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J Virol, July 1998, p. 6237-6243, Vol. 72, No. 7
Interdepartmental Graduate Program in
Genetics1 and
Department of
Entomology,2 University of
California, Riverside, California 92521
Received 10 November 1997/Accepted 13 April 1998
Substitution of granulin from the Trichoplusia ni
granulosis virus (TnGV) for polyhedrin of the Autographa
californica multinucleocapsid nuclear polyhedrosis virus
(AcMNPV) yielded a few very large (2 to 5 µm) cuboidal
inclusions in the cytoplasm and nucleus of infected cells. These
polyhedra lacked the beveled edges characteristic of wild-type
AcMNPV polyhedra, contained fractures, and occluded few
virions. Placing a nuclear localization signal (KRKK) in granulin directed more granulin to the nucleus and resulted in more structurally uniform cuboidal inclusions in which no virions were observed. A
granulin-polyhedrin chimera produced tetrahedral occlusions with more
virions than granulin inclusions but many fewer than wild-type
polyhedra. Despite the unusual structure of the granulin and
granulin-polyhedrin inclusions, they interacted with AcMNPV p10 fibrillar structures and electron-dense spacers that are precursors of the polyhedral calyx. The change in inclusion shape obtained with
the granulin-polyhedrin chimera demonstrates that the primary amino
acid sequence affects occlusion body shape, but the large cuboidal
inclusions formed by granulin indicate that the amino acid sequence is
not the only determinant. The failure of granulin or the
granulin-polyhedrin chimera to properly occlude AcMNPV virions suggests that specific interactions occur between polyhedrin and other viral proteins which facilitate normal virion occlusion and
occlusion body assembly and shape in baculoviruses.
Most baculoviruses produce
occlusions of a characteristic size and shape, but little is known
about the specific interactions between the occlusion matrix protein
and other viral components that control virion occlusion and occlusion
body morphology. In nuclear polyhedrosis viruses (NPVs), it has been
hypothesized that virion occlusion and polyhedral growth are initiated
by specific interactions between polyhedrin molecules and the virion
envelope (17). Ultrastructural evidence suggests that
polyhedron formation is initiated by nucleation of polyhedrin on the
virion surface (9, 10, 16), after which polyhedra grow by
the addition of more virions and polyhedrin. In granulosis viruses
(GVs), granulin deposition begins at one end or one side of the virion
envelope and proceeds around the particle (2). It has been
suggested that the size and shape of mature occlusion bodies are
determined to a large extent by the sequence or secondary structure of
the occlusion protein (4, 6), and a number of polyhedrin
mutations have been described for which this is the case
(3-8), including a mutant of the Autographa
californica multinucleocapsid NPV (AcMNPV), in which
leucine rather than proline occurs at residue 58, producing large
cuboidal polyhedra that often lack virions (4). Information on potential functional domains of AcMNPV polyhedrin has
been provided by fusion of different polyhedrin regions to
The studies described above suggest that specific regions or amino
acids of polyhedrin are critical to interactions with polyhedrin and
other viral molecules. Substitution of a heterologous occlusion protein
for AcMNPV polyhedrin separates the effects of interactions between occlusion matrix proteins from the effects of virion
interactions with polyhedrin, assisting identification of the molecular
determinants of these traits. Expression of Spodoptera frugiperda
MNPV (SfMNPV) polyhedrin, which has 93% similarity to
AcMNPV polyhedrin, in an occlusion-negative (occ
mutant) AcMNPV strain (14) resulted in small
polyhedra that contained fewer virions than wild-type polyhedra.
SfMNPV polyhedrin was expressed at a low level in the recombinant compared with expression in wild-type
AcMNPV, and the recombinant virus had reduced expression
of the 25K gene, which also could have affected the polyhedron
phenotype. However, interactions between virions and the heterologous
polyhedrin occurred, even if at a reduced level.
