Abstract
Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the type I alphabaculoviruses, is able to transduce and deliver a functional gene to a range of non-host cells, including many mammalian lines and primary cells, a property mediated by the envelope fusion protein GP64. AcMNPV is non-cytopathic and inherently replication deficient in non-host cells. As such, AcMNPV represents a possible new class of gene therapy vector with potential future clinical utility. Whilst not a problem for in vitro gene delivery, the broad tropism displayed for non-host cells is less desirable in a gene therapy vector. The fusion protein F of type II alphabaculoviruses can substitute functionally for GP64, and such pseudotyped viruses display a severely impaired capacity for non-host-cell transduction. Thus, surface decoration of such an F-pseudotyped AcMNPV with cell-binding ligands may restore transduction competence and generate vectors with desirable cell-targeting characteristics. By seamlessly swapping the native gp64 coding sequence with each of five sequences encoding different F proteins, a set of F-pseudotyped AcMNPV was generated. This report details their relative abilities both to functionally replace GP64 in viral growth and to transduce human Saos-2 and HeLa cells. All five supported viable infections in insect cell cultures and one, the Mamestra configurata NPV (MacoNPV) F pseudotype, could be amplified to titres close to those of native AcMNPV. In contrast, none was able to transduce the Saos-2 and HeLa cell lines. The robust support provided by MacoNPV F in virus production makes the corresponding pseudotype a viable scaffold to display surface ligands to direct selective mammalian cell targeting.
Highlights
The family Baculoviridae comprises large, rod-shaped, enveloped dsDNA viruses infective to insects belonging to the orders Lepidoptera, Hymenoptera and Diptera
We describe the construction of new Dgp64 Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vectors pseudotyped with five additional type II alphabaculovirus F proteins and report on their respective abilities in supporting virus production and the delivery of a reporter to mammalian cells
Using our modified recombineering protocol (Westenberg et al, 2010), the F coding sequences (CDSs) were PCR amplified and, following addition of extended terminal sequences homologous to the gp64 59 and 39 flanking regions, via an intermediate subcloning step, each sequence was introduced into a Dgp64-bMON14272 bacmid (Fig. 2)
Summary
The family Baculoviridae comprises large, rod-shaped, enveloped dsDNA viruses infective to insects belonging to the orders Lepidoptera, Hymenoptera and Diptera. A number of studies (Table 1) have demonstrated that the majority of type II alphabaculoviral F proteins, apparently binding to a different insect cell receptor (Westenberg et al, 2007), can substitute functionally for GP64 by supporting AcMNPV production in culture, albeit with reduced titres.
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