Download Replication and morphogenesis of Amsacta moorei entomopoxvirus

Journal of General Virology (1993), 74, 1457 1461. Printed in Great Britain
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Replication and morphogenesis of Amsacta moorei entomopoxvirus in
cultured cells of Estigmene acrea (salt marsh caterpillar)
Susan A. M a r l o w , Lara J. Billam, Christopher P. Palmer and Linda A. King*
School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, U.K.
A study of the sequence of morphogenic events
occurring within Amsacta moorei entomopoxvirusinfected Estigmene acrea cells is presented. Stages in
virion development, and the various cytopathic effects
observed in these cells between 0 and 120h postinfection (p.i.) are described. Events in the early stages
of virion assembly (24 to 48 h p.i.), the formation of the
outer viral membrane (48 to 72 h p.i.) and the de-
velopment of occlusion bodies or spheroids (72 to
120 h p.i.) were identified. Cells grown in TC100 culture
medium supported production of mature virus particles,
the majority of which were either of the intracellular
naked virion form, or mature virions incorporated into
occlusion bodies. Only limited production of the
extracellular enveloped form was observed in these cells.
The Amsacta moorei entomopoxvirus (AmEPV) is one of
the most well studied members of the subfamily
Entomopoxviridae. It is currently classified with the
intermediate-sized entomopoxviruses (genus B) that have
been isolated from Lepidoptera and Orthoptera
(Matthews, 1982). In common with the other entomopoxviruses (Bergoin et al., 1969; Goodwin & Filshie, 1969;
Henry et al., 1969) and some vertebrate poxviruses
(Ichihashi et al., 1971), AmEPV produces three forms of
virion, the non-occluded intracellular naked virion, the
extracellular enveloped virion (EEV) and occluded
virions. The spheroid occlusion bodies are primarily
composed of spheroidin protein (Langridge et al., 1977;
Langridge & Roberts, 1982; Hall & Hink, 1990). The
spheroidin gene has been identified and sequenced, and
encodes a polypeptide of 114-8K (Hall & Moyer, 1991).
Spheroidin forms a crystalline matrix which protects the
mature virions during transmission between insects
(Goodwin et al., 1990; Granados & Roberts, 1970).
Relatively little is known about the AmEPV replication cycle, although cell lines from Lymantria dispar
(Goodwin et al., 1990), Heliothis zea, Spodoptera
frugiperda, Bombyx mori and Estigmene acrea (Granados
& Naughton, 1975; Granados, 1981) have been reported
to support virus replication. From observations made in
AmEPV-infected E. acrea larval tissues at 9 and 14 days
post-infection (p.i.), various stages in virion maturation
have been identified (Granados & Roberts, 1970).
Because these infections were asynchronous, a sequence
of virion morphogenesis was proposed using the vertebrate poxviruses as a model (Bergoin & Dales, 1971).
These studies identified primary sites of virus assembly
within distinct regions of the cytoplasm adjacent to the
nucleus, termed viroplasms and designated type I or II
depending upon the arrangement and composition of the
granular material contained within. Immature virus
particles that formed in the viroplasms were shown to be
formed from sections of membrane, which circularized
and filled with nuclear material. This condenses during
virus maturation to form a characteristic 'brick-shaped'
core. In cross-section, the core was observed to contain
rodlike structures, suggesting that the nuclear material
forms a continuous, filamentous coiled rod (Granados &
Roberts, 1970). The outer virus membrane, of both
naked and occluded virions, has been shown to possess
a beaded surface (McCarthy et al., 1974) similar to that
observed in Melolontha melolontha EPV (Bergoin et al.,
1971) and vaccinia virus (VV) (Dales, 1963; Medzon &
Bauer, 1970; Dales & Pogo, 1981).
In this study, AmEPV-infected E. acrea cells at times
from 0 to 120 h.p.i, were examined using transmission
electron microscopy (TEM), and an ordered sequence of
morphogenic and cytopathic events was established in
cultured cells.
E. acrea cells (EAA-BTI) were kindly supplied by Dr
W. F. Hink (Ohio State University Columbus, Ohio,
U.S.A.). Cells were grown and infected as previously
described (Marlow et at., 1992), and were examined
under the light microscope from 24 to 96 h p.i. for
cytopathic signs of infection. All samples were fixed in a
mixture of 2-5 % glutaraldehyde/2 % paraformaldehyde,
post-fixed in 1% osmium tetroxide, washed several times
in cacodylate buffer and then dehydrated through a
graded alcohol series before infiltration with Spurr's
resin. Ultrathin sections were stained with uranyl acetate
and lead citrate, and were examined by TEM.
