3.2 | AcMNPV FP had higher budding efficiency than wt
AcMNPV in Hi5 cells
In order to understand why the AcMNPV FP viruses accumulated at such a
high budding frequency compared to the wt AcMNPV, we compared the
budding kinetics between the AcMNPV FP virus AcP13 and the wt AcMNPV
AcP3 in Hi5 cells. We monitored the virus titers of the wt-virus
infection in Hi5 cells during the first 72 h. It was found that AcP13
had a higher budding efficiency than that of AcP3. At 72 h post
infection, there was a significant 3-fold higher BV titer of AcP13 than
AcP3 (Fig. 3A).
3.3 | AcP3-FPSDM delayed FP formation and improved
polyhedra production in Hi5 cells. As we displayed that serial passage
of AcP3 showed rapid fp25k mutation in Hi5 cells (Fig. 2 B), we
then became interested in comparing polyhedra production stabilities
between AcP3 and AcP3-FPSDM that eliminated the 2 A7 MNR sites and the
10th TTAA site of the AcMNPV fp25k . We
conducted 10 passages of AcP3 and AcP3-FPSDM passages in Hi5 cells. AcP3
showed a continuous reduction of polyhedra yield during the 10 passages,
whereas AcP3-FPSDM showed moderate reduction of polyhedra from P0 to P2,
but starting at P3 no significant reduction of polyhedra reduction was
observed (Fig. 3B and D). The reduction of polyhedra yield of AcP3 at
P10 compared to P0 was about 11-fold whereas the reduction of polyhedra
yield of AcP3-FPSDM at P10 compared to P0 was about 2.5-fold. (Fig. 3
D).
3.4 | AcP3-FPSDM had more stable virion occlusion than
AcP3 during Hi5 cell passage. Since fp25k mutations often lead
to reductions in polyhedra yield and virion occlusion [17, 18], and
we found polyhedra yield reduced starting at passage 3, we asked the
question if there was an accompanied virion occlusion reduction of
AcP3-FPSDM. Polyhedra of AcP3 and AcP3-FPSDM from the above polyhedra
yield study were used for the comparison of virion occlusion between the
two viruses. SDS-PAGE and Western blot were carried out with equal
numbers of polyhedra used for each loading. A monoclonal antibody
against AcMNPV major capsid protein VP39 was used to detect virion
contents inside the polyhedra. We found that the anti-VP39 antibody
detected more AcMNPV VP39 (an indication of virions in polyhedra) in the
polyhedra of AcP3-FPSDM than that of AcP3 produced in Hi5 cells during
passage (Fig. 4 A, B). No virion occlusion reduction of AcP3-FPSDM
during the 10 passages conducted in Hi5 cells, whereas about 50% virion
occlusion reduction of AcP3 was observed at P10 compared to P0 (Fig. 4
C).
3.5 | RFLP analysis of AcP3-FPSDM genome. In order to
find out the genetic causes for the FP formation and polyhedra yield
reduction of AcP3-FPSDM passage in Hi5 cells, EcoRI and HindIII RFLP
comparison was performed using DNA purified from BVs at P10 and P0. An
8.7 kbp EcoRI fragment of AcP3-P0 appeared shifted to be 11 kbp in
AcP3-P10, and a 4.98 kbp HindIII fragment in AcP3-P0 shifted to a 5.3
kbp HindIII fragment in the AcP3-P10 genome (Fig. 5A left panel). To
ultimately confirm the copy number of the 287 host DNA insertion in the
AcMNPV genome, Southern blot hybridization should provide the answer.