2 | EXPERIMENTAL SECTION
2.1 | Insect cells lines and viruses. Two insect cell lines BTI-TN-5B1-4 (Hi5) derived from Trichoplusia ni and -SF21AE (Sf21) derived from Spodoptera frugiperda were cultured in TNM-FH medium with 10% fetal bovine serum at 27 °C. AcP3 was a plaque purified strain of wt AcMNPV E2. AcP13 was a FP isolate [17].
2.2 | AcP3 fp25k gene engineering. In order to mutate the hyper-mutable “hot” spots of the fp25k, we carried out two rounds of site-directed mutagenesis (SDM) on a transfer vector (pGEMT25k) containing the wild type fp25k sequence which was PCR cloned from AcP3 with the primer pair 25kPU (5’-GAATTCGAGAAACACGTTGGACGG-3’, F25kPD-R (5’-AAGCTTGTTCGTCTTCGGTTGTACTG-3’) (Fig. 1). The primers used for the SDM and the “hot” spots mutated plasmid was named pAcFPSDM. All the newly constructed plasmids described above were sequenced to ensure desired sequence without any mutation. To produce the virus with the “hot” spots eliminated, Sf21 cells were co-transfected with pAcFPSDM and AcP3-FPGFP DNA to produce AcP3-FPSDM that was purified by three rounds plaque assay in Sf21 cells (Fig. 1).
2.3 | AcMNPV BVs budding assay. AcP3 and fp25kmutant virus AcP13 were used to infect equal number of Hi5 cells (5×105) at a multiplicity of infection (MOI) of 10 p.f.u/cell in the 6-well dishes and the budded viruses were harvested at 2 hr p.i., 24 hr p.i., 48 hr p.i. and 72 hr p.i. from which each 300 µl BVs were used to extract viral DNA [19]. Equal volume of ddH2O (10 µl) was used to dissolve the extracted DNA and equal volume of completely dissolved DNA was used as templates in the real-time PCR quantification of AcMNPV genome copies with a pair of specific primers pp34F (5’-GTT GGA AAC CGC TAA AGA TGT-3’) and pp34R (5’-AAG TCG GGT AAC GAG TCT GTA A-3’) and a SYBR®green Supermix (BIO-RAD, Hercules, USA) in a Bio-RAD iCycler iQTM system [20]. AcMNPV DNA of known concentrations were serially diluted to build a standard curve for the estimate of AcMNPV genome copy numbers and each real-time PCR was performed in triplicates (n=3).
2.4 | Serial passage of AcP3 and AcP3-FPSDM in Hi5 cells. The engineered virus AcP3-FPSDM and the wt AcP3 virus were serially passaged in Hi5 cells. At each passage, 50 µl cell culture media containing BVs from a previous cell infection were used to infect cells at a density of 5×105 cells/dish in 2 cm cell culture dishes. The development of FP was monitored by phase contrast microscopy until almost all the cells were infected. The BVs were harvested for infecting Hi5 cells in the next passage. A total of 10 passages have been conducted, and at each passage, the cells in the dishes were lysed with 1 ml 0.5% SDS to release the polyhedra for later polyhedra yield and virion occlusion analyses.
2.5 | Comparison of polyhedra yield and polyhedra virion content between AcP3 and AcP3-FPSDM. A hemocytometer was used to enumerate the polyhedra harvested from each passage and the yield differences were analyzed by the Student’s T test in Excel (Microsoft). The polyhedra were purified by a 30% (w/v) sucrose cushion centrifuge at 16,100× g for 3 min followed by washing once in the 0.5% SDS by centrifugation at 5,000 × g for 5 min each. An equal number of polyhedra (1×106) were processed for SDS-PAGE and Western blot. The polyhedra were dissolved in 0.1 M Na2CO3 to dissolve the polyhedra to release the virions. SDS-PAGE was used to determine polyhedra loading and Western blot with an anti VP39 monoclonal antibody was used to detect virion present in polyhedra to calculate relative virion content in polyhedra (Wang et al., 2009). The gels and membranes were photo-documented and the signal intensity of each bands or integrated density values (IDV) was quantified by the AlphaImager® HP Imaging System (Cell Biosciences). Each data was read 4 times in order to calculate the average (n=4). Relative ratio IDV of VP39/polyhedrin were tested by the Student’s T test using Excel (Microsoft)
2.6 | PCR and RFLP analysis of DNA from AcP3-FPSDM. A primer pair (FP-F, 5’-ATGCATAGCAATGTCTTC -3’ and FP-R, 5’-TTTACGCACCATATACGC-3’) was used to identify fp25k mutants by PCR analysis. DNA from BVs harvested at different passages was used as template in the PCR. Un-passaged AcP3 DNA was used as positive control with an expected amplicon of 1.2 kbp of an intact fp25k . The amplified DNA products were analyzed by agarose gel electrophoresis. Amplified DNA resolved by agarose gel electrophoresis was purified by the glassmilk method for direct DNA sequencing using primer FP25-R. In order to PCR sequence the fp25k promoter and 3’UTR region, the two primer pairs 25kPU-F/25kPU-R and 25kPD-F/25kPD-R were used for PCR sequencing that were originally used for the fp25k upstream and downstream fragments PCR cloning for the transfer vector pGEMT25kGFP. Restriction Fragment Length Polymorphism (RFLP) was employed to detect DNA insertion or deletion in the genome of serially passaged AcP3-FPSDM by comparing to the original AcP3-FPSDM without passage. Viral genomic DNAs of passaged and un-passaged AcP3-FPSDM (0.4 µg/each digestion) were cleaved by EcoRI or HindIII individually for 17 h. The digested DNAs were separated by 0.6 % agarose gel electrophoresis at 90 v for 1 h for analysis of large viral DNA fragments. In order to investigate if smaller EcoRI and HindIII fragments contained fp25k , more DNA (0.8 µg/each digestion) was digested with EcoRI or HindIII individually using conditions recommended by NEB (Ipswich, MA), and separated on an 1% agarose gel at 80 v for 40 min to prevent REN fragments running out of the agarose gel. Ethidium bromide staining was used to visualize the DNAs using an Imaging system (BioDoc-It™). The separated DNA in the agarose gels were transferred to a nylon membrane for Sothern hybridization analysis.