The possibility of functional conservation between granulins and
polyhedrins has not previously been explored, however. To determine
whether the protein domains conserved between the Trichoplusia ni GV (TnGV) granulin (1) and AcMNPV
polyhedrin (18) included those regions important to virion
occlusion, we expressed the granulin gene in occ mutant
AcMNPV and studied virion occlusion and occlusion body
formation. We selected TnGV granulin because its amino acid similarity
to AcMNPV polyhedrin is only 72% and because, in wild-type
TnGV infection, occlusions are markedly different in size and shape
from AcMNPV polyhedra. Since wild-type TnGV granulin has no
nuclear localization signal and therefore might not reach as high a
concentration in the nucleus as does AcMNPV polyhedrin, and
since a low concentration of occlusion protein in the nucleus has been
suggested as the proximal cause of the few-polyhedra (FP) mutant
phenotype (21), we constructed a granulin mutant that
contained the AcMNPV polyhedrin nuclear localization signal
(KRKK). A hybrid granulin-polyhedrin gene was also constructed and
expressed to determine whether the highly conserved C-terminal region
of polyhedrin plays a role in virion occlusion.
Viruses and cells.
Viruses were grown in BTI-TN-5B1-4 (Tn5)
cells (Invitrogen, Carlsbad, Calif.) maintained as monolayer cultures
in TC-100 (12) supplemented with 10% fetal bovine serum
(Gibco BRL, Gaithersburg, Md.). Recombinant strains of
AcMNPV were constructed with the Bac-to-Bac system (Gibco
BRL) and maintained in Escherichia coli as described in the
Bac-to-Bac manual. The TnGV was obtained from John D. Paschke (Purdue
University, West Lafayette, Ind.) and propagated in T. ni
larvae.
Cloning of polyhedrin and granulin open reading frames (ORFs).
TnGV DNA was digested with EcoRV and ligated into the
EcoRV site of pcDNAII (Invitrogen) to produce a genomic
library. This library was screened with a digoxigenin-labeled probe
(Genius system; Boehringer Mannheim, Indianapolis, Ind.) made from
a 444-bp HindIII-EcoRI fragment of the
granulin gene (1). The probe fragment was purified from an
EcoRI digest of a HindIII genomic clone by
agarose gel electrophoresis and glass bead purification (Geneclean; BIO
101, La Jolla, Calif.). An EcoRV clone of about 7 kb was
identified and digested with NheI and AseI to
yield a 918-bp fragment containing the granulin gene. The ends of this fragment were filled with Klenow fragment (Boehringer Mannheim), and
the fragment was purified by gel electrophoresis and recovered with a
Qiaquick column (Qiagen, Chatsworth, Calif.) and then ligated (Fastlink
kit; Epicentre, Madison, Wis.) into a modified pFASTBAC (Bac-to-Bac
kit; Gibco BRL) that lacked the AcMNPV polyhedrin promoter
and that had been digested with StuI. The polyhedrin promoter was deleted from the modified pFASTBAC by digestion with BamHI and SnaBI, followed by blunting with Klenow
fragment and self-ligation to produce the plasmid pFBd. All restriction
enzymes were from New England Biolabs (Beverly, Mass.) unless otherwise specified.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Substituting Granulin or a
Granulin-Polyhedrin Chimera for Polyhedrin on Virion Occlusion and
Polyhedral Morphology in Autographa californica
Multinucleocapsid Nuclear Polyhedrosis Virus
ABSTRACT
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-galactosidase (20). Polyhedrin contains a nuclear
localization signal at residues 32 to 35 (KRKK). Mutation of this
signal to NGNN abolished nuclear localization, and large cuboidal
polyhedrin crystals formed in the cytoplasm. Additionally, amino acids
19 to 130 were shown to be required for formation of polyhedron-like
crystals in the nucleus, and amino acids 30 to 130 were shown to be
required for stable localization of fusion proteins to the nucleus,
though the latter fusion did not form crystals (20).
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Construction of granulin with a nuclear localization signal. Alignment of the predicted amino acid sequences of selected polyhedrin and granulin genes available from GenBank (Fig. 2) showed that most of these contain a motif at amino acids 30 to 33 similar to the AcMNPV polyhedrin basic amino acid sequence KRKK, which has been demonstrated to function as a nuclear localization signal (20). The homologous region of the predicted TnGV granulin protein, however, is the non-consensus sequence RHKE. Accordingly, to determine whether the presence of this sequence affected granulin nuclear localization and occlusion body formation, RHKE was replaced by mutagenesis with the equivalent sequence, KRKK, that occurs in AcMNPV polyhedrin.
