Download Nuclear F-actin: a functional component of

Journal of Cell Science 103, 15-22 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
15
Nuclear F-actin: a functional component of baculovirus-infected
lepidopteran cells?
LOY E. VOLKMAN1-*, SALMA N. TALHOUK2, DANIEL I. OPPENHEIMER1 and CAROL A.
CHARLTON1
'Department of Entomology and 2Department of Plant Biology, University of California, Berkeley, California 94720, USA
•Author for correspondence
Summary
Cellular functions known to involve actin are thought to
occur in the cytoplasm. Even though actin has frequently been found in the nucleus, systems well-suited
for studying the function of such nuclear actin are rare.
We observed filamentous (F) actin within nuclei of
IPLB-Sf-21 cells infected with Autographa californica M
nuclear polyhedrosis virus (AcMNPV) as detected by
laser confocal microscopy using fluorescent phalloidin
probes. The nuclear F-actin co-localized with the major
capsid protein of the virus during normal infections.
Cytochalasin D, known to interfere with nucleocapsid
morphogenesis, uncoupled the apparent co-localization
of F-actin and the capsid protein and inhibited infectious
progeny production. Inhibition was reversible throughout infection (even in the presence of a protein synthesis
inhibitor) and the nuclear co-localization of F-actin and
the capsid protein was re-established upon removal of
the drug. These observations suggest that nuclear Factin plays a role in virus replication, and that
AcMNPV-infected cells may constitute a useful system in
which to expand our understanding of nuclear actin
transport and function.
Introduction
al., 1977), and mRNA capping, in cytoplasmic polyhedrosis virus-infected cells (Furuichi and Miura, 1975).
Within this tradition, the study of Autographa californica M nuclear polyhedrosis virus (AcMNPV), a
nuclear-replicating DNA virus, could provide fundamental information regarding the significance of nuclear actin.
In all virus-host cell systems studied so far, viral
components have been shown to associate with cellular
filaments in at least some processes necessary for viral
replication (Penman, 1985; Ciampor, 1988; Simard et
al., 1986; Eaton and Hyatt, 1989; Knipe, 1990).
Accordingly, several nuclear-replicating viruses such as
herpes and adenoviruses are thought to depend on
nuclear matrices for their respective assembly processes, but no nuclear association with F-actin has been
reported for these viruses. In AcMNPV-infected cells,
actin microfilaments undergo a series of changes
throughout the course of infection that appear to
correlate with the sequential expression of virus gene
classes (Charlton and Volkman, 1991). During late gene
expression, at the time of nucleocapsid morphogenesis,
actin microfilaments form within the nuclear region.
Herein we show laser confocal microscopic evidence of
the co-localization of p39, the major AcMNPV capsid
The participation of actin in nuclear functions of cells
(with the possible exception of amphibian oocytes) is
still regarded as a controversial issue (Clark and
Merriam, 1977; Clark and Rosenbaum, 1979; Sheer et
al., 1984). Although cell fractionation studies of many
different organisms have indicated that actin can be
found in the nucleus (LeStourgeon, 1978), unless the
nuclear actin is an isoform distinct from that found in
the cytoplasm, as reported by Bremer et al. (1981),
contamination is difficult to rule out. In addition, no
information is available on how actin gets transported
to the nucleus in the absence of any obvious nuclear
transport signals. Recently, however, the case for actin
as a valid nuclear component has been bolstered by
reports of the discovery of nucleus-specific, actinbinding proteins (Rimm and Pollard, 1989; Ankenbauer et al., 1989). Even so, systems well suited for
addressing questions regarding nuclear actin function
are rare.
Historically, viruses have proven useful for imparting
information regarding the activities of their hosts. For
example, mRNA splicing was first documented in
adenovirus-infected cells (Berget et al., 1977; Chow et
Key words: Autographa californica M nuclear polyhedrosis
virus, baculovirus, nuclear F-actin, nucleocapsid
morphogenesis, cytochalasin D.
16
L. E. Volkman and others
protein, and F-actin within the nucleus of infected insect
cells. Treatment of infected cells with cytochalasin D
(CD), known to interfere with nucleocapsid morphogenesis (Volkman et al., 1987; Volkman, 1988; Hess et al.,
1989), uncoupled the nuclear co-localization of p39 and Factin, and inhibited infectious virus production. Removal
of CD from infected cultures in both the presence and
absence of cycloheximide (CH), a protein synthesis
inhibitor, resulted in the rapid re-establishment of F-actin
within the nucleus, its co-localization with p39, and the
onset of progeny production. These results are consistent
with the hypothesis that nuclear F-actin plays a critical
role in AcMNPV nucleocapsid morphogenesis, and
suggest that the study of this virus may provide a window
for enhancing our understanding of nuclear actin function
in general.
Materials and methods
Cells, inoculum