Plutella xylostella granulovirus (PlxyGV) contains homologs of 15 Autographa californica MNPV (AcMNPV) late expression factor (lef) genes. The prospective products of 14 PlxyGV lef genes (ie-0 is not included) share 13%-53% amino acid similarity with their corresponding homologs of AcMNPV, among which LEF-9, LEF-8 and P47, three subunits of the virus-encoded RNA polymerase, share 49%, 53% and 46% sequence identity, respectively. In this study, an established transient expression system was used to test the ability of the PlxyGV LEFs to activate an AcMNPV vp39 promoter-driven reporter gene in SF9 cells. It was shown that PlxyGV le f-2 replaced the corresponding AcMNPV gene and exhibited partial activity in the context of the remaining set of AcMNPV le fs. PlxyGV LEF-2 was found to contain additional 100aa and 70aa at the C-terminus in comparison with the LEF-2 of other GVs and lepidopteran NPVs respectively.
Amino Acid Sequence ; Animals ; Gene Expression Regulation, Viral ; Granulovirus ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Moths ; virology ; Nucleopolyhedrovirus ; genetics ; metabolism ; Sequence Alignment ; Viral Proteins ; genetics ; metabolism

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.
Bacteriophage T4 ; Bacteriophages ; Biological Warfare Agents ; DNA ; Electrophoresis ; Freeze Drying ; Genome ; Granulovirus ; Hantaan virus ; Levivirus ; Nitrogen ; Polymerase Chain Reaction ; Reverse Transcription ; Variola virus ; Yellow fever virus

Protein-protein interactions between viruses and hosts are common during viral infection and replication. In this study, a cDNA library from larvae of Plutella xylostella was constructed and used for screening of genes encoding proteins interacting with Plutella xylostella granulovirus (PlxyGV) proteins. Two cDNA clones containing genes encoding proteins interacting with PlxyGV PP31 were identified by yeast two-hybrid assays. Sequence analysis showed that the genes encoded homologues of receptor for activated protein C kinase (RACK) and methionine aminopeptidase 2 (MetAP2), respectively. The P. xylostella rack gene and the PlxyGV pp31 was expressed in an E. coli strain to produce proteins fused with a 6-His or a GST tag. It was shown that the rack was expressed as a 38kD peptide as prospected. The 38kD His-tagged peptide was co-purified with GST-PP31 by GST-bind resin in GST-pulldown assays, confirming interaction between the PlxyGV PP31 and the RACK protein of P. xylostella.
Aminopeptidases ; genetics ; physiology ; Animals ; Gene Library ; Granulovirus ; physiology ; Metalloendopeptidases ; genetics ; physiology ; Moths ; virology ; Receptors for Activated C Kinase ; Receptors, Cell Surface ; genetics ; physiology