USDA/ARS, Insect Biocontrol Laboratory, BARC-West, Building 011A, Room 214, Beltsville, MD 20852-2350, USA
Correspondence
Jeffrey M. Slack
slackj{at}ba.ars.usda.gov
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Department of Entomology, Soils and Plant Sciences, Clemson University, Clemson, SC 29634-0315, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the following investigation, we sought to determine whether the AgMNPV v-trex gene was expressed and whether the V-TREX protein product functions as a 3' to 5' exonuclease. RT-PCR was used to detect v-trex transcripts in the context of AgMNPV infection. The AgMNPV v-trex ORF was also cloned into the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) under the control of a polyhedrin (polh) promoter. A fluorescence-based assay was then used to examine the exonuclease activity of the overproduced V-TREX protein.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Constructs and recombinant baculoviruses.
The 693 bp ORF of v-trex was amplified from AgMNPV genomic DNA by PCR and cloned into the baculovirus transfer vector plasmid pBacPAK8 (Clontech). The PCR primers TREX-LP-XbaI-NcoI (5'-AAACATCTAGAGTTCACCATGGCTGTCGTCAAGAC-3') and TREX-RP-NotI-NcoI del (5'-TAATAAGCGGCCGCTTATTCCCCCATAGGGATGAC-3') were used to engineer 5' XbaI and 3' NotI restriction sites onto the ends of the v-trex ORF such that it could be cloned downstream of the polh promoter of the pBacPAK8 plasmid. The resulting construct, pBacPAK8-v-trex, was co-transfected with Bsu36I-digested BacPAK6 viral DNA into Sf-9 cells, as described by Kitts & Possee (1993). Transfections were facilitated by using the lipid transfection agent Cellfectin (Invitrogen). BacPAK-v-trex virus clones were isolated by plaque purification.
Preparing cell lysates for TREX assays.
T-75 tissue-culture flasks of Sf-9 cells (1x107 cells per flask) were infected at an m.o.i. of 1. At 72 h post-infection (p.i.), cells were collected in 50 ml Corning tubes and counted. Cells were pelleted at 1000 g for 1 min and suspended in 25 ml chilled PBS/EDTA (125 mM NaCl, 10 mM NaH2PO4, 5 mM EDTA, 2·5 mM KCl, pH 6·2). Cells were pelleted again at 1000 g for 1 min and suspended in 5 ml chilled PBS/EDTA. Finally, cells were pelleted at 1000 g and suspended at a concentration of 5x104 cells µl1 in TREX dilution buffer [75 mM NaCl, 50 mM Tris/HCl (pH 8·0), 5 mM NaH2PO4, 2·5 mM EDTA, 2 mM DTT, 5 % (v/v) glycerol, 2 % ethanol, 0·25 mM Ac-Leu-Leu-norleucinal (cysteine protease inhibitor)]. Suspended cells were frozen overnight at 20 °C and then thawed on ice and disrupted by sonication for 30 s using a Microson XL ultrasonic cell disrupter (Heat Systems). Cell lysates were centrifuged at 4500 g for 5 min at 10 °C. Supernatants were collected into 1·5 ml Eppendorf tubes and assayed for total protein by using a Coomassie Plus Protein Assay kit (Pierce). Supernatants were diluted in TREX dilution buffer to a protein concentration of 1 mg ml1. The resulting soluble lysates were used in exonuclease assays.
Budded virus (BV) purification and processing.
Cell-culture supernatant volumes of 33 ml from 4x107 infected Ag-286 cells were collected at 52 h p.i. Cellular debris was removed by centrifuging twice at 1000 g for 5 min. BV-containing cell-culture supernatants were centrifuged for 1·5 h at 100 000 g at 10 °C through a 5 ml cushion of 20 % (w/w) sucrose in PBS (pH 7·4) containing 5 mM iodoacetamide (cysteine protease inhibitor), 5 mM EDTA. BV pellets were suspended in 300 µl TREX dilution buffer and frozen at 20 °C. BV samples were thawed on ice and sonicated for 15 s. BV lysates were assayed for total protein by using a Coomassie Plus Protein Assay kit. BV lysates were diluted in TREX dilution buffer to a protein concentration of 0·9 mg ml1.
