1 | INTRUDUCTION
Baculoviruses are insect specific DNA viruses with a double-stranded
circular genome in the size range of 80-180 kbp, which were grouped into
four genera, alpha-, beta-, gama- and deltabaculovirus [1, 2]. They
are widely used in insect pest control in agriculture and forestry to
reduce chemical reliance for food and fiber production [3]. They are
also used in exogenous gene expression in insect cells for vaccine
production [4]. Infection of a susceptible insect cell by a
baculovirus starts by viral entry and transport to the nucleus where the
viral replication takes place. Early genes are transcribed by the host
RNA polymerase II. After early gene transcription, the viral genome is
replicated by the viral DNA polymerase. Late genes are transcribed by
the viral RNA polymerase after DNA replication. Some of the late gene
products are dedicated to the assembly of nucleocapsids that acquire an
envelope from the host cell to form the infectious virions. Some late
genes such as the polyhedrin gene (polh ) are highly expressed in
the late phase of cell infection. High polyhedrin expression leads to
the formation of polyhedra that occlude the infectious virions and
provide protection of virions.
Baculoviruses during cell infection produce two phenotypically distinct
virion forms: those that are retained in the nuclei and normally
occluded in a proteinaceous matrix or polyhedron are called
occlusion-derived virus (ODV), and those that bud out of the cell are
called budded virus (BV). BV acquires an envelope from the plasma
membrane while ODV acquires an envelope which is derived from the inner
nuclear membrane (INM) [5, 6].
The ODV containing polyhedra have been commercially produced to combat
insect pests in agriculture and forestry. Polyhedra protect the ODV from
inactivation by UV irradiation and desiccation in the natural
environment. Non-occluded virions, although infectious, are prone to
inactivation by UV irradiation and desiccation in the field due to the
lack of protection from polyhedra. Several genes have been reported to
be involved in virion occlusion such as the fp25k of AcMNPV,
LdMNPV and HearSNPV [7-9]. AcMNPV is deemed as the model virus for
baculovirus since it has been extensively studied due to availability of
cell lines and generation of high virus titers. The genome of AcMNPV was
also the first baculovirus sequenced and it has a circular genome of 134
kbp [10].
The fp25k gene of AcMNPV and other baculoviruses is often mutated
during passage in insect cells. Reported mutations of fp25k are
caused by host DNA transposition insertion, or nucleotide(s) deletion,
insertion or conversion [7, 8, 11, 12]. The fp25k mutations
lead to polyhedron yield reduction and formation of polyhedra with
reduced virion occlusion [13]. Mutations of fp25k produce the
few polyhedra phenotype, called FP, which is characterized by fewer
cells containing polyhedra, fewer polyhedra per cell and fewer or no
virions in polyhedra, leading to reduced infectivity to insect pests
[14]. In addition, mutations of fp25k also leads to increased
BV production, altered intra-nuclear envelopment [13] and decreased
rates of polyhedrin gene transcription and its nuclear importing [15,
16]. Therefore, the fp25k is a multi-functional gene of
baculovirus and its mutations produce multiple effects to the virus.
Early we found AcMNPV fp25k mutations at the two A7 MNRs and a
TTAA site with a 287 bp host transposable DNA insertion [17, 18]. In
this report, we hypothesized that mononucleotide repeats (MNR) of AcMNPV
can be eliminated by site directed mutagenesis. We further hypothesized
that virion occlusion can be improved by the site directed mutagenesis
method. We demonstrated that elimination of the three hypermutable sites
result in more reduced mutation rates of the fp25k of AcMNPV
during passage in insect cells, which lead to improvement of polyhedra
production and virion occlusion for better bio-insecticide quality in
insect pest control to reduce cancer cases caused by chemical
applications in agriculture and forestry.