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Baculoviridae is a family of rod-shaped viruses with circular, covalently closed, double-stranded DNA genomes that range in size from 81.7 kb to 161 kb[11, 19, 20]. There are two genera, Nucleopolyhedroirus (NPV) and Granulovirus(GV), in this family[34]. NPV genes are subdivided into four groups: immediate early, delayed early, late, and very late [4]. Immediate early and delayed early genes are transcribed using host RNA polymerase Ⅱ after infection and some of them code for transcriptional transactivators of viral genes[7, 12, 14, 23, 32].
Baculovirus IE1 is a putative multifunctional protein coded by the viral immediate early 1(ie1) gene[1, 3, 37]. It was shown, in transient expression experiments, to be essential in viral DNA replication [16, 31]. Baculovirus IE1 can transactivate all known early genes and late genes of the virus [2]. Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 transactivates a cell housekeeping gene promoter [22], Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) IE1 can self stimulate their expression[18, 33]. Furthermore, the baculovirus IE1 gene down regulates the expression of some other genes such as ie0, ie2[17] and pe38[21]. NPV genomes contain several regions of homologous DNA of 76 to 800 bp that are interspersed throughout the genome.The larger homologous regions (hrs) were shown to have the ability to cis-activate expression of the early generomoters. Recent studies showed that heterologous IE1 had a negative effect on late gene expression and truncated BmNPV IE1 strongly inhibited activation of the hr5-dependent p35 promoter[38]. These suggest that IE1 functions as a regulatory factor in different modes.
The functional domain of the baculovirus IE1 has been well characterized by mutant analysis. There is an acidic activation domain (AAD) which contains a virus specific domain for DNA replication, a highly conserved basic domain which is required for DNA binding and transactivation[29, 31], and a domain for possible second transactivation in the N-terminal region. An DNA binding domain and oligomerization domain are in the C-terminal region[9, 31].
The baculovirus IE1 was found to be functional in mammalian cells by transient expression assay. The viral early 39k promoter activated by the IE1 is dependent on the hr sequence in mammalian cells but is independent of the hr sequence in insect cells, which suggests that IE1 might exhibit different transcriptional modes[25]. Since BmNPV IE1 can transactivate several viral and heterologous core promoters this suggests that baculovirus IE1 has the potential to activate a core promoter in mammalian cells[24].
Phylogenetic analysis indicates that the nucleopolyhedroviruses are comprised of groupⅠand group Ⅱ NPV[6, 13]. Although the SeMNPV belong to group Ⅱ NPV, the amino acid sequence of SeMNPV IE1 has diverged considerably from others of group Ⅰ and group Ⅱ NPV IE1[15, 35, 36]. So far, the characterization of baculovirus IE1 had only been performed in group Ⅰ NPV. In this paper, we reported the function of SeMNPV IE1 in mammalian cells. This was first work for groupⅡ NPV IE1 function. By studying the expression of SeMNPV IE1-green fluorescent protein (IE1-EGFP) in HEK 293 cells, the IE1-EGFP fusion protein was localized in the nucleus of mammalian cells. SeMNPV IE1 could stimulate the F protein promoter but not affect the gp64 promoter in HEK 293 cells.
Localization and Functional Analysis of SeMNPV IE1 in Mammalian Cells
- Received Date: 19 November 2009
- Accepted Date: 02 March 2010
Abstract: In this paper, the function of the ie1 gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), belonging to group II nucleopolyhedrovirus, was studied in mammalian cells. We amplified the SeMNPV ie1 gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn’t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.