1 State Key Laboratory of Virology, Key Laboratory of Molecular Virology, Joint Laboratory of Invertebrate Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
2 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430071, China
3 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
4 Laboratory of Virology, Wageningen University, 6709 PD Wageningen, The Netherlands
Correspondence
Zhihong Hu
huzh{at}pentium.whiov.ac.cn
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many proteins seem to be affected by FP25K mutations at the molecular level. It has been reported that mutations in FP25K result in (i) a decreased amount of polyhedrin mRNA and altered transport of polyhedrin protein into the nucleus (Harrison et al., 1996), (ii) significantly decreased amounts of ODV-E66 and impaired transportation of ODV-E66 into the nucleus (Braunagel et al., 1999
, 2004
; Rosas-Acosta et al., 2001
), and (iii) a significant increase in the synthesis of some structural viral proteins of BVs such as membrane fusion protein GP64, BV/ODV-E26 and VP39 (Braunagel et al., 1999
). Although the effects produced by FP25K mutants are known, the function of this protein is still unknown. As most of the molecular effects of FP25K were generated from AcMNPV, it remains unclear whether the same effects exist in other baculoviruses.
The HearNPV FP mutant was first reported by Chakraborty & Reid (1999). Lua et al. (2002)
reported the rapid accumulation of FP mutants in HearNPV. During passage of wild-type HearNPV on HzAM1 cells, the FP phenomenon was observed by passage 6 in all the infected cells. Electron microscopy studies revealed that very few polyhedra were produced in cells that were infected with the FP mutant and in most cases the polyhedra did not contain virions (Lua et al., 2002
). The fp25k genes of these mutants contained point mutations, insertions or deletions (Lua et al., 2002
).
In this work, we first analysed the transcription and expression of fp25k in HearNPV-infected cells. We then attempted to study the function of the fp25k gene by replacing it with a gene encoding an enhanced green fluorescent protein (GFP) in HaBacHZ8, an infectious polyhedrin null HearNPV bacmid (Wang et al., 2003). The production of BV and intranuclear enveloped nucleocapsid (equivalent to ODV) of the recombinant virus was compared with a control virus HaBacHZ8GFP, which contained the native fp25k gene and an inserted enhanced gfp gene. The fp25k gene was reintroduced into the recombinant virus to ascertain that no other function was affected by the deletion. In addition, we analysed the expression of BV membrane fusion protein (Ha133) and ODV-E66 in the fp25k null mutant. Our results indicated that the deletion of fp25k caused a higher BV yield and a decrease in progeny ODVs. Unlike AcMNPV, in which the deletion of fp25k would result in an increase of the BV membrane fusion protein GP64 and a decrease of ODV-E66 (Braunagel et al., 1999
; Rosas-Acosta et al., 2001
), the expression of the membrane fusion protein and ODV-E66 in HearNPV were not significantly affected when fp25k was deleted.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcriptional analysis of HearNPV fp25k gene.
HzAM1 cells (3x105) were infected with HearNPV-G4 at an m.o.i. of 5. At appropriate time points post-infection (p.i.), the infected cells were collected and the total RNAs were extracted by RNA-solv reagent (Omega). 3'RACE experiment was performed using 3'-full RACE core set (TaKaRa) according to the manufacturer's instructions. Total RNA (1 µg) from each time point was transcribed by AMV reverse transcriptase and an oligo(dT) three-site adaptor primer to synthesize the first strand cDNA. The PCR reaction was followed using the cDNA as template; the primers were a three-site adaptor primer and an fp25k gene-specific forward primer with sequence of 5'-ACTCGTGACGCCCTATTACCG-3' (424445 nt in the fp25k gene). PCR products were analysed by agarose gel electrophoresis. The PCR product derived from the 48 h p.i. sample was purified and cloned into pGEM-T easy vector (Promega) and then sequenced with M13 primers to determine the 3'end of fp25k gene transcript.
Expressional analysis of HearNPV fp25k.
HzAM1 cells (3x105) were infected with HearNPV-G4 at an m.o.i. of 5. At appropriate time points p.i., the infected cells were collected and rinsed with PBS. Protein samples were separated on 10 % SDS-PAGE and transferred onto nitrocellulose membrane using Semi-Dry Transfer Cell (Bio-Rad) as recommended by the manufacturer. The primary antibody was a rabbit polyclonal antibody against HearNPV FP25K (Wu et al., 2004). The secondary antibody was goat anti-rabbit IgG conjugated with alkaline phosphatase. NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) detection was performed as described (Sambrook et al., 1989
).
