A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag

IE1, a principal transcriptional activator of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), is an essential factor for viral DNA replication. During viral infection, IE1 accumulates in discrete subnuclear structures where viral DNA replication occurs. To analyse the dynamic properties of IE1, we monitored green fluorescent protein-tagged IE1 (IE1-GFP) in BmNPV-infected B. mori cells by live-cell microscopy. Time-lapse imaging showed that IE1-associated structures gradually expanded and occasionally fused with one another, while photobleaching experiments revealed that IE1-GFP was relatively immobile inside the IE1-associated structures. To investigate the spatial relationships between IE1 and viral structural proteins in infected cells, three GFP-tagged viral components were expressed together with DsRed-tagged IE1. Two structural proteins that constitute the occlusion-derived virus (ODV), P91-GFP and GFP-ODV-E25, localized to the periphery of the IE1-associated structures. While local accumulations of these proteins were often in contact with the IE1-associated structures, they did not extend beyond the boundaries of the structures. In contrast, the major capsid protein VP39-GFP predominantly accumulated within the IE1-associated structures. These data indicated, in conjunction with the finding of a high DNA content in the structures, that IE1 localizes to the virogenic stroma and therefore support the prediction previously proposed that the virogenic stroma is a site for viral DNA replication as well as for the assembly of nucleocapsids.

Section: Two Odv-associated Proteins P91-gfp and Gfp-odv-e25 Become mentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins

Kawasaki

Matsumoto

Nagamine

2004

“…Until recently the only baculovirus protein demonstrated to be involved in oral infectivity by ingestion of polyhedra was the ODV-specific P74 (Kuzio et al, 1989), which contains a hydrophobic C terminus involved in protein localization and transmembrane anchoring. None of the other proteins of the ODV envelope, such as ODV-E18, -EC27, -E35, -E25, -E56 and -E66, were proven to participate in the oral infectivity process (Slack et al, 2001). Recently, however, a conserved baculovirus gene encoded by ORF7 of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was also found to be required for the oral infection of S. littoralis.…”

Section: Introductionmentioning

confidence: 99%

Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Pijlman

Pruijssers

Vlak

2003

127

Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29-35. In this paper we have shown that SeMNPV ORFs 15-35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15-35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviridae. INTRODUCTIONSpodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infects the single insect species S. exigua (Smits & Vlak, 1988) and belongs to the group II NPVs. Baculoviruses of this group do not contain a GP64 homologue, but have a functionally homologous F protein as a structural element of the budded virus (BV) . BVs are required for the systemic spread of infection throughout the insect, whereas occlusion-derived viruses (ODVs) are involved in the horizontal spread of the virus in insect populations (Blissard & Rohrmann, 1990). Occlusion bodies (OBs) are ingested orally and the alkaline environment of the midgut causes the release of the ODVs. The ODVs first pass the peritrophic membrane and subsequently fuse to the midgut epithelial cells, thereby causing the initial infection (Funk et al., 1997). Large-scale production of SeMNPV for biological control is carried out in vivo using insect larvae, since SeMNPV infection in cell culture leads to the rapid generation and predominance of deletion mutants. These mutants, with deletions up to 25 kb, often lack the ability to infect S. exigua larvae by oral ingestion of OBs (Heldens et al., 1996). Therefore, the genetic engineering of SeMNPV via recombination in cell culture is complicated (Dai et al., 2000) and precludes the in vitro production of biologically active SeMNPV in insect cell bioreactors.Deletions in the SeMNPV genome predominantly occur within the XbaI-A restriction fragment and these deleted genotypes are present in SeMNPV wild-type isolates where they may act as parasitic genotypes (Muñoz et a...

“…The plasmids, pBAC-5-EGFP and pBAC-5-p74-EGFP, were previously described as pBAC-5-GFP and pBAC-5-p74-GFP (Slack et al, 2001). pBAC-5-p74-EGFP contains the AcMNPV p74 ORF fused at the 39 end in-frame with the jellyfish EGFP ORF (Zhang et al, 1996).…”

Section: Methodsmentioning

confidence: 99%

“…2a). These plasmids were previously used to study P74 localization in infected cells (Slack et al, 2001). Though designed as baculovirus shuttle vectors, pBAC-5-based plasmids are excellent for transient gene expression in insect cell culture (Ogay et al, 2006), due to the presence of an AcMNPV gp64 early/late promoter (Whitford et al, 1989;Blissard & Rohrmann, 1991).…”

Section: Development Of a Transient Expression-based Assay For Characmentioning

confidence: 99%

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection

Slack

Lawrence²,

Krell

et al. 2008

Self Cite

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin. INTRODUCTIONBaculoviruses are a group of arthropod-specific viruses (Zanotto et al., 1993) that have been applied as insecticides and as vectors for the expression of exogenous genes. They have a biphasic replication strategy producing two distinct viral phenotypes: the budded virion (BV) and the occlusion-derived virion (ODV). Both virion phenotypes have bilayer lipid envelopes surrounding bacillus-shaped nucleocapsids and contain double-stranded, circular DNA genomes. However, the integral protein composition of their envelopes and their roles in infection are distinct.BVs spread viral infection throughout host tissues by attaching to and entering host cells via receptor-mediated endocytosis (Volkman & Goldsmith, 1985). Endosomal acidification triggers envelope fusion with the endosomal membrane to release the viral nucleocapsid into the host cell (Leikina et al., 1992). In the case of group I alphabaculoviruses (Jehle et al., 2006), budding, attachment and envelope fusion are mediated by the viral protein GP64 (Blissard & Wenz, 1992) and for other baculovirus types these processes are mediated by an F protein (Pearson et al., 2000; Westenberg et al., 2002).ODVs are required in the horizontal transmission of baculoviruses between insect hosts (Kozlov et al., 1986). The ODVs of all baculoviruses are occluded into proteinaceous occlusion bodies (OBs) prior to release from the host (Rohrmann, 1986). The distinctive protein structure of OBs has a dual function. It serves to protect virions from deleterious environmental factors, and also acts as a delivery mechanism to transport the ODVs to the alkaline midgut where the cells are susceptible to infection.ODV envelopes are derived from the inner nuclear membrane (Braunagel & Summers, 1994) and contain envelope proteins that can survive the protease-rich environment of the insect midgut. ODVs attach to midgut columnar epithelial cells and fuse th...