1 RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
2 Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Saitama City, Saitama 338-8570, Japan
Correspondence
Toshihiro Nagamine
tnaga{at}postman.riken.jp
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ABSTRACT |
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INTRODUCTION |
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In Bombyx mori (BmN) cells, DNA replication of the baculovirus B. mori nucleopolyhedrovirus (BmNPV) occurs at discrete regions within the nucleus where IE1 and two DNA-binding proteins (DBP and LEF3) co-localize throughout the duration of DNA replication [<8 to approx. 24 h post-infection (p.i.); Okano et al., 1999]. Among these three proteins, IE1 initially localizes at small foci within the nucleus and, subsequently, the other two proteins associate with these foci prior to the onset of DNA replication to assemble viral DNA replication factories (Okano et al., 1999
). During viral DNA replication within these IE1-associated structures, the structures gradually enlarge and ultimately occupy a large proportion of the infected-cell nuclei at the end of the replication period (2436 h p.i.). Hence, temporal changes of IE1 localization seem to be correlated with viral DNA synthesis (Okano et al., 1999
). Consequently, extensive studies on IE1 localization may serve to facilitate our understanding of the multifunctional roles of IE1 in virus replication. Previous experiments examining IE1 localization were performed using fixed cell samples; however, live-cell imaging has become a powerful tool for measuring intracellular dynamics and therefore would greatly facilitate analysis of IE1 and its dynamic properties. In living cells, time-lapse studies can track the movement of larger cellular structures, whereas fluorescence recovery after photobleaching (FRAP) is ideal for measuring the intracellular mobility of fluorescently tagged proteins (Lippincott-Schwartz & Patterson, 2003
).
Observations of baculovirus-infected cells by electron microscopy have shown that progeny nucleocapsids assemble in a virus-induced subnuclear structure, called the virogenic stroma (reviewed by Williams & Faulkner, 1997). This stroma consists of two distinct regions: one is a fibrillar electron-dense area, while the other is an electron-lucent intrastromal space in which progeny nucleocapsids are formed (Summers, 1971
; Harrap, 1972
; Young et al., 1993
). A major capsid protein of 39 kDa, VP39 (Pearson et al., 1988
; Blissard et al., 1989
; Thiem & Miller, 1989
), has been shown to accumulate in this intrastromal space (Braunagel et al., 1996a
). In contrast to the intrastromal space, the electron-dense region is considered to be DNA rich and can be intensively stained with DNA-specific fluorescent dyes (Williams & Faulkner, 1997
; Rosas-Acosta et al., 2001
). In addition to its role in the maturation of nucleocapsids, the virogenic stroma is assumed to be the most probable site of viral DNA replication because of its high DNA content (Williams & Faulkner, 1997
). However, to date, no study has established a direct relationship between DNA replication sites and the virogenic stroma.
Following maturation within the virogenic stroma, the nucleocapsids leave the stroma, with some of them acquiring their envelopes within the peristromal regions of the nucleus (Kawamoto et al., 1977a, b
; Fraser, 1986
). This type of virion, called an occlusion-derived virus (ODV), is eventually encapsulated in an occlusion body within the nucleus after intranuclear envelopment (reviewed by Funk et al., 1997
). Baculoviruses can also produce another type of virion, termed a budded virus (BV). Nucleocapsids destined to become BVs are transported from the nucleus to the plasma membrane and obtain BV envelopes by budding through the plasma membrane instead of the intranuclear envelopment (reviewed by Funk et al., 1997
). In studies examining the intracellular localization of viral proteins by fluorescent microscopy, a number of ODV-associated proteins (e.g. ODV-E25, ODV-E66, P91 and P74) were shown to localize to the periphery of the nucleus (Russell & Rohrmann, 1997
; Braunagel et al., 1999
; Rosas-Acosta et al., 2001
; Slack et al., 2001
). More detailed observations with immunoelectron microscopy, however, have revealed that these proteins are mainly localized to the peristromal region in association with ODVs and intranuclear microvesicles (Russell & Rohrmann, 1993
, 1997
; Braunagel et al., 1996a
, b
; Hong et al., 1997
). The intranuclear microvesicles are the proposed source of ODV envelopes (Hong et al., 1997
). Thus, the peripheral regions of the nucleus in which ODV-associated proteins often accumulate are considered to be the peristromal regions for intranuclear envelopment and are assumed to be a subnuclear compartment distinct from the virogenic stroma (Hong et al., 1997
).
