The Rep gene open reading frame (ORF) (GenBank: U57457.1) was amplified by PCR as shown in Figure 1a. Lines 2–4 (Figure 2a) show a band of approximately 1,047 bp corresponding to PepGMV Rep protein gene. After PCR amplification, Rep was cloned into the expression vector pTracer-SV40. Ligation was confirmed by double digestion with SpeI and KpnI enzymes. Figure 1b shows the gel electrophoresis of the double digestion product. As expected, two bands were observed in each of the samples. One corresponds to the linearized pTracerSV40 vector (Figure 1b, lines 2–4) which has a size of 4.2 kb and a second band corresponding to Rep (Figure 1b, lines 2–4).

These results confirmed the ligation of PepGMV-Rep gene into the pTracer-SV40 expression vector, obtaining the following constructions: pTracer-SV40:Rep and pTracer-SV40:RepATG^-.

In order to indicate if Geminivirus PepGMV is able to replicate in mammalian cells, the PepGMV A component was transfected into 3T3L1 fibroblast cells (as described in the Experimental section). PepGMV A DNA presence was confirmed by PCR amplification using the primers for the Rep gene and the JM23 and JM24 primers (Table 1). Figure 2 shows the PCR amplification of PepGMV A DNA-transfected cells. Again, a band of 1,047 bp corresponding to Rep is observed in samples taken at 24 and 144 h post transfection. Additionally, a band of 280 bp corresponding to the common region of PepGMV is observed, which confirms the presence of Geminivirus DNA inside the cells. In addition, RT-PCR was performed in order to verify Rep gene transcription in transfected cells (Figure 3a). The level of Rep transcription was too low to be clearly seen. Thus a re-amplification was made to corroborate the presence of the Rep transcript (Figure 3b). A band corresponding to Rep gene was observed after re-amplification, indicating that Rep transcription was taking place in transfected cells but with a low efficiency. Therefore geminivirus PepGMV was recognized by mammalian cell machinery and Rep transcription occurred.

To determine if the Geminivirus affects the cell growth, transfected cells with PepGMV A component were counted. Figure 4 shows the number of cells at 0, 48 and 144 h after transfection. A difference in the cell number was observed in cells transfected with the PepGMV in contrast with control cells, indicating that the presence of PepGMV is affecting cell growth.

Expression of green fluorescent protein (GFP) was detected in order to verify the construction transfection into cells (Figure 5). Cells transfected with the pTracer-SV40:Rep (Figure 5a) reacted and fluoresced in contrast with control cells in which no fluorescence was observed (Figure 5b). This indicates the insertion of the construction and the expression of the GFP correlates with Rep expression.

Additionally, Rep expression was confirmed by RT-PCR. Figure 6 shows the RT-PCR analysis of transfected cells at 72 h post transfection. A band corresponding to Rep gene was observed in cells transfected with both pTracer-SV40:Rep and pTracer-SV40:RepATG⁻ constructs. In contrast, with control and vector-transfected cells (Figure 6). This result indicates that Rep gene is being transcribed in transfected cells (Figure 6).

The dimeric clone of Pepper Golden Mosaic Virus (PepGMV) (Tamaulipas isolate) was used in this work. PepGMV genome has been previously described [6].

The Rep gene open reading frame (ORF) was amplified by PCR using the oligonucleotides shown in Table 1. The PepGMV Rep sequence used was obtained from the National Center for Biotechnology Information (NCBI) database (GenBank: U57457.1). To clone the Rep gene in the expression vector pTracer™-SV40, the sites for SpeI (extreme 3') and KpnI (extreme 5') restriction enzymes were used. PCR amplification conditions were as follows: 95 °C for 1 min, 30 cycles of 95 °C for 30 s and 68 °C for 2 min, and a final extension of 68 °C for 2 min. In addition, a negative translation control was generated by changing the Rep-gene initiation codon sequence (ATG) for the sequence ACT.

After PCR amplification, the 1,047 bp gene was cleaned with the Wizard® PCR Preps DNA Purification System (Promega) and cloned in the intermediate pGem®-T Easy vector (Promega) using the manufacturer’s procedures. The pTracer™-SV40 vector (Invitrogen) was double digested with SpeI and KpnI enzymes (Fermentas) in order to linearize it and clone the Rep gene. Insert and vector purification was performed using the Silica Bead DNA Gel Extraction kit (Fermentas) and ligation reaction was done using the ligase T4 (Invitrogen) and a 4:1 insert/vector molar ratio. The insert presence was confirmed by double digestion with SpeI and KpnI enzymes (Fermentas).Gene correct orientation was confirmed by sequencing (Genetic Analyzer 3130, Applied Biosystems).

3T3L1 mice fibroblast cells (ATCC No. CL-173) were grown in Dubelco’s Modified Eagle Medium (DMEM, Gibco) supplemented with penicillin (62.1 mg/L; Sigma), streptomycin (100 mg/L; Sigma), anphotericin (250 µg/L; Gibco), and 5% of calf serum (Biowest). The cells grew in an incubator at 37 °C and a 5% CO₂ atmosphere.

Cell transfection was performed by calcium-phosphate precipitation method [20]. Twenty four hours before transfection, cells were seeded into 60 mm plates at a concentration of 1 × 10⁵ cells/mL [20]. The culture medium was replaced with fresh medium one hour before transfection. The phosphate-DNA precipitation was performed as follows: 25 µg of plasmid DNA were mix with 100 µL of 2.5 M CaCl₂. This solution was diluted with 1/10 TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) to a final volume of 1 mL. One volume of this 2× Ca/DNA solution was added drop by drop to an equal volume of 2× HEPES buffered saline solution. The resulting solution was incubated at room temperature for one minute prior to adding to the cells medium. One hundred microliters of precipitate per milliliter of culture medium were added to the cells Cells were exposed to the precipitate for 6 h at 37 °C and 5% CO₂ atmosphere. After this time, the culture medium was replaced with fresh medium and the cells were incubated for 6 days at 37 °C and 5% CO₂ atmosphere. Cells were transfected with pTracerSV40:Rep and pTracerSV40:RepATG^-constructions. Additionally, the PepGMV A component was transfected in order to probe if PepGMV is able to replicate in mammalian cells.

3T3L1 cells transfection with pTracerSV40:Rep and pTracerSV40:RepATG⁻ was verified by detection of green fluorescent protein (GFP) by fluorescent microscopy. Additionally, Rep gene expression was verified by RT-PCR for treatments72 h after transfection. Total RNA was extracted from transfected cells using the SV total RNA isolation system (Promega).DNA first strand was generated by the First strand cDNA synthesis kit (Fermentas). The same treatment was applied to cells transfected with PepGMV A component. cDNA PCR amplification was performed with the oligonucleotides shown in Table 1 and the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GADPH) and β actin were used as expression controls [21,22].