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Construction of a hybrid granulin-polyhedrin ORF. The AcMNPV polyhedrin and TnGV granulin genes contain a conserved HindIII site in predicted amino acid 84 of polyhedrin and amino acid 87 of granulin. To construct a hybrid gene, pFB/APO was digested with HindIII, and a 0.6-kb fragment was purified by agarose gel electrophoresis and ligated into the gel-purified vector fragment of HindIII-digested pFB/TGO. Clones were screened for orientation by restriction enzyme digestion. The resulting construct, pFB/AT, contained amino acids 1 to 87 of granulin in frame with amino acids 84 to 245 of polyhedrin (Fig. 1).
Each construct was transferred into a polyhedrin-negative AcMNPV strain with the Bac-to-Bac kit, following the recommendations of the manufacturer (Gibco BRL). The final viral constructs were as follows: Ac/FB/TGO, with the TnGV granulin gene; Ac/FB/TGO/K, with the modified TnGV granulin gene, encoding a nuclear localization signal; Ac/FB/AT, with the chimeric granulin-polyhedrin; and Ac/FB/APO, with the AcMNPV polyhedrin gene. All occlusion protein genes were expressed under control of the AcMNPV polyhedrin promoter to ensure equivalent expression. All constructs were confirmed by sequencing, phenotype of infected cells, and, in the case of the granulin constructs, dot blotting of viral DNA with the granulin probe described above.Transfection of insect cells and T. ni larvae. Viral DNA for the recombinant constructs was isolated from E. coli by alkaline lysis miniprep and transfected into Tn5 cells (Invitrogen) in 75-cm2 tissue culture flasks with Insectin (Invitrogen) or Cellfectin (Gibco BRL) according to the manufacturers' instructions. Recombinant viruses were maintained in Tn5 cells. T. ni larvae were infected by puncture with a pin that had been dipped in virus-containing cell culture medium and were examined at 6 days postinfection.
Light and electron microscopy. After transfection, Tn5 cells were monitored in 75-cm2 tissue culture flasks with phase microscopy involving a Zeiss Invertoscope D or as wet mounts involving a Zeiss Photomicroscope III. Infected cells to be embedded for electron microscopy were rinsed from the bottom of the flask with a Pasteur pipette and pelleted at approximately 500 × g. They were then transferred to a microcentrifuge tube, prefixed for 15 to 30 min in 1.5% glutaraldehyde-0.25% sucrose, resuspended in 3% glutaraldehyde-0.25% sucrose, fixed for at least 2 h, and processed for light and electron microscopy as described previously (9).
Expression of the TnGV granulin gene by AcMNPV. The recombinant AcMNPV (Ac/FB/TGO) expressing the TnGV granulin gene typically produced a few large cuboidal "polyhedra," averaging 2 to 5 µm on edge, in each infected cell, although in some cells only a single large inclusion was produced (Fig. 3A). The granulin inclusions occurred in both the cytoplasm and the nucleus. In a sample of 30 cells, the mean number of granulin inclusions in the nucleus was 2.5 (range, 0 to 12); in the cytoplasm, it was 2.0 (range, 0 to 6).
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Expression of the TnGV granulin gene with a nuclear localization signal. In cells infected with Ac/FB/TGO/K (producing KRKK-granulin), cuboidal inclusions were evident in the cell nucleus by three days postinfection. Most nuclei contained only one or a few large cuboidal granulin inclusions, whereas others contained many smaller inclusions (Fig. 3B). Occasionally, two inclusions appeared to be in the process of combining to form a large crystal.
When examined by electron microscopy, the inclusions produced by Ac/FB/TGO/K were homogeneous, contained no virions, and did not appear fractured (Fig. 4A and B). As in Ac/FB/TGO, large numbers of virions accumulated at the periphery of the nucleus. The granulin inclusions were fewer, larger, and less evenly spaced around the periphery of the nucleus than typical polyhedra in cells infected with wild-type AcMNPV. Association of the granulin inclusions with fibrillar networks was common, and electron-dense spacers could be seen aligned with the outer surface. Although more than 95% of the KRKK-granulin inclusions occurred in the nucleus, an occasional inclusion, also homogeneous and unfractured, was observed in the cytoplasm.
