4 | DISCUSSION
Historically, the mechanism of fp25k mutations was reported as
transposon insertion [7, 11, 12, 22]. Since AcMNPV genome does not
appear to have a transposase gene, the enzymes that catalyze the
insertion of host DNA to fp25k has to be provided by the host
cells (Ayres et al., 1994). These host derived transposable elements
have been identified as TED [23], IFP2 or PiggyBac [24], TFP3 or
tagalong [25], Hitchhiker [26]. Of the 13 TTAA sites of the
AcMNPV fp25k , only four TTAA sites have been reported with host
DNA insertions [27, 28]. Some of these TTAA insertion sites were
found in the fp25k of Galleria mellonella nuclear
polyhedrosis virus (GmMNPV).
This 10th TTAA is also the site of host DNA insertion
of AcMNPV FP virus M5 that was isolated from Sf9 cells [11]. Further
confirmation by site directed mutagenesis demonstrated that this
10th TTAA site is responsible for host DNA insertion
(Fig. 2 B). Since the host transposase has to recognize the TTAA site
first before it cleaves the site for DNA insertion, different cells may
have different transposases with different recognition sequences for
cleavage.
The first TTAA transposition recognition site was found in thefp25k of GmMNPV IFP2 that was obtained during passage in Tn368
cells derived from T. ni ovaries [29]. The recognition site
is determined to be 5’-gggtta-3’ in the fp25k of GmMNPV by
mutagenesis studies [30]. In our studies, the only host DNA
insertion site is at the 10th TTAA of the fp25kof AcMNPV during passage in Hi5 cells (Fig. 2). The upstream sequences
of the 10th TTAA site of AcMNPV fp25k is TAC.
It is highly like that the TACTTA of the 10th TTA is
the recognition site of the transposase active in Hi5 cells. Event
though, both the Hi5 cells and Tn368 cells are derived from T. nithat may have different transposase genes in the genome, only certain
transposases are specifically expressed in different cells. This
transposition site is currently under studies by the site-directed
mutagenesis method.
In addition to host transposon insertions at the TTAA site of thefp25k gene of certain NPVs, insertion of a single nucleotide,
base substitution, and deletion of nucleotides in the promoter regions
or the coding region of fp25k [7, 8, 11, 12]. To our
surprise, in our AcMNPV passage studies in three commonly used insect
cell lines (Sf21, Sf9 and Hi5) only three types of mutations were found:
an A nucleotide insertion at the two A7 MNRs and a 287 bp host
transposable DNA insertion. It is possible for the existence of large
DNA insertions in the fp25k of AcMNPV so we tried to extend the
amplification time in the hope of getting PCR products larger than the
1.5 kbp product for cloning and DNA sequencing. Faint PCR products
larger than 1.5 kbp and smaller than 1.2 kbp were observed but cloning
and PCR sequencing failed to identify any large DNA insertions and other
types of deletions. Plague purification of more FP viruses is required
to pursue in more FP mutation identification.
The Hi5 cell line was originally cloned from T. ni embryos for
propagation of betabaculoviruses that are often more difficult than
alphabaculoviruses to have cells for infection [31]. Later, the Hi5
cells are used for high protein expression due to the larger cell size
than Sf9 and Sf21 cells. As soon as AcMNPV enters Hi5 cells, the
transposase in the cells recognizes the TTAA site and insert the 287
host DNA in the fp25k to produce the FP virus that results in
lower polyhedrin expression and higher BV production than the wt AcMNPV
(Fig. 2 B. Fig. 3). It is likely that the BV from the FP virus
outcompeted that from the wt AcMNPV. This high BV budding efficiency of
AcMNPV FP is likely the cause of the rapid accumulation of FP and
diminishing wt AcMNPV during Hi5 cell infection (Fig. 3).
The success of obtaining AcP3-FPSDM virus that does not have the 2 A7
MNRs and the 10Th TTAA site of the fp25 is a
major achievement in this project. We also demonstrated that during Hi5
cell passage, the fp25k showed no MNR mutations and host DNA
insertions by PCR sequencing the fp25k locus as well as other
deletion mutations. The A adding at the A7 MNRs is likely due to
replication slippage as the viral DNA polymerase pauses at the MNR
[32]. The isolation of AcP2, AcFP1and Acp13 in Sf21 [17] and
only host DNA insertion and no MNR mutations in this paper during AcMNPV
passage in Hi5 cells may suggest that host DNA replication and error
repair systems are involved in AcMNPV DNA replication. This notion is
supported by a report that host DNA polymerase is involved in AcMNPV
replication [33]. If this is true, in some Sf21 cells, AcMNPV may
use its own DNA polymerase for genome replication that leads to the MNR
slippage replication errors in the fp25k , whereas in Hi5 cells,
it may use Hi5 host cellular DNA polymerase for genome replication. The
property of the Hi5 DNA polymerase is unknown but cellular DNA
polymerases of Saccharomyces cerevisiae and human have a minimum
of a ray of 8 base monopolymer for slippage replication [34, 35].
Although no fp25k mutations of AcP3-FPSDM have been detected in
Hi5 cell passage, this does not eliminate other DNA mutations in the
viral genome.
Comparing viral genome between AcP3-FPSDM P0 and P10 showed difference
in EcoRI and HindIII RFLP (Fig. 5). However, no major DNA deletion or
insertion was deduced from the RFLP comparison. It is likely that the
small difference in EcoR1 and HindIII fragment mobility changes are due
to base substitution that introduced more EcoRI and HindIII restriction
sites into the AcP3-FPSDM P10 genome. Virion occlusion essays also
supports that no fp25k mutations of AcP3-FPSDM P10 since an
increased polyhedra yield was observed compared to AcP3 P10 (Fig. 4).
This also supports that virion occlusion occurred in Hi5 cells infected
with AcMNPV FP viruses discovered early [18]. The reduction of
polyhedra yield in Hi5 cell infection by AcP3-FPSDM P10 may suggest
mutations in other AcMNPV genes such as da26 and 94k for
few polyhedra generation [22, 36, 37].
The hallmark of the FP phenotype is its ability to increase virus titer
in cell culture [9, 13, 38-40]. In this paper, we confirmed the
reports of high virus budding from these authors. In addition, we showed
our AcP3-SDM virus has high virion occlusion efficiency for insect pest
control. We are currently investigating the mechanism of this high virus
budding mechanism.