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Fig. 1. Sections through E. acrea cells infectecd with AmEPV showing events in virus replication and morphogenesis. (a) At 24 h p.i.,
cells contained type I viroplasms (vpI), crescent-shaped membranes (cm) and membranes filling with nuclear material (arrowheads).
(b) At 48 h p.i., cells contained various forms of immature particle (im), some acquiring an outer membrane (arrow). (c) By 72 h p.i.,
virus particles have an outer membrane (arrow) and cytopathic effects are present, including crystalline bodies (nb) within the nucleus
(arrowheads) and cytoplasmic fibrils (cf). (d) Inset showing beaded morphology of the outer membrane at 72 h p.i. (e) At 96 h p.i.,
occlusion bodies (ob) were also present. Bar markers represent 0.1 gm.
Control, uninfected E. acrea cells p r o d u c e d m o n o - or
bipolar processes. Following infection with A m E P V the
cells r o u n d e d up between 12 and 2 4 h p . i . , and by
48 h p.i. the c y t o p l a s m was visibly grainy and highly
vacuolated as previously described ( M a r l o w et al., 1992).
Between 96 and 120 h p.i., infected cells contained
varying n u m b e r s of spheroids, with an estimated 95 % o f
the cells containing m a t u r e spheroids at 120 h p.i.
By using a high multiplicity o f infection (m.o.i. = 20),
we were able to achieve synchronous infection o f the cells
so that the sequence o f morphological events during
virus m a t u r a t i o n could be ordered with respect to time.
Various stages in the replication cycle of A m E P V that
were observed confirmed previous findings ( G o o d w i n et
al., 1990), but additional intermediate stages in virion
assembly between 48 to 72 h p.i. were demonstrated.
The first signs o f virus infection at 24 h p.i. were the
a p p e a r a n c e o f discrete type I viroplasms within the
cytoplasm, often adjacent to the nucleus (Fig. 1 a). These
electron-dense areas, previously described by G r a n a d o s
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Fig. 2. Intermediate stages of virus morphogenesis. (a) Immature virus cores in association with cytoplasmic vesicles (V) become
enveloped in a double membrane. (b) The outer membrane acquired between 48 and 72 h p.i. is formed from short curved sections
(arrowheads), in severallayers around the core (im). Polyribosome-likestructures were often present in association with the assembling
particles (arrows). (c) Core maturation proceeds during acquisition of the outer membrane (arrow), which is cytoplasmic in origin
(arrow); the core membrane has an outer spicule-coated layer (arrowheads). Bar markers represent 0"1 p.m. (d) The outer membrane
appears to consist of several layers, with distinct subunits undergoing differentiation. Cytoplasmic fibrils are present, associated with
assembling virions (cf). Bar marker represents 0.5 ~tm.
& Roberts (1970) in infected larval tissue, are the sites of
D N A replication and early virion assembly events, by
analogy with VV-infected cells (Bergoin & Dales, 1971).
Between 12 and 24 h p.i., crescent-shaped membranes
began to appear within, and at the periphery of, the
viroplasms and filled with viroplasmic material to form
immature viral particles. The majority of these particles
appeared to form within the first 24 h after infection, but
were also present in cells examined at 48 h p.i., in close
association with the viroplasmic areas. In addition, at
48 h p.i. multiple membranes were observed around
some of the cores (Fig. 1 b). Infected cells sampled at 72
(Fig. 1 c, d) and 96 (Fig. 1 e) h p.i. contained various
forms of virus particle. Although the immature forms
were still present at 72 h p.i., m a n y particles had acquired
an outer m e m b r a n e (intermediate assembly stage) (Fig.
1 c, d). The section in Fig. 1 (d) shows the characteristic
beaded surface of this outer membrane. At 72 and
96 h p.i., the cells contained characteristic bundles of
fibrils and developing occlusion bodies. The sections of
the outer membrane were cytoplasmic in origin, and
were often found in association with cytoplasmic vesicles
(Fig. 2a). As with VV and other EPVs, the m e m b r a n e is
probably derived from the Golgi apparatus. The mem-
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(b)
V
Fig. 3. Intracellular maturing virus particles. (a) Cells contained virions
grouped close to the plasma membrane (pm). (b) Early nucleation of
virions at 72 h p.i., showing the maturing virions during occlusion into
the spheroidin protein. (c) Spheroid development at 120h p.i.,
containing numerous particles, with the characteristic halo around the
mature particles. Bar markers represent 0-5 gin.
