P34.8 (GP37) is not essential for baculovirus replication

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...

Section: Discussionmentioning

confidence: 99%

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

Pijlman

Pruijssers

Vlak

2003

127

“…Per os infection studies of vThGFP on P. includens, S. frugiperda, S. exigua and H. zea followed Cheng et al (2001).…”

Section: Methodsmentioning

confidence: 99%

“…vThGFP was propagated in Hi5 cells. An AcMNPV bacmid (AcBacmid, bMON14271; Invitrogen) containing no polyhedrin gene and an AcMNPV with an EGFP expression cassette at the gp37 locus (AcGFP) (Cheng et al, 2001) were also included for viral infection enhancement and OB formation and were propagated in Sf21 cells. A recombinant AcMNPV containing the red fluorescent protein (RFP) gene (DsRed2; Clontech) was generated using the bacmid system (Invitrogen) for tissue tropism study (vAcDsRed2).…”

Section: Methodsmentioning

confidence: 99%

“…Other cells used in the in vitro infection were from the USDA/ARS Insect Biocontrol Laboratory (Beltsville, MD, USA) (Supplementary Table S1). Viruses used in this project included ThorMNPV containing an enhanced green fluorescent protein (EGFP) gene expression cassette at the gp37 locus (vThGFP), constructed according to Cheng et al (2001). vThGFP was propagated in Hi5 cells.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Slow cell infection, inefficient primary infection and inability to replicate in the fat body determine the host range of Thysanoplusia orichalcea nucleopolyhedrovirus

Wang

Salem

Lynn

et al. 2008

Self Cite

Thysanoplusia orichacea multicapsid nucleopolyhedrovirus (ThorMNPV) carrying an enhanced green fluorescent protein (EGFP) gene expression cassette (vThGFP) was used to study host-range mechanisms. Infection kinetics showed that vThGFP replication in Sf21 cells was too slow to suppress cell growth. Wide-host-range Autographa californica MNPV (AcMNPV) could speed up vThGFP infection and enhance the vThGFP infection rate in Sf21 cells. The enhancement was not due to recombination, as no recombinant virus was isolated from coinfection by plaque assay. No improvement of vThGFP infection in Sf21 was found by AcMNPV cosmid transactivation assay. However, culture medium from Sf21 cells infected with AcMNPV did enhance vThGFP replication in Sf21. Third-instar larvae of Spodoptera frugiperda, S. exigua and Helicoverpa zea were not killed by feeding with vThGFP polyhedra but were killed by intrahaemocoelic injection using budded viruses (BVs). This suggested that insufficient BVs were generated during the primary infection in the midgut. vThGFP infected haemocytes, tracheae and Malpighian tubules but not fat bodies of larvae of S. frugiperda, S. exigua and H. zea. Third-instar S. frugiperda larvae co-infected by injection with vThGFP and vAcDsRed2, an AcMNPV expressing a red fluorescent protein gene, showed EGFP expression in the fat body. This result suggests that vAcDsRed2 could help vThGFP to replicate in the fat body or trans-activate EGFP expression in the fat body. All these results suggested that slow cell infection, insufficient primary infection and inability to replicate in the fat body control the host range of ThorMNPV.

“…The OB concentrations were estimated using a haemocytometer. To isolate AcDef, the OBs purified from larvae were fed to third instar T. ni larvae at a dose of 30 000 polyhedra per larva by using the diet plug method (Cheng et al, 2001); mortality was recorded.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes

Wang

Salem

Campbell

et al. 2009

Self Cite

Nucleopolyhedroviruses (NPVs), family Baculoviridae, are insect-specific viruses with the potential to control insect pests in agriculture and forestry. NPVs are occluded in polyhedral occlusion bodies. Polyhedra protect virions from inactivation in the environment as well as assisting virions in horizontal transmission in the insect population. The process of virion occlusion in the polyhedra is undefined and the genes that regulate the virion occlusion process have not been well investigated yet. An Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant (AcDef) that has a 2136 bp DNA deletion, including p26, p10 and p74 genes, has been isolated. No virions were detected in the polyhedra of AcDef. Restoration of all the missing sequences into AcDef led to proper virion occlusion. Individual gene deletion of either p10 or p26 could not abolish virion occlusion in the polyhedra of AcMNPV, but p10 deletion reduced virion occlusion efficiency more than threefold compared with the wild-type AcMNPV. Previous studies by other research groups on deletion of AcMNPV gene p74 suggested that p74 is a per os infectivity factor, and deletion of the p74 gene did not eliminate virion occlusion. Collectively, the three genes (p26, p10 and p74) may act in concert to regulate the virion occlusion process. Therefore, p26, p10 and p74 are all required for proper virion occlusion in the polyhedra of AcMNPV.