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.