Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells

Abstract

The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. IMPORTANCEBaculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.

Gene delivery and gene expression in vertebrate using baculovirus Bombyx mori nucleopolyhedrovirus vector

“…Baculoviruses have been also explored as a possible gene delivery vector system for vertebrate cells [ 9 – 12 ]. These viruses may be ideal for gene therapy because their viral promoters are almost silent in mammalian cells, and the budded viral form is harmless to the environment [ 13 – 16 ]. Autographa californica multicapsid nucleopolyhedrovirus ( Ac MNPV), a widely studied baculovirus, is as a promising gene therapy vector [ 17 ].…”

Identification of Multiple Replication Stages and Origins in the Nucleopolyhedrovirus of Anticarsia gemmatalis

“…Apart from hr s, other ORIs containing tandemly repeated sequences have been characterized in AcMNPV [39,42], SpliMNPV [70], SeMNPV [58,71,72], OpMNPV [33,59], and CrleGV [60]. Also, in AcMNPV, two other kinds of non- hr ORIs have been described: Early promoters [34], and a segment of the DNA helicase ORF [35]. In our work, 12 different sequences of AgMNPV classified as intergenic and intragenic regions were detected and named ORI-igrs and ORI-grs, according to the presence of putative functional early promoters or the presence of fragments of ORFs, respectively.…”

“…With this approach, homologous regions ( hr s) [30,31], non- hr sequences [32,33] and early promoters [34] have been identified as causing the infection-dependent replication of plasmids. Wu et al [35] identified a different non- hr ORI inside a core gene of AcMNPV ( p143 or DNA helicase), broadening the spectrum of sequences involved in this function.…”

“…However, when given time, after plasmid transfection prior to infection, it has been observed that only the plasmids containing viral promoter or hr s replicate [23,34]. Following this strategy, the previously mentioned non- hr direct repetitions located into the coding region of the p143 gene were functional as ORI in insect and mammalian cells [35].…”

The Homologous Regions ofSpodoptera litura(Lepidoptera: Noctuidae) Nucleopolyhedrovirus II Have Both the Function as Origin of DNA Replication and Enhancer

“…Only one non- hr ori was found in the AcMNPV genome, and it showed some distant structural homology to consensus eukaryotic ori s ( DePamphilis 1993 , Lee and Krell 1994 ). Surprisingly, the p143 -3.2a ori of AcMNPV was found to function as an efficient ori in mammalian cells without the need for any viral proteins ( Wu et al. 2014 ).…”