Construction of fp25k-deleted HearNPV bacmid.
A pair of primers was designed to amplify the coding sequence of HearNPV fp25k, forward primer fp-F with an EcoRI site (underlined): 5'-GGCGAATTCAAAATGGAAACTGATCTAATTAATG-3' (the italic nucleotides are complementary to 4401744041 nt in HearNPV G4 genome) and reverse primer fp-R with a NotI site (underlined): 5'-GGCGCGGCCGCTATGACGGGCAAGGGGGCG-3' (the italic nucleotides corresponded to 4335543373 nt in HearNPV G4 genome). The PCR fragment was inserted into pGEM-T-easy vector (Promega). Based on this recombinant pGEM-T-easy vector, a 480 bp coding sequence of fp25k digested by HpaI (4349043969 nt in HearNPV-G4 genome) was replaced with a gene cassette with a chloramphenicol resistance (Cmr) gene and an enhanced GFP gene under the hsp70 promoter. This recombinant plasmid was named pFPdel. The 2·4 kb PCR fragment amplified from pFPdel using primers fp-F and fp-R was transformed into Escherichia coli BW25113 containing HaBacHZ8 DNA. The fp25k-deleted bacmid was generated by homologous recombination in E. coli, and was screened by kanamycin and chloramphenicol resistance as described previously (Hou et al., 2002). The positive recombinant was named HaBacWD11. For a control, an enhanced GFP gene was introduced to HaBacHZ8 by using the HearNPV bac-to-bac system (Wang et al., 2003
) and this generated the bacmid HaBacHZ8GFP. The constructs of the recombinant HaBacWD11 and HaBacHZ8GFP are shown in Fig. 1
.
|
All bacmids, identified by PCR and restriction enzyme analysis, were transfected into HzAM1 cells according to O'Reilly et al. (1992) and the supernatants of transfection were used as primary stocks of BV.
Detection of fp25k expression of the recombinant viruses.
HzAM1 cells were infected with HearNPV-G4, HaBacHZ8GFP, HaBacWD11, HaBacWD13 and HaBacWD14. The cells were collected at 48 h p.i., and cellular proteins were separated by 10 % SDS-PAGE. Immunoblotting was performed as described above.
Comparison of BV growth curves between recombinant viruses.
HzAM1 cells were infected with HaBacHZ8GFP, HaBacWD11, HaBacWD13 or HaBacWD14 at an m.o.i. of 5. At the appropriate time points p.i., supernatants were collected and the titres were detected using the end-point dilution method. The green fluorescence was used as the infectious marker during the assay. Each virus infection was repeated three times and the growth curves were generated by arithmetic mean data of three infections. The titre values of the four viruses were compared with one-way ANOVA (SPSS).
Electron microscopic observations of cells infected with the recombinant viruses.
HzAM1 cells were infected with HaBacHZ8GFP, HaBacWD11, HaBacWD13 or HaBacWD14 at an m.o.i. of 5. Infected cells were fixed at 96 h p.i. and processed for electron microscopic analysis. The arithmetic mean numbers of ODV particles were calculated from 20 to 25 cells that contained ODV particles.
Expression of Ha133 and ODV-E66.
HzAM1 cells were infected with HaBacHZ8GFP and HaBacWD11 at an m.o.i. of 5. At the appropriate time points p.i., infected cells were collected and rinsed with PBS. SDS-PAGE and immunoblotting were performed as described above. Anti-HaF1, a polyclonal rabbit antiserum against the N-terminal fragment of Ha133 (F1), was used as the primary antibody to detect Ha133, and a polyclonal rabbit antiserum anti-HaE66 was used for detecting ODV-E66.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Construction of recombinant viruses
Four recombinant viruses, HaBacWD11, HaBacHZ8GFP, HaBacWD13 and HaBacWD14 were constructed according to Methods. Their structures are shown in Fig. 1. All these recombinants were authenticated by PCR and restriction enzymes analysis. The recombinants were infective to HzAM1 cells where green fluorescence was evident (data not shown). The expression of FP25K in the cells infected with these viruses was detected by Western blots. As shown in Fig. 2(c)
, two forms of FP25K (26 and 23 kDa) were detected at 48 h p.i. in the cells infected with HearNPV-G4, HaBacHZ8GFP and HaBacWD14, but not in those infected with HaBacWD11 and HaBacWD13. Our results indicate that the recombinants were constructed correctly.