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Expression of hybrid granulin-polyhedrin gene. Cells infected with the recombinant AcMNPV producing the granulin-polyhedrin chimera produced one or a few large tetrahedral inclusions per cell, most of which occurred in the nucleus (Fig. 3C and D). Like the granulin inclusions, these were unevenly spaced in the nucleus.
Viewed by electron microscopy, the tetrahedral crystals appeared dense and unfractured and contained AcMNPV virions, although at a much lower concentration than wild-type polyhedra (Fig. 4D to F). As in the AcMNPV recombinants that produced granulin, unoccluded virions accumulated at the nuclear membrane of Ac/FB/AT-infected cells. Fibrillar p10 networks and electron-dense spacers were also associated with the tetrahedral occlusions.Expression of granulin and KRKK-granulin by AcMNPV in T. ni larvae. Light microscopy of plastic sections showed that granulin inclusions in both Ac/FB/TGO infection and Ac/FB/TGO/K infection were mostly nuclear, smaller, and fewer in number than the granulin inclusions produced in cell culture infections. The Ac/FB/TGO/K virus produced larger inclusions than Ac/FB/TGO. Only a few inclusions were present per fat body cell, and typically only one inclusion was present per hemocyte (Fig. 4C).
Electron microscopy of larvae infected with Ac/FB/TGO/K showed essentially similar results to those described above for Tn5 cells. Fat body tissue contained inclusions that were still cuboidal but had edges more rounded than the inclusions produced in tissue culture cells (not shown). These inclusions contained no virions, associated with p10, and were aligned with electron-dense spacers as described above. In some cases, nucleocapsids could be seen aligned end-on with granulin inclusions. Ac/FB/TGO was not examined by electron microscopy because, by light microscopy, visible inclusions were rare.Expression of AcMNPV polyhedrin by recombinant AcMNPV. In contrast to the results obtained with the AcMNPV-expressed granulin constructs, the Ac/FB/APO control construct, which expressed the AcMNPV polyhedrin gene under control of the AcMNPV polyhedrin promoter, produced what appeared to be typical AcMNPV polyhedra (Fig. 4G to I). These polyhedra were of normal size, formed in the zone around the virogenic stroma, and occluded many virions in patterns characteristic of wild-type AcMNPV polyhedra.
The results of the present study demonstrate that TnGV granulin cannot substitute efficiently for polyhedrin in the AcMNPV occlusion process. Granulin without the KRKK nuclear localization signal occluded small numbers of virions in some cells, but no occluded virions were observed in KRKK-granulin inclusions. Moreover, both types of granulins formed large cuboidal crystals, resembling neither characteristic AcMNPV polyhedra nor TnGV granules. A granulin-polyhedrin hybrid was able to partially rescue occlusion, suggesting that the C-terminal region of polyhedrin is involved in occlusion but is not sufficient for normal occlusion. Occlusions produced by the hybrid protein resembled neither polyhedra nor granulin crystals. These results provide evidence that initiation and continuation of virion occlusion require specific domain-domain interactions between the occlusion body molecule and the virion envelope, probably proteins on the envelope surface, as suggested previously (4, 6-8, 16, 17), and that these interactions failed to occur because the homologous domains of granulin and AcMNPV virions were poorly compatible. In the absence of normal interactions between the AcMNPV virion and granulin, large granulin crystals formed by default in the nucleus and cytoplasm of the infected cells. Cuboidal or other aberrant crystals which occlude virions poorly if at all have been reported for several mutant AcMNPV polyhedrins (3-6). The mutations in some of these polyhedrins had potentially disruptive effects on the folding of the polyhedrin protein and therefore most likely accounted for the lack of characteristic virion occlusion and formation of typical AcMNPV polyhedra. In the present study, however, at least the wild-type granulin, when produced by AcMNPV, should have yielded a granulin molecule with a normally folded structure capable of normal granulin-granulin interactions. If so, the failure of the wild-type granulin to occlude most AcMNPV virions further supports the hypothesis that poor occlusion was due to a lack of compatible interactions between granulin and the AcMNPV virion. Nevertheless, the occurrence of virions in some of the granulin crystals and the exclusion of other structural viral components, such as fibrillar structures, nucleocapsids, or electron-dense spacers, suggest that some weak but specific interaction occurred