brane formed several layers around the core (Fig. 2b),
and this process may correspond to the second wrapping
stage in VV assembly (Dr G. Griffiths, EMBL, University of Heidelberg, Germany, personal communication). This second membranous wrapping of the virus
core appeared to proceed simultaneously with the
maturation events occurring within the core between 72
and 96hp.i. (Fig. 2b to d). The outer membrane
complex appeared to be composed of a unit membrane
(Fig. 2d), with distinct subunits, undergoing differentiation. The area between the core and the outer
membrane, shown in Fig. 2 (c), may be the site of lateral
body development. Similar membrane structures have
also been observed in AmEPV-infected Gatleria mellonella larvae (Roberts, 1970), but their function was not
discussed. Our observations suggest that these membrane
complexes form the outer viral envelope, and may
undergo differentiation to give rise to the beaded surface
morphology commonly observed in negatively stained
virion preparations (McCarthy et al., 1974). The transverse section in Fig. 2 (c) shows the core surrounded by
a double membrane with a smooth inner layer and an
outer, spicule-coated layer. Polyribosome-like structures
were often observed in association with the assembling
virions (Fig. 2b), and occlusion bodies in AmEPV-cells
were similar to those found by Ichihashi & Dales (1973)
in cowpox virus infections, and are probably involved in
the assembly process.
As in previous reports (Dales, 1963; Bergoin et al.,
1971; Granados, 1973; Kurstak & Garzon, 1977;
Goodwin et al., 1990), a number of cytopathic effects
were observed in infected cells between 72 and 96 h p.i.
These included in the presence of spherical, paracrystalline bodies within the nucleus (Fig. 1 c), and
bundles of cytoplasmic fibrils which often appeared to be
associated with immature virus particles (Fig. 1 c, d). The
latter may be modified host cell cytoskeleton proteins,
such as actin, or tubulin, which have been implicated in
the assembly process of poxviruses (Hiller et al., 1979).
This is currently being investigated further using
immunolabelling techniques.
The routinely low titres (5 x l06 TCIDs0 maximum) of
extraceUular virus produced by AmEPV-infected E.
acrea cells may be partially explained by the retention of
large numbers of mature virus particles within the
cytoplasm, as the intracellular naked form (Fig. 3a).
Although many of these were observed at the periphery
of the cell, there was little evidence of exocytic release.
This may also partially explain the difficulties involved in
obtaining multiple rounds of infection when using a low
m.o.i., and in obtaining a reliable plaque assay in E.
acrea cells. Virions were released passively from the cell
after 144 h p.i., following disintegration of the plasma
membrane (not shown). Virus released passively fol-
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lowing breakdown of the plasma membrane, or via
artificial cell disruption, will not acquire the outer
envelope derived from the plasma membrane. In VV, the
outer envelope contains Vp37 (Blasco & Moss, 1991), a
glycoprotein important for cell-to-cell transmission. In
one of the alternative cell lines, L. dispar, that supports
AmEPV replication, extracellular enveloped virions are
produced (C. P. Palmer, S.A. Marlow & L. A. King,
unpublished) and provide a reliable plaque assay and
secondary infection (Goodwin et aL, 1990; Hall &
Moyer, 1991). This would suggest that the cell line
appears to influence the form of virion produced.
Between 72 and 96 h p.i., nucleating groups of mature
virions became occluded into the spheroidin protein in a
random arrangement (Fig. 3 b, c). The immature spheroids were small and round, but as they matured (Fig. 3 b)
they adopted the characteristic oval shape (Fig. 3c).
Virions undergoing occlusion at 72hp.i. exhibited
structural features characteristic of the mature virions,
suggesting that occlusion of mature virions occurs, as
well as post-occlusion virion maturation, as has been
previously reported (Granados, 1981).
In conclusion, the series of events observed in the
maturation of AmEPV virions in vitro appear to be
closely related to that reported for VV (Dales, 1963;
Dales and Kajioka, 1964; Ichihashi et al., 1971; Dr G.
Griffiths, personal communication) and confirm most of
the maturation stages proposed by Granados & Roberts
(1970) from their in vivo replication studies.
This work was funded by the SERC Biotechnology Directorate and
ICI Agrochemicals. C. P. Palmer was supported by a SERC studentship. We would like to thank Dr G. Griffiths, EMBL, Heidelberg, for
communicating results prior to publication.
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