In this report, we have described the use of green fluorescent protein-tagged IE1 (IE1GFP) to demonstrate the dynamic behaviour of IE1 in BmN cells infected with BmNPV. We have also shown, using other GFP-tagged proteins, P91GFP, GFPODV-E25 and VP39GFP with DsRed-tagged IE1 (IE1DsRed), that IE1 localized to subnuclear structures that were separate from the ODV-associated proteins, P91 and ODV-E25, but were associated with the major capsid protein, VP39. These subnuclear structures were also shown to contain large amounts of DNA. These data revealed the specific localization of IE1 in the virogenic stroma and this conclusion was consistent with a previous theory, which proposed that the virogenic stroma is a site for viral DNA replication, as well as for the assembly of nucleocapsids (Williams & Faulkner, 1997).
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METHODS |
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Plasmid constructions.
To make a plasmid expressing GFP-tagged IE1 under the control of the ie1 promoter, its open reading frame (ORF) and 760 bp upstream region were inserted into a promoterless GFP vector, pEGFP-1 (Clontech). The termination codon of the ie1 coding sequence and the next 3 bp (TGAATT) in a plasmid containing the EcoRI-G fragment [map unit (m.u.) 90·596·4] of the BmNPV genome were replaced with the DNA sequence CTCGAG by means of site-directed mutagenesis for XhoI digestion. The mutagenized plasmid was digested with EcoRI and XhoI and the resulting fragment, which contained the ie1 ORF and its 760 bp upstream region, was inserted into the EcoRISalI sites of pEGFP-1. To align the two coding sequences of ie1 and GFP in the same reading frame, the recombinant plasmid was digested with BamHI, treated with mung bean nuclease and ligated intramolecularly. The resulting plasmid, pKm-IE1-GFP was partially sequenced to confirm that it was the desired construct. Plasmid pKm-IE1-DsRed was constructed by replacement of the KpnINotI fragment in pKm-IE1-GFP with the corresponding fragment of pDsRed (Clontech) and by alignment of the ie1 and DsRed coding sequences to the same reading frame. This was performed by AgeI digestion, mung bean nuclease treatment and intramolecular ligation. To construct plasmids expressing P91GFP and P91DsRed under the control of the p91 promoter (pPK-p91-GFP and pPK-p91-DsRed, respectively), the coding sequences of GFP and DsRed were inserted into the PstI-K fragment (m.u. 48·452·3) of the BmNPV genome that contains the p91 gene. The termination codon and the 3'-flanking region of the p91 coding sequence (TAAACATTTTATGT) in the PstI-K fragment were replaced with a DNA sequence containing XhoI and SpeI sites (CTCGAGTTACTAGT) by two cycles of site-directed mutagenesis. The resulting plasmid was digested with XhoI and SpeI and ligated with the XhoIXbaI fragments of pEGFP-1 and pDsRed2-1 (Clontech) containing the coding sequences of GFP and DsRed, respectively. Plasmids expressing GFPODV-E25 and VP39GFP under the control of their own promoter (pPES-oe25-GFP and pPES-vp39-GFP, respectively) were generated in a procedure similar to that for P91GFP. The PstISphI subfragment (m.u. 52·357·0) in the PstI-E fragment of the BmNPV genome was mutagenized. For the production of pPES-oe25-GFP, the 12 bp 5'-flanking region (immediately in front of the initiation codon) of the ODV-E25 coding sequence (TTAAAACAAATC) was replaced with the NheIXhoI-cutting sequence (GCTAGCCTCGAG). The NheISalI fragment of pEGFP-C1 (Clontech) containing the GFP ORF was subsequently inserted into the NheIXhoI site. Plasmid pPES-vp39-GFP was constructed by replacing the termination codon of the vp39 coding sequence and the next 9 bp (TAAAAATGGAGT) with the XhoINheI-cutting sequence (CTCGAGGCTAGC) followed by insertion of the XhoIXbaI fragment of pEGFP-1 containing the GFP ORF into the XhoINheI site.