Exonuclease assays.
Exonuclease assays were done in 96-well U-bottomed plates. Plates were placed on ice while reagents were combined. Lysate volumes of 10 µl were combined with 30 µl TREX assay buffer [20 mM Tris/HCl (pH 7·5), 5 mM MgCl2, 2 mM DTT, 100 µg BSA ml1]. During assays, plates were covered with aluminium foil and incubated for 1 h at 37 °C. Assays were stopped with 20 µl TREX stop buffer [50 % (v/v) formamide, 3x TBE, 15 % (w/v) sucrose] and assays were stored at 4 °C until analysis. All TBE solutions were made from a 10x TBE stock (890 mM H3BO3, 450 mM Tris-base, 20 mM EDTA, pH 8·0). Exonuclease assay sample volumes of 20 µl were fractionated by electrophoresis (3 h, 25 mA, 200300 V) in 13 % (w/v) acrylamide : N,N'-methylene-bis-acrylamide (20 : 1), 1x TBE, 5 M urea gels. Electrophoresis was done by using a Hoeffer SE600 vertical gel unit and 0·7 mmx18 cmx16 cm gels. Gels were scanned in their plates by using a Typhoon fluorescent scanner (Amersham Biosciences) that had been set to 3 mm above the focal plane. For exonuclease assays, two 35 nt, fluorescently labelled DNA oligomers were synthesized at a 25 µmol scale (Integrated DNA Technologies). One oligomer (5HEX-oligo) was covalently linked at its 5' end to hexamethylfluorescein (5'-HEX-GCTCACCACTCCTGCAGCTCTAGATTCCCACCATC-3'). The other oligomer (3FAM-oligo) was covalently linked at its 3' end to 6-carboxymethylfluorescein (5'-AGCAACATAGATCTAGAGCTGCAGGAGTGGTGAGC-FAM-3'). In some experiments, the 5HEX-oligo and the 3FAM-oligo partially annealed to each other such that 25 nt annealed, leaving 10 mismatched nucleotides on the non-labelled ends that did not anneal (see Fig. 7a). Assays containing the 5HEX-oligo were scanned at excitation 532 nm/emission 555 nm BP 20 nm. Assays containing the 3FAM-oligo were scanned at excitation 532 nm/emission 526 nm SP. Assays containing both oligomers were scanned at dual wavelengths and images were separated by using Fluorsep 2.2 software (Amersham Biosciences). All Typhoon-scanned images were analysed on ImageQuant 5.0 (Amersham Biosciences).
|
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence for v-trex expression
From the AgMNPV DNA sequence, it was predicted that the v-trex gene would be expressed at early times during infection. This prediction was based on the presence of an ATCAGT motif 7 bp upstream of the v-trex translation start point (Fig. 1). In addition, the v-trex gene promoter region has a TATAA box and the eukaryotic transcription factor-binding motifs CACGTG and GATA. These elements have been shown to be important for the transcription of early baculovirus genes (Kogan & Blissard, 1994
; Shippam-Brett et al., 2001
). The GATA element may not be ideal, as it overlaps the TATAA box. With RT-PCR, we were able to detect the presence of v-trex RNA transcripts from 3 to 72 h p.i (Fig. 2
). This result confirmed the prediction that v-trex is an early gene. There were no late promoter (A/T)TAAG motifs in the vicinity of v-trex. The presence of v-trex RNA transcripts late in infection may be the result of v-trex transcript stability. The end of the v-trex gene contains a strong polyadenylation signal motif, ATAATAAA, which would promote the production of more stable, polyadenylated transcripts.