between granulin and the AcMNPV virion that was enhanced by partial replacement of granulin in the granulin-polyhedrin chimera. The low level of virion occlusion obtained when wild-type granulin was substituted for polyhedrin in the AcMNPV resembled the AcMNPV FP mutant phenotype, caused by a deletion of the 25-kDa protein gene (11). FP mutants produce a small number of polyhedra, which contain few or no virions, and it has been suggested that the FP phenotype is caused by slower accumulation of polyhedrin, so that a critical concentration of polyhedrin required for occlusion is not reached (21). To eliminate this as a cause of the low level of virion occlusion obtained with granulin, we constructed the KRKK-granulin, which clearly resulted in a higher concentration of granulin in the nucleus (Fig. 3C and D). That we observed no virions in the crystals formed by KRKK-granulin again suggests a lack of compatible interaction between granulin and the AcMNPV virion. Though granulin failed to interact compatibly with AcMNPV virions, it did interact with another AcMNPV protein involved in polyhedron formation. The p10 protein is a component of fibrillar networks in the nucleus and cytoplasm of infected cells (22, 24, 26, 29, 30) that is not required for production of infectious polyhedra (26). Fibrillar networks were associated with granulin crystals and with hybrid crystals in our recombinant AcMNPV strains, indicating that AcMNPV p10 is able to interact with TnGV granulin. This result is similar to the observation that Spodoptera exigua MNPV (SeMNPV) p10 formed fibrillar networks in infected cells when expressed by AcMNPV (25). It is less likely that the polyhedron envelope protein (PEP) interacted compatibly with granulin, KRKK-granulin, or the hybrid granulin-polyhedrin. PEP occurs in electron-dense spacers associated with the p10-containing fibrillar structures found in the nucleus of baculovirus-infected cells as well as in the polyhedrin envelope (13, 23-25, 28, 30). Electron-dense spacers did align along the occlusion surfaces in Tn5 cells infected with the recombinant viruses (Fig. 3F and 4A, B, and D). However, in some cells in advanced stages of disease, the granulin or mutant polyhedra were clumped together, forming a single large irregular crystal, suggesting that the crystals had no delimiting envelope. Thus, it appears that p10 can interact with granulin to bring it into close proximity with AcMNPV PEP, as occurs normally during occlusion formation, but that interactions between AcMNPV PEP and granulin may not be sufficient for envelope formation. The cuboidal shape of the granulin occlusions may also reflect an inability to be "sealed" with a polyhedron envelope, as Orgyia pseudotsugata MNPV (OpMNPV) with a deleted PEP gene produced polyhedra with a cuboidal shape distinct from the more beveled wild-type OpMNPV polyhedra (15). Individual NPV species produce polyhedra of a characteristic shape, providing some evidence that occlusion body shape is determined in part by the primary amino acid sequence of the occlusion protein (4, 6, 8, 10, 27). The formation of large cuboidal crystals by wild-type granulin reported here, however, makes it clear that occlusion body shape is determined by more than the primary amino acid sequence alone. Nevertheless, the formation of cuboidal crystals by granulin and of tetrahedral crystals by the granulin-polyhedrin chimera provides experimental evidence that the amino acid sequence of the occlusion protein affects occlusion body shape. Previous studies on the molecular basis of baculovirus virion occlusion and occlusion body formation have focused on polyhedrin mutants or polyhedrin fusion proteins with unusual phenotypes (3-8, 20, 21). Except for the production of the closely related SfMNPV polyhedrin in AcMNPV (14), heterologous expression of occlusion proteins has not been used to study the occlusion process. Our results indicate that substituting full and partial regions of occlusion body proteins among distantly related viruses will be useful for defining the molecular determinants of virion occlusion and occlusion body formation and shape.| |
ACKNOWLEDGMENTS |
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We thank Dennis Bideshi for his constructive review of the research and the manuscript.
This research was supported in part by USDA Competitive Grant 95-37312-1634 to B.A.F.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Entomology, University of California, Riverside, CA 92521. Phone: 909-787-5006. Fax: 909-787-3086. E-mail: brian.federici{at}ucr.edu.
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J Virol, July 1998, p. 6237-6243, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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