Plasmid transfections and viral infections.
BmN cells (8x105) were seeded onto 27 mm glass-bottomed dishes (Matsunami) and left to stand for several hours to allow cell attachment. To introduce plasmids, the cells were transfected with 0·5 µg of each plasmid DNA sample using Lipofectin reagent (Invitrogen). The transfected cells were incubated at 28 °C for 24 h and directly analysed with a confocal microscope or infected with BmNPV at an m.o.i. of 10. In all experiments, time zero was defined as the time point at which fresh medium was added following the 1 h virus adsorption period. For DNA staining, BmN cells were plated onto 22x22 mm coverslips (no. 1; Matsunami) and allowed to attach over a period of several hours. Cells were transfected using Lipofectin reagent (Invitrogen) and subsequently infected with BmNPV. Infected cells were fixed in pre-chilled methanol at 20 °C for 6 min, incubated with propidium iodide (PI; 1 µg ml1) and RNase A (100 µg ml1) in PBS at 37 °C for 30 min and mounted with the Slow Fade light anti-fade kit (Molecular Probes).
Confocal microscopy and FRAP.
Confocal images were obtained with a Leica TCS NT using a 488 nm laser line for GFP and a 568 nm laser line for DsRed and PI. For double-labelling experiments, only one laser line was used for any single-channel recording to avoid cross talk between channels. To obtain merged figures, single-channel images were digitally superimposed with Photoshop 6.0 software. In FRAP experiments, Leica software (TCS NT version 1.6) was used to bleach a spot with a 100 ms pulse at high laser intensity and to acquire time-lapse images of the bleached cells. Quantification of fluorescence intensity was also done using Leica software.
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RESULTS |
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Time-lapse imaging of IE1GFP during viral infection demonstrates the dynamics of IE1-associated structures
To follow the temporal changes of IE1 localization in a single cell, we performed time-lapse recording of IE1GFP-expressing cells using a confocal microscope. At 45 h p.i., small IE1 foci were clearly observed in most of the BmNPV-infected BmN cells (see Fig. 1). As seen in Fig. 2
, time-lapse monitoring of these cells revealed a progressive expansion of IE1-associated structures. The onset time of this expansion varied considerably (i.e. some started at 56 h p.i., while others started at 1011 h p.i.) in cells that were infected for 1 h at an m.o.i. of 10. Our employment of an asynchronous culture for this experiment may have been the cause of the observed variation (Braunagel et al., 1998
; Ikeda & Kobayashi, 1999
). Typically, a stationary (pre-expansion) phase of the bright IE1-associated structures required 23 h, after which the IE1-associated structures began to expand. In cells possessing multiple IE1-associated structures, we observed a tendency among the structures to undergo simultaneous expansion, which often resulted in their fusing with one another. This highly dynamic stage of expansion and fusion usually persisted for 34 h and the subsequent enlargement of the IE1-associated structures was thereafter maintained, although the structure appeared to shrink after polyhedron formation (data not shown).