|
|
Soluble protein lysates from insect cells were diluted serially and incubated at 37 °C with the 5'-fluorescently labelled 5HEX-oligo. Exonuclease assays included lysates from control uninfected Sf-9 cells and Sf-9 cells that had been infected with wt AcMNPV, BacPAK--gal or BacPAK-v-trex. Exonuclease assays were analysed in denaturing acrylamide/urea gels. Lysates from Sf-9 cells produced a gradient of faster-migrating 5HEX-oligonucleotides when protein amounts were 11003300 ng (Fig. 4
, top panel). Lysates from wt AcMNPV and BacPAK-
-gal produced faster-migrating 5HEX-oligonucleotides when protein amounts were 3703300 ng (Fig. 4
, middle panels). Our interpretation of these results was that the increased mobility of 5HEX-oligo was the result of exonuclease activity decreasing the size of the oligomers. Lysates from BacPAK-v-trex caused the 5HEX-oligo to shift to a faster-migrating species when protein amounts were 4·53300 ng (Fig. 4
, bottom panel). At protein amounts between 0·17 and 1·5 ng, there was a gradient of 5HEX-oligo sizes.
|
Effects of pH, oligomer competitors, EDTA and divalent cations on V-TREX
V-TREX-associated exonuclease activity on the 5HEX-oligo substrate was inhibited when unlabelled oligomers were added in molar excess (Fig. 5a). Activity was also inhibited in the presence of EDTA (Fig. 5b
), indicating that V-TREX is a metalloenzyme that requires the presence of divalent cations.
|
Exonuclease assays were carried out over a range of pH values (Fig. 6). V-TREX activity was optimal between pH 6·1 and 7·4. This differentiates V-TREX from the more alkaline-active baculovirus exonuclease AN (Li & Rohrmann, 2000
). V-TREX also has a more acidic activity profile than mammalian TREX proteins (Mazur & Perrino, 2001
).
|
V-TREX produced different results when acting on 5'-labelled and 3'-labelled ssDNA substrates. As in earlier assays, increasing amounts of V-TREX extracts generated a gradient of smaller 5HEX-oligo fragments (Fig. 7b, panel 1). In contrast, the 3FAM-oligo abruptly dropped to a very small size when treated with V-TREX extracts (Fig. 7b
, panel 3). This was interpreted to be the result of V-TREX cleaving off the labelled terminal nucleotide on the 3' end of the 3FAM-oligo. These results are as would be predicted for a 3' to 5' exonuclease and are the converse of what others have observed for the baculovirus 5' to 3' exonuclease AN (Mikhailov et al., 2003
).
To examine the effects of dsDNA on V-TREX exonuclease activity, the 5HEX-oligo and 3FAM-oligo were annealed. The HEX and FAM fluorescent labels could be seen separately in the same gel, due to different emission spectra (see Methods). The annealed 5HEX-oligo and 3FAM-oligo substrates required more TREX extract in order to be digested (Fig. 7b, panels 2 and 4). This indicated that V-TREX exonuclease activity has some ssDNA specificity. The 5HEX-oligo and 3FAM-oligo design was such that when these 35 nt oligomers were annealed, 10 bp mismatched ends would be present (Fig. 7a, 5H
/3F). An intermediate-sized 5HEX-oligo product was generated at protein extract concentrations of 41123 ng (Fig. 7b
, panel 2). No such intermediate-sized products were generated from the 3FAM-oligo (Fig. 7b
, panel 4). V-TREX thus exhibited characteristics of a 3' repair exonuclease by targeting the misannealed 3' end. This type of activity has been observed for mammalian TREX proteins (Mazur & Perrino, 2001
).
Exonuclease activity associated with AgMNPV BV
Experiments were done using the 5'-labelled 5HEX-oligo to determine whether there was 3' to 5' exonuclease activity associated with AgMNPV infection. Soluble protein lysates from Ag-286 cells that had been infected with AgMNPV were compared with lysates from uninfected Ag-286 cells. The relative amount of 3' to 5' exonuclease activity associated with AgMNPV-infected Ag-286 cells was not significantly different from that of uninfected Ag-286 cells (data not shown).