BV growth curves of recombinant viruses
HzAM1 cells were infected with HaBacHZ8GFP, HaBacWD11, HaBacWD13 or HaBacWD14. The one-step growth curves of BV are shown in Figure 3. Before 24 h p.i., the replication kinetics of the four viruses were very similar (F3,11=0·96; P=0·48). After 24 h p.i., the fp25k-deleted viruses (HaBacWD11 and HaBacWD13) always had a higher BV titre than those of non-deleted virus (HaBacHZ8GFP) and rescued virus (HaBacWD14) (Fig. 3
), although the differences were not significant. At 96 h p.i., the production of BVs was more significant from fp25k-deleted viruses than that of the fp25k-containing viruses (F3,11=7·416; P=0·011).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In cells infected with HearNPV, FP25K was expressed in two major molecular forms, 26 and 23 kDa (Fig. 2b and c), which is similar to the two forms in AcMNPV-infected Sf21 cells (Braunagel et al., 1999
). The smaller molecule may be the second product translated from the internal AUG codon present at 81 nt downstream of the first AUG codon in HearNPV. In AcMNPV, there is also an internal Met at aa 32 of FP25K. As not all sequenced baculovirus fp25k genes contain the second internal ATG codon, the functional significance of the small protein has not yet been determined. A third molecular form of FP25K, with the size of 24·5 kDa, was detected in HearNPV-infected cells after 48 h p.i. (Fig. 2b
), and it was not reported in AcMNPV-infected cells. The function of the 24·5 kDa protein is not known. We cannot rule out at present that the 24·5 kDa protein is a degradation product, even though protease inhibitors were used in the preparation of the infected-cell lysates.
Previously, most investigations on FP mutants were carried out on viruses with active polyhedrin. Since one of the effects of the FP25K mutation was decreased levels of polyhedrin mRNA together with an altered transport of polyhedrin into the nucleus, it raised the question if other effects of the FP25K mutation were the consequences of polyhedrin alteration. In this study, we used a polyhedrin-null bacmid system to study the function of FP25K. Our results indicate that FP25K mutants could cause increased production of BV and decreased amount of ODV production in the absence of a polyhedrin gene. It has been reported that AcFPgalCAT, a recombinant AcMNPV without polyhedrin and FP25K-negative, could release more infectious particles than Ac360CAT, which was occlusion-negative and FP25K-positive (Harrison & Summers, 1995b
). Our data and those of Harrison & Summers (1995b)
support each other with respect to the fact that the effects of FP25K mutants may not be related to polyhedrin.
HearNPV belongs to group II NPV (Chen et al., 1999), which use a homologue of Ld130 as a membrane fusion protein instead of GP64 present in group I nucleopolyhedroviruses (Pearson et al., 2000
). The homologue of Ld130 in HearNPV is Ha133 (Chen et al., 2001
). It was reported that AcMNPV fp25k mutants had enhanced accumulation of GP64 and decreased accumulation of ODV-E66 (Braunagel et al., 1999
; Rosas-Acosta et al., 2001
). The decrease of AcMNPV ODV-E66 was regulated either directly or indirectly at the translation level by FP25K (Rosas-Acosta et al., 2001
). The deletion of FP25K also altered the transportation and localization of ODV-E66 during infection (Rosas-Acosta et al., 2001
). However, we did not observe significant changes in the level or temporal expression of Ha133 or ODV-E66 in HaBacWD11 (FP25K mutant)-infected cells in comparison to the wild-type parental virus HaBacHZ8GFP. We suggest that the transportation and localization of ODV-E66 in FP25K-deleted mutants need to be investigated.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Braunagel, S. C., Burks, J. K., Rosas-Acosta, G., Harrison, R. L., Ma, H. & Summers, M. D. (1999). Mutations within the Autographa californica nucleopolyhedrovirus FP25K gene decrease the accumulation of ODV-E66 and alter its intranuclear transport. J Virol 73, 85598570.
Braunagel, S. C., Williamson, S. T., Saksena, S., Zhong, Z., Russell, W. K., Russell, D. H. & Summers, M. D. (2004). Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: facilitated trafficking to the inner nuclear membrane. Proc Natl Acad Sci U S A 101, 83728377.
Chakraborty, S. & Reid, S. (1999). Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures. J Invertebr Pathol 73, 303308.[CrossRef][Medline]
Chen, X., IJkel, W. F., Dominy, C. & 10 other authors (1999). Identification, sequence analysis and phylogeny of the lef-2 gene of Helicoverpa armigera single-nucleocapsid baculovirus. Virus Res 65, 2132.[CrossRef][Medline]
Chen, X., IJkel, W. F., Tarchini, R. & 8 other authors (2001). The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 82, 241257.