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IE1-associated structures expand with progressive DNA concentration
As described above, the IE1-associated structures were surrounded by accumulations of the ODV-associated proteins, P91 and ODV-E25 (Fig. 4 jo). Furthermore, previous studies have shown that the ODV-associated proteins accumulate at the periphery of the virogenic stroma (Russell & Rohrmann, 1993
, 1997
; Hong et al., 1997
). Taken together, these two independent observations lead to speculation that IE1 associates with the virogenic stroma. To test this possibility, we stained IE1GFP-expressing cells with the DNA-specific fluorescent dye, PI, which allows for visualization of the virogenic stroma given its high DNA concentration (Williams & Faulkner, 1997
; Rosas-Acosta et al., 2001
). In most of the uninfected interphase cells, both IE1GFP and DNA were distributed throughout the nucleoplasm (Fig. 5ac
). A certain proportion of the uninfected cells were present in the mitotic phase, which could be distinguished by the alignment of condensed DNA at the metaphase plate (Fig. 5b
, arrow). In these cells, IE1GFP also concentrated at the same place (Fig. 5a and c
, arrows), suggesting that IE1 could co-localize with cellular chromosomes independent of specific viral DNA sequences, such as hr (homologous region). When compared with the uninfected cells, the infected cells at 4 h p.i. showed no difference in the DNA staining pattern (Fig. 5e
), whereas small IE1GFP foci were visible (Fig. 5d
). At 8 h p.i., DNA staining in the marginal regions of the nuclei was slightly emphasized (Fig. 5h
). This pattern seemed to be consistent with previous observations by electron microscopy, which showed that the heterochromatin becomes progressively marginalized along the inner nuclear membrane (Williams & Faulkner, 1997
). Although DNA replication in the IE1-associated structures occurs at this time, the increased concentration of DNA in the structures could not be detected by PI staining. At 24 h p.i., however, the increase in DNA within the structures was clearly evident (Fig. 5jl
). This result suggested that IE1 becomes associated with the virogenic stroma, i.e. as the DNA progressively accumulates, the IE1-associated structure develops into the virogenic stroma.
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DISCUSSION |
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The virogenic stroma is a virus-induced nuclear structure in which progeny nucleocapsids are assembled (Williams & Faulkner, 1997). In this study, we have provided a number of lines of evidence showing that IE1 localizes to the virogenic stroma (i.e. that IE1-associated structures are identical to the virogenic stroma): (i) ODV-associated proteins P91 and ODV-E25 accumulated at the periphery of IE1-associated structures; (ii) the IE1-associated structures could be intensively stained with a DNA-specific dye; and (iii) the major capsid protein, VP39, was almost exclusively distributed in the IE1-associated structures. While this is the first report describing the localization of IE1 to the virogenic stroma, it has been shown previously that IE1 localizes to the site of viral DNA replication (Okano et al., 1999
). These two independent findings are consistent with the previously proposed idea that the virogenic stroma is the site of viral DNA replication (Williams & Faulkner, 1997
). The virogenic stroma begins to form around the time when viral DNA replication is initiated (Williams & Faulkner, 1997
) and the IE1-associated structure expands coincidently with DNA synthesis (Okano et al., 1999
). These observations may also support the identity of the two subnuclear structures, the virogenic stroma and the IE1-associated structure. Hence, we concluded that the virogenic stroma provides the site not only for nucleocapsid assembly but also for viral DNA replication.
Formation of the virogenic stroma (IE1-associated structure) may serve as an intranuclear compartmentalization to promote baculovirus replication. We have recently found that IE1 focus formation is induced by the binding of IE1 to the hr sequences of viral genomes (Nagamine et al., 2004). If this is the case, then the IE1 foci containing parental genomes may provide initial scaffolds to facilitate assembly of the DNA replication complex or factory (Okano et al., 1999
). With the onset of DNA synthesis, the IE1 foci begin to expand and develop into the virogenic stroma, which can hold the newly synthesized DNA within its own structure, thereby making it possible to accelerate the chain reaction of DNA replication. Furthermore, the concentration of DNA within the virogenic stroma may be advantageous to the assembly of nucleocapsids within the structure. After DNA synthesis, the nucleus is most likely divided into at least two subnuclear compartments, one being the virogenic stroma and the other the periphery of the virogenic stroma, namely the peristromal compartment (Williams & Faulkner, 1997
). Formation of this compartment seems to be dependent on the virogenic stroma, since P91GFP and GFPODV-E25 often accumulate at the edge of the IE1-associated structure (data not shown). At this late stage of the infection cycle, the virogenic stroma becomes the supplier of progeny nucleocapsids, while the peristromal compartment serves as the site of intranuclear envelopment of nucleocapsids and subsequently as the site of polyhedron formation. Hence, the partitioning of the nucleus into these compartments may be necessary for spatial segregation of the two steps of virion formation: nucleocapsid assembly and intranuclear envelopment. These two events may be unable to occur at the same place for as yet unknown reasons. The intranuclear membrane formation is a unique feature of baculoviruses and, therefore, nuclear partitioning may also be a unique requirement for baculovirus infection. The virogenic stroma may have evolved from this requirement but still maintained its roles for providing the site of DNA replication and nucleocapsid assembly.