We also looked for exonuclease activity associated with AgMNPV BV. Ag-286 cells were infected with AgMNPV or AcMNPV. It was ensured that similar levels of infection had been achieved (Fig. 8a) and that sucrose-cushion ultracentrifugation-purified virion preparations were diluted to contain similar amounts of total protein (Fig. 8b
). The 5HEX-oligo substrate was incubated with sonicated BV preparations from AgMNPV and AcMNPV. Significantly more exonuclease activity was present in AgMNPV BVs than in AcMNPV BVs (Fig. 8c
).
|
Conclusions
The evidence presented in this study leads to the conclusion that the v-trex gene product is a functional 3' to 5' exonuclease and that V-TREX belongs to the TREX family of exonucleases. At 23·7 kDa, V-TREX is one of the smallest functional 3' to 5' exonucleases to be described. V-TREX showed remarkable stability throughout this study, with activity varying little over several months of repeated freezing and thawing. Recently, a v-trex gene homologue appeared in GenBank as ORF 119 of the C. fumiferana defective NPV (CfDEFNPV) baculovirus genome (accession no. AY327402.1). The CfDEFNPV v-trex homologue is predicted to encode a protein that is 148 aa in size and appears to be missing one-third of its C-terminus.
The v-trex gene has not been identified in the genomes of most other sequenced baculovirus species. There are no v-trex homologues in other virus families and v-trex is most similar to eukaryotic genes. This suggests that the v-trex gene was probably acquired recently in baculovirus evolution. Studies are currently being done to determine whether the v-trex gene is essential for the replication of AgMNPV and what biological function v-trex may have. We anticipate that v-trex will be classified as a baculovirus auxiliary gene, along with such genes as v-cath (Slack et al., 1995) and ChiA (Hawtin et al., 1995
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hink, W. F. (1970). Established insect cell line from the cabbage looper, Trichoplusia ni. Nature 226, 466467.
Kitts, P. A. & Possee, R. D. (1993). A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810817.[Medline]
Kogan, P. H. & Blissard, G. W. (1994). A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements. J Virol 68, 813822.[Abstract]
Li, L. & Rohrmann, G. F. (2000). Characterization of a baculovirus alkaline nuclease. J Virol 74, 64016407.
Mazur, D. J. & Perrino, F. W. (1999). Identification and expression of TREX1 and TREX2 cDNA sequences encoding mammalian 3'5' exonucleases. J Biol Chem 274, 1965519660.
Mazur, D. J. & Perrino, F. W. (2001). Excision of 3' termini by Trex1 and TREX2 3'5' exonucleases. J Biol Chem 276, 1702217029.
Mikhailov, V. S., Okano, K. & Rohrmann, G. F. (2003). Baculovirus alkaline nuclease possesses a 5'3' exonuclease activity and associates with the DNA-binding protein LEF-3. J Virol 77, 24362444.
Mikhailov, V. S., Okano, K. & Rohrmann, G. F. (2004). Specificity of the endonuclease activity of the baculovirus alkaline nuclease for single-stranded DNA. J Biol Chem 279, 1473414745.
Moscardi, F. (1999). Assessment of the application of baculoviruses for control of lepidoptera. Annu Rev Entomol 44, 257289.[CrossRef]
Serafini, D. M. & Schellhorn, H. E. (1999). Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation. Can J Microbiol 45, 632637.[CrossRef][Medline]
Shapiro, M., Farrar, R. R., Jr, Domek, J. & Javaid, I. (2002). Effects of virus concentration and ultraviolet irradiation on the activity of corn earworm and beet armyworm (Lepidoptera: Noctuidae) nucleopolyhedroviruses. J Econ Entomol 95, 243249.[Medline]
Shippam-Brett, C. E., Willis, L. G. & Theilmann, D. A. (2001). Analysis of sequences involved in IE2 transactivation of a baculovirus immediateearly gene promoter and identification of a new regulatory motif. Virus Res 75, 1328.[CrossRef][Medline]
Sieburth, P. J. & Maruniak, J. E. (1988). Susceptibility of an established cell line of Anticarsia gemmatalis (Lepidoptera: Noctuidae) to three nuclear polyhedrosis viruses. J Invertebr Pathol 52, 453458.
Slack, J. M., Kuzio, J. & Faulkner, P. (1995). Characterization of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus. J Gen Virol 76, 10911098.[Abstract]
Slack, J. M., Ribeiro, B. M. & Lobo de Souza, M. (2004). The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus contains a 3' repair exonuclease homologue and lacks v-cath and ChiA genes. J Gen Virol 85, 211219.
Received 12 March 2004;
accepted 30 June 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |