Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus

Kikhno, I. M.; Gutiérrez, Serafín; Croizier, L.; Croizier, G.; Ferber, Miguel López

doi:10.1099/0022-1317-83-12-3013

Cited by 145 publications

(141 citation statements)

References 46 publications

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“…2). These results also support the hypothesis that Se36 (pif; Kikhno et al, 2002) was still functional in our mutants and that Se36 transcription is likely to start from the TAAG at position 212. We did not compare transcription levels of Se36 between the various mutants, since this may only have a quantitative but not a qualitative effect on the identification of Se35 as essential gene for oral infectivity.…”

Section: Discussionsupporting

confidence: 79%

“…Polyhedra from this repair mutant SeBACphD15235*rep35 also resulted in restoration of oral infectivity, indicating that the 18 bp sequence (including a TAAG motif) upstream of the ATG start site of Se36 serves as an active promoter in the mutant bacmid SeBACD15-35*. Alternatively, Se36 may not be essential for oral infectivity, which would be in contrast to other results (Kikhno et al, 2002). …”

contrasting

confidence: 52%

“…AcMNPV ORF22) in other baculoviruses. Kikhno et al (2002) showed by Western blot analysis that PIF is a structural protein of the ODV envelope and they speculated on a putative interaction between PIF and P74, but now PIF-2 must be considered as well. The overlap of the Se35 (pif-2) 39UTR and the promoter of the neighbouring Se36 (pif) may indicate that these two genes in SeMNPV have co-evolved and are closely associated, although their homologues in other baculoviruses (including SpliNPV) are located in separate gene clusters.…”

Section: Discussionmentioning

confidence: 99%

“…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. This gene, which is homologous to SeMNPV ORF36 (Se36), was designated per os infectivity factor (pif) (Kikhno et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

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

Pijlman

Pruijssers

Vlak

2003

Journal of General Virology

127

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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...

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Section: Discussionsupporting

confidence: 79%

contrasting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Pijlman

Pruijssers

Vlak

2003

Journal of General Virology

127

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“…ODVs attach to midgut columnar epithelial cells and fuse their envelopes directly with the cell membrane (Kawanishi et al, 1972;Tanada et al, 1975;Granados, 1978;Horton & Burand, 1993). A number of ODV envelope proteins are essential per os infectivity factors (PIFs), including P74 , PIF1 (Kikhno et al, 2002), PIF2 (Pijlman et al, 2003) and PIF3 (Ohkawa et al, 2005). PIF1, PIF2 and P74 have been implicated to be involved in ODV attachment to midgut cells (Haas-Stapleton et al, 2004;Yao et al, 2004;Ohkawa et al, 2005).…”

Section: Introductionmentioning

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

Journal of General Virology

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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...

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Comparative biological activities of a plaque‐purified variant and a Turkish native isolate of SpliNPV‐B against Spodoptera littoralis (Lepidoptera: Noctuidae)

Toprak

Bayram

Gurkan

2005

Pest Management Science

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The noctuid moth Spodoptera littoralis (Boisduval) is an important pest of many cultivated plants worldwide and five different geographical Nucleopolyhedrovirus (NPV) strains of this pest have been isolated to date. Two of these, a plaque-purified variant of the S. littoralis NPV from Morocco (SpliNPV-M2) and a SpliNPV isolated from field-infected S. littoralis larvae found in Turkey (SpliNPV-TR1), were compared biologically in terms of infectiveness (median lethal dose, LD50) for third instars and in terms of virulence (median lethal time, LT50) for neonates and third-instar S. littoralis larvae. The LD50 values of SpliNPV-TR1 and SpliNPV-M2 were 20.73 and 185.21 occlusion bodies (OBs)/larva, respectively, with non-overlapping confidence limits indicating they were significantly different. Thus, SpliNPV-M2 was found to be significantly less infective (about nine times higher LD50) than SpliNPV-TR1. The LT50 values of neonates for SpliNPV-M2 and SpliNPV-TR1 were 37 and 43.9 h at a concentration of 10(6) OBs ml(-1), respectively. For these same isolates, the LT50 values at a concentration of 3 x 10(6) OBs ml(-1) were calculated as 35.6 and 41.7 h, respectively. The LT(50) values of third instars for SpliNPV-M2 and SpliNPV-TR1 were 147.4 and 160.5 h, respectively, at a dose of 3000 OBs/larva and 145.4 and 152.4 h, respectively, for the same isolates at a dose of 20,000 OBs/larva. On the other hand, Helicoverpa armigera (Hübner) and Agrotis ipsilon (Hufnagel) revealed a lack of lethality of the SpliNPV-TR1 isolate.

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Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus

Cited by 145 publications

References 46 publications

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

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

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

Comparative biological activities of a plaque‐purified variant and a Turkish native isolate of SpliNPV‐B against Spodoptera littoralis (Lepidoptera: Noctuidae)

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