IE1GFP within the IE1-associated structure exhibited slow fluorescence recovery in FRAP experiments. This immobility of IE1GFP may imply a structural role for IE1 in the virogenic stroma. While further experiments are still needed, our results may provide new insight into the structural aspect of the virogenic stroma and show the multifunctional nature of the nuclear structure that provides the site for DNA replication and nucleocapsid assembly.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Blissard, G. W. & Rohrmann, G. F. (1991). Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1. J Virol 65, 58205827.[Medline]
Blissard, G. W., Quant-Russell, R. L., Rohrmann, G. F. & Beaudreau, G. S. (1989). Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding vp39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata. Virology 168, 354362.[Medline]
Braunagel, S. C., Elton, D. M., Ma, H. & Summers, M. D. (1996a). Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56. Virology 217, 97110.[CrossRef][Medline]
Braunagel, S. C., He, H., Ramamurthy, P. & Summers, M. D. (1996b). Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35 and ODV-EC27. Virology 222, 100114.[CrossRef][Medline]
Braunagel, S. C., Parr, R., Belyavskyi, M. & Summers, M. D. (1998). Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 244, 195211.[CrossRef][Medline]
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.
Fraser, M. J. (1986). Ultrastructural observations of virion maturation in Autographa californica nuclear polyhedrosis virus infected Spodoptera frugiperda cell cultures. J Ultrastruct Mol Struct Res 95, 189195.
Friesen, P. D. (1997). Regulation of baculovirus early gene expression. In The Baculoviruses, pp. 141170. Edited by L. K. Miller. New York: Plenum.
Funk, C. J., Braunagel, S. C. & Rohrmann, G. F. (1997). Baculovirus structure. In The Baculoviruses, pp. 732. Edited by L. K. Miller. New York: Plenum.
Guarino, L. A. & Smith, M. (1992). Regulation of delayed-early gene transcription by dual TATA boxes. J Virol 66, 37333739.[Abstract]
Guarino, L. A. & Summers, M. D. (1986). Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol 57, 563571.[Medline]
Guarino, L. A. & Summers, M. D. (1987). Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol 61, 20912099.
Harrap, K. A. (1972). The structure of nuclear polyhedrosis viruses. III. Virus assembly. Virology 50, 133139.[CrossRef][Medline]
Hong, T., Summers, M. D. & Braunagel, S. C. (1997). N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus. Proc Natl Acad Sci U S A 94, 40504055.
Ikeda, M. & Kobayashi, M. (1999). Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovirus. Virology 258, 176188.[CrossRef][Medline]
Kawamoto, F., Kumada, N. & Kobayashi, M. (1977a). Envelopment of the nuclear polyhedrosis virus of the oriental tussock moth, Euproctis subflava. Virology 77, 867871.[CrossRef][Medline]
Kawamoto, F., Suto, C., Kumada, N. & Kobayashi, M. (1977b). Cytoplasmic budding of a nuclear polyhedrosis virus and comparative ultrastructural studies of envelopes. Microbiol Immunol 21, 255265.[Medline]
Kool, M., Ahrens, C. H., Goldbach, R. W., Rohrmann, G. F. & Vlak, J. M. (1994). Identification of genes involved in DNA replication of the Autographa californica baculovirus. Proc Natl Acad Sci U S A 91, 1121211216.
