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.