Insect Control: Biological and Synthetic Agents - Index of

348 10: Genetically Modified Baculoviruses for Pest Insect Control
(3) expressing multiple toxins that target different
sites within the insect sodium channel. Related to
this last point, studies on synergistic interactions
between the expressed toxin and chemical pesticides
are also discussed.
10.3.4.1. Alteration of the timing and level of
expression In general, the first generation of
recombinant baculovirus constructs used a single
baculovirus derived very late (e.g., polh or p10) or
late (e.g., p6.9) promoter to drive expression of the
toxin gene (or other foreign gene). These promoters
generally drive exceptionally high levels of protein
expression (i.e., they are very strong promoters), but
they require the products of viral early genes for
activation. Thus, the very late/late promoters are
not activated until a late stage of the baculovirus
replication (Lu and Milller, 1997). At this late stage
of infection, the translational machinery and processing
pathways of the cell may be compromised
(Jarvis et al., 1990). Thus, a large amount of protein
may be produced but not all of the protein will
be functionally active due to poor folding or poor
posttranslational modifications and the protein may
also be retained within the cell due to poor secretion.
Baculovirus early gene promoters, alternatively,
are recognized by host transcription factors
and are activated very soon after the viral DNA is
uncoated in the nucleus. At this early stage of infection,
the processing and secretory pathways of the
cell should be in a less compromised state (in comparison
to the late stage of infection). Thus, it is
likely that a higher percentage of the expressed proteins
will be properly folded, modified, and secreted.
Earlier expression may be especially important for
the proper folding of neurotoxins because they possess
a relatively large number of disulfide bonds
(e.g., the 70 amino acid-long AaIT peptide contains
four disulfide bonds). Early gene promoters,
however, generally drive very low levels of protein
expression (i.e., they are weak promoters).
Several groups have attempted to circumvent the
potential problems associated with protein expression
under very late promoters using: (1) early gene
promoters; (2) dual or chimeric promoters; and/or
(3) constitutively expressed promoters to drive the
expression of a foreign gene. Jarvis et al. (1996) have
constructed a recombinant AcMNPV (ie1-AaIT)
expressing the AaIT gene under the control of an
immediate early gene (ie1) promoter of AcMNPV. In
cultured Sf-9 cells infected with the ie1-AaIT construct,
AaIT was detected as early as 4 h p.i. and
continued to accumulate until at least 24 h p.i. At
24 h p.i., the amount of the expressed AaIT peaked
and was about equal to that expressed by AcAaIT
(AaIT expressed under the very late p10 promoter).
In vitro expression assays indicated that there was
early and abundant expression of AaIT under the ie1
promoter. These findings suggested that the ie1-
AaIT construct should kill infected larvae more
quickly than the AcAaIT construct. The results of
the insect bioassays using neonate H. virescens,
however, did not support this. Time-mortality bioassays
showed that the ie1-AaIT virus killed neonate
H. virescens only about 10% faster than the wildtype
AcMNPV (LT50 of 87.0 versus 96.5 h). In comparison,
AcAaIT killed the neonate H. virescens
about 22% faster than AcMNPV (LT 50 of 75.0 versus
96.5 h) in parallel experiments. Although Jarvis
et al. (1996) were unclear as to the reason(s) for
the slower speed of kill of ie1-AaIT in comparison
to AcAaIT, they speculated that: (1) ie1-AaIT
does not produce a threshold dose of AaIT that
must be reached before kill rates can be enhanced;
(2) duplication of the ie1 promoter usurps limited
cellular transcription factors needed by the endogenous
ie1 promoter for expression of the IE1 protein;
(3) early expression of AaIT in midgut epithelial
cells induces these cells to be shed thereby reducing
the primary infection in these cells and/or dissemination
of the virus into the hemocoel; and (4) the ie1
promoter of AcMNPV has a narrower host range
and/or tissue specificity in comparison to the virus
as a whole. Interestingly, although ie1-AaIT infected
larvae did not die as quickly as AcAaIT infected
larvae, the relative growth rate of the ie1-AaIT
infected larvae was about 13% lower (0.8 versus
0.92 mg per larva per day) than that of AcAaIT
infected larvae. This suggested that even though
they do not die as quickly, the ie1-AaIT infected
larvae may consume less food.
van Beek et al. (2003) have compared the efficacy
of recombinant AcMNPVs expressing either AaIT
or LqhIT2 under various baculoviral early (ie1 or
lef3), early/late (39K), or very late (p10) gene promoters
in larvae of H. virescens, T. ni, and S. exigua.
The larvae were inoculated at both low (approximately
LD50) and high (approximately 100 times
the LD50) doses of each recombinant virus. By
time-to-response bioassays using neonate and third
instar H. virescens or T. ni, and second instar
S. exigua, they found that the ET50 of the recombinant
AcMNPV expressing AaIT under the early hr5/
ie1 promoter led in the majority of cases to a faster
response, but a generalized pattern of response in
terms of the effect of viral dose and instar was not
found. The ‘‘hr5’’ designation indicates that the
baculovirus hr5 enhancer sequence was placed
immediately upstream of the promoter sequence. By
bioassays using third instar larvae of H. virescens,