Kovacs, G. R., Guarino, L. A. & Summers, M. D. (1991). Novel regulatory properties of the IE1 and IE0 transactivators encoded by the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus. J Virol 65, 52815288.[Medline]
Lippincott-Schwartz, J. & Patterson, G. H. (2003). Development and use of fluorescent protein markers in living cells. Science 300, 8791.
Lu, A. & Carstens, E. B. (1993). Immediate-early baculovirus genes transactivate the p143 gene promoter of Autographa californica nuclear polyhedrosis virus. Virology 195, 710718.[CrossRef][Medline]
Lu, A. & Miller, L. K. (1995). The roles of eighteen baculovirus late expression factor genes in transcription and DNA replication. J Virol 69, 975982.[Abstract]
Maeda, S. & Majima, K. (1990). Molecular cloning and physical mapping of the genome of Bombyx mori nuclear polyhedrosis virus. J Gen Virol 71, 18511855.[Abstract]
Nagamine, T., Kawasaki, Y., Iizuka, T. & Matsumoto, S. (2005). Focal distribution of baculovirus IE1 triggered by its binding to the hr DNA elements. J Virol (in press).
Nissen, M. S. & Friesen, P. D. (1989). Molecular analysis of the transcriptional regulatory region of an early baculovirus gene. J Virol 63, 493503.[Medline]
Okano, K., Mikhailov, V. S. & Maeda, S. (1999). Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories. J Virol 73, 110119.
Pearson, M. N., Russell, R. L., Rohrmann, G. F. & Beaudreau, G. S. (1988). p39, a major baculovirus structural protein: immunocytochemical characterization and genetic location. Virology 167, 407413.[Medline]
Prikhod'ko, E. A. & Miller, L. K. (1999). The baculovirus PE38 protein augments apoptosis induced by transactivator IE1. J Virol 73, 66916699.
Pullen, S. S. & Friesen, P. D. (1995). Early transcription of the ie-1 transregulator gene of Autographa californica nuclear polyhedrosis virus is regulated by DNA sequences within its 5' noncoding leader region. J Virol 69, 156165.[Abstract]
Ribeiro, B. M., Hutchinson, K. & Miller, L. K. (1994). A mutant baculovirus with a temperature-sensitive IE-1 transregulatory protein. J Virol 68, 10751084.[Abstract]
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, L. Q. & Rohrmann, G. F. (1993). A 25-kDa protein is associated with the envelopes of occluded baculovirus virions. Virology 195, 532540.[CrossRef][Medline]
Russell, L. Q. & Rohrmann, G. F. (1997). Characterization of P91, a protein associated with virions of an Orgyia pseudotsugata baculovirus. Virology 233, 210223.[CrossRef][Medline]
Slack, J. M., Dougherty, E. M. & Lawrence, S. D. (2001). A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag. J Gen Virol 82, 22792287.
Summers, M. D. (1971). Electron microscopic observations on granulosis virus entry, uncoating and replication processes during infection of the midgut cells of Trichoplusia ni. J Ultrastruct Res 35, 606625.[Medline]
Theilmann, D. A. & Stewart, S. (1991). Identification and characterization of the IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 180, 492508.[Medline]
Thiem, S. M. & Miller, L. K. (1989). Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 63, 20082018.[Medline]
Williams, G. V. & Faulkner, P. (1997). Cytological changes and viral morphogenesis during baculovirus infection. In The Baculoviruses, pp. 61107. Edited by L. K. Miller. New York: Plenum.
Young, J. C., Mackinnon, E. A. & Faulkner, P. (1993). The architecture of the virogenic stroma in isolated nuclei of Spodoptera frugiperda cells in vitro infected by Autographa californica nuclear polyhedrosis virus. J Struct Biol 110, 141153.[CrossRef]
Received 5 July 2004;
accepted 25 August 2004.