Construction of vectors
To construct the d3 vectors AatII and SphI were used to carry out a restriction digest of proA8m1. The enzyme sites were blunted using a DNA Blunting Kit (TaKaRa, Shiga, Japan), after which blunt-end ligation was performed. To construct the d3 + 1026 vector, the HindIII–BglII fragment of proA8m1 was cloned by PCR using a forward primer containing an AatII restriction site in the 5′ terminus, 5′-GAGACGTCAGCTTTAGCAGTAGAC-3′, and a reverse primer containing the SphI restriction site in the 5′ terminus, 5′-TGGCATGCTTTTCAGCTTCCCTCA-3′. The ArtII–SphI fragment was recombined using the restriction site of proA8m1.
The vectors B1, B2, B3, B2-a, B2-b, B2-c, B3-d, B3-e, B3-f, B3-d1, B3-d2, B3-d3 and B3-d4, which have serially truncated HindIII–BglII regions of proA8m1, were constructed using a KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan). Point mutations, G to T (2608 nt), G to T (2614 nt) and G to T (2629 nt), were introduced into the pol gene of proA8m1 in order to suppress production of progeny . The mutations A to T (2126 nt) and T to A (2777 nt) were also introduced into proA8m1. To construct the B1 vector, the 879–1182 bp fragment was deleted from proA8m1 by inverse PCR using the primer sets listed in Table 1. The B2, B3, B2-a, B2-b, B2-c, B3-d, B3-e, B3-f, B3-d1, B3-d2, B3-d3 and B3-d4 vectors were constructed by deleting the 1183–1541, 1542–1904, 1183–1296, 1297–1443, 1444–1541, 1542–1649, 1650–1770, 1771–1904, 1542–1569, 1570–1591, 1592–1611 and 1612–1649 bp fragments, respectively, from proA8m1, using the same method as for B1. The primers used for generation of these vectors are listed in Table 1. To construct the B3-d4inv38 vector, two-stage inverse PCR was performed using the two sets of primers shown in Table 1.
Table 1. List of primers used for construction of vectors
|Name of vector||Forward (5′ 3′) primer||Reverse (5′ 3′) primer|
|d3 + 771||CCACAGGTATTGGGAACCGA||GCGCTTTGGAGACCCGCTAGGA|
|d3 + 293||CACTATCGCCAGTTGCTCCTAG||ACGTCTCCCAGGGTTGCGGC|
|d3 + 467||CCTGACTCTTCCCCAATGGTATC||ACGTCTCCCAGGGTTGCGGC|
|d3 + 353||TACTGGCCATTTTCCTCCTCTG||ACGTCTCCCAGGGTTGCGGC|
|d3 + 206||ACCCTGCTGACGGGAGAAGAAA||ACGTCTCCCAGGGTTGCGGC|
|d3 + 108||TAATGATGCTTTTCCCTTGGAACGTCC||ACGTCTCCCAGGGTTGCGGC|
|d3 + 80||GACTGGGACTACAACACCCAAC||ACGTCTCCCAGGGTTGCGGC|
|d3 + 58||GAGGTAGGAACCACCTAGTCCA||ACGTCTCCCAGGGTTGCGGC|
To construct 57 MLV, MSV, FeLV, XMRV and CasBrE vectors, point mutations were inserted into d3 + 1026 by inverse PCR using the primer pairs shown in Table 1.
To construct the d3 + 771 vector, the 1650–1904 bp fragment of d3 + 1026 was deleted by inverse PCR using the primer set shown in Table 1. The d3 + 293, d3 + 467, d3 + 353, d3 + 206, d3 + 108, d3 + 80 and d3 + 58 vectors were constructed by deleting the 879–1611, 879–1182, 879–1296, 879–1443, 879–1541, 879–1569 and 879–1591 bp fragments, respectively, from d3 + 1026, using the same method as for d3 + 771. The primer pairs listed in Table 1 were used to construct the vectors. To construct d3 + 38, a 38 bp fragment with an AatII restriction site in the 5′ terminus and a SphI restriction site in the 3′ terminus was prepared by annealing the following synthetic single-stranded DNAs: 5′-CCACTATCGCCAGTTGCTCCTAGCGGGTCTCCAAAGCGCGCATG-3′ and 5′-CGCGCTTTGGAGACCCGCTAGGAGCAACTGGCGATAGTGGACGT-3′. To construct d3 + inv38 vectors, a fragment with the inverse sequence of the 38 bp fragment and with an AatII restriction site in the 5′ terminus and a SphI restriction site in the 3′ terminus was prepared by annealing the following synthetic single-stranded DNAs: 5′-CCGCGAAACCTCTGGGCGATCCTCGTTGACCGCTATCACGCATG-3′ and 5′-CGTGATAGCGGTCAACGAGGATCGCCCAGAGGTTTCGCGGACGT-3′. These fragments were inserted into the AatII and SphI restriction sites of proA8m1.
To construct the d4 + 58 and d4 + 1026 vectors, SphI and NdeI were used to carry out a restriction digest of d3 + 58 and d3 + 1026, respectively. The enzyme sites were blunted using a DNA Blunting Kit (TaKaRa), after which blunt-end ligation was performed.
Cell cultures and transfections
NIH3T3 cells were grown in Dulbecco modified eagle medium (Cellgro, Manassas, VA, USA) supplemented with 10% (v/v) FCS (MP Biomedicals, Santa Ana, CA, USA), 50 U penicillin (Gibco, Gaithersburg, MD, USA)/mL, and 50 µg streptomycin (Gibco) at 37°C in a 7% CO2 atmosphere. Cells were seeded with 1 × 106 cells in a 6 cm dish with growth medium minus penicillin–streptomycin. They were transfected the next day with 8 µg of vectors using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) diluted with OPTI-MEM (Invitrogen) according to the manufacturer's instructions.
RT-PCR analysis of viral spliced mRNA in transcripts
Total cellular RNA was isolated from transfected cells using an RNeasy Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer's instructions. After treatment with RNase-free DNase (Qiagen), 2 µg of RNA were added to the RT reaction, which used an oligo (dT) primer (Invitrogen). The spliced mRNA was detected by PCR using Go Taq (Promega, Madison, WI, USA) and specific primers. The primers for detecting spliced mRNA containing the splicing junction region were: s1 forward primer 5′-GAGACCCTTGCCCAGGGA-3′ and s2 reverse primer 5′-TGCCGCCAACGTCTCC-3′. The env coding region was detected by PCR, using KOD-plus (Toyobo) and the s1 forward primer and the u3 reverse primer 5′-TGCGGCTATCAGGCTAAGCAACTTGGT-3′. PCR products were separated on 2% or 1% agarose gels in tris-borate-EDTA or tris-acetate-EDTA buffer, respectively, and stained with ethidium bromide. Negative control samples without the cDNA synthesis step did not yield specific bands.
Sequence analysis of splice variants
To analyze the sequences of splice variants, the relevant electrophoretic bands were extracted from the agarose gel using a QIAquick Gel Extraction Kit (Qiagen), and cloned into a pGEM-T-easy vector (pGEM-T-easy Vector System; Promega). The sequences of the cloned fragment in T-easy vector were amplified using T7 (5′-GTAATACGACTCACTATAGGGC-3′), or sp6 (5′-ATTTAGGTGACACTATAGAA-3′) primers, and a BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA). The sequences were analyzed using an ABI PRISMOR 3100 Genetic Analyzer (Applied Biosystems).