum-free DMEM medium, and then Lipofectine reagent (1 μL) was added. The DNA-Lipofectin mix was incubated for 30 min for DNA-Lipofectin complex formation. Then the DNA-Lipofectin complex solution was transferred to the cells at a total volume of 500 μL by adding serum-free medium. After 5 h, the medium was removed and 500 μL fresh medium with 10% fetal bovine serum was added. One day post-transfection, these transfected cells were observed under fluorescence microscopy (Nikon, Shinagawa, Tokyo, Japan).