Fraser, M. J. & Hink, W. F. (1982). The isolation and characterization of the MP and FP plaque variants of Galleria mellonella nuclear polyhedrosis virus. Virology 117, 366378.[CrossRef]
Fraser, M. J., Smith, G. E. & Summers, M. D. (1983). Acquisition of host cell DNA sequences by baculoviruses: relationship between host DNA insertions and FP mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses. J Virol 47, 287300.
Harrison, R. L. & Summers, M. D. (1995a). Biosynthesis and localization of the Autographa californica nuclear polyhedrosis virus 25K gene product. Virology 208, 279288.[CrossRef][Medline]
Harrison, R. L. & Summers, M. D. (1995b). Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. J Gen Virol 76, 14511459.[Abstract]
Harrison, R. L., Jarvis, D. L. & Summers, M. D. (1996). The role of the AcMNPV 25K gene, "FP25", in baculovirus polh and p10 expression. Virology 226, 3446.[CrossRef][Medline]
Hink, W. F. & Vail, P. V. (1973). A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line. J Invertebr Pathol 22, 168174.[CrossRef]
Hou, S., Chen, X., Wang, H., Tao, M. & Hu, Z. (2002). Efficient method to generate homologous recombinant baculovirus genomes in E. coli. Biotechniques 32, 783784.[Medline]
Katsuma, S., Noguchi, Y., Zhou, C. L., Kobayashi, M. & Maeda, S. (1999). Characterization of the 25K FP gene of the baculovirus Bombyx mori nucleopolyhedrovirus: implications for post-mortem host degradation. J Gen Virol 80, 783791.[Abstract]
Lua, L. H., Pedrini, M. R., Reid, S., Robertson, A. & Tribe, D. E. (2002). Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. J Gen Virol 83, 945955.
McIntosh, A. H. & Ignoffo, C. M. (1983). Characterization of 5 cell lines established from species of Heliothis. Appl Entomol Zool 18, 262269.
O'Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992). Baculovirus Expression Vectors: a Laboratory Manual. New York: W. H. Freeman.
Pearson, M. N., Groten, C. & Rohrmann, G. F. (2000). Identification of the Lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J Virol 74, 61266131.
Potter, J. N., Faulkner, P. & MacKinnon, E. A. (1976). Strain selection during serial passage of Trichoplusia ni nuclear polyhedrosis virus. J Virol 18, 10401050.[Medline]
Rosas-Acosta, G., Braunagel, S. C. & Summers, M. D. (2001). Effects of deletion and overexpression of the Autographa californica nuclear polyhedrosis virus FP25K gene on synthesis of two occlusion-derived virus envelope proteins and their transport into virus-induced intranuclear membranes. J Virol 75, 1082910842.
Russell, R. L. Q. & Rohrmann, G. F. (1993). A 25-kDa protein is associated with the envelopes of occluded baculovirus virions. Virology 195, 532540.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn, vol. 1. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Slavicek, J. M., Podgwaite, J. & Lanner-Herrera, C. (1992). Properties of two Lymantria dispar nuclear polyhedrosis virus isolates obtained from microbial pesticide Gypchek. J Invertebr Pathol 59, 142148.[CrossRef]
Sun, X. L., Zhang, G. Y., Zhang, Z. X., Hu, Z. H., Vlak, J. M. & Arif, B. M. (1998). In vivo cloning of Helicoverpa armigera single nucleocapsid nuclear polyhedrosis virus genotypes. Virologica Sinica 13, 8388.
Wang, H., Deng, F., Pijlman, G. P., Chen, X., Sun, X., Vlak, J. M. & Hu, Z. (2003). Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome. Virus Res 97, 5763.[CrossRef][Medline]
Wu, D., Deng, F., Hu, Z. H., Yuan, L. & Sun, X. L. (2004). Molecular cloning and expression of the fp25k gene of HearNPV in E. coli and preparation of its antiserum. Virol Sin 19, 380384.
Zhang, G., Zhang, Y., Ge, L. & Shan, Z. (1981). The production and application of the nuclear polyhedrosis virus of Heliothis armigera (Hubei) in biological control. Acta Phytophylacica Sin 8, 235240.
Received 14 April 2005;
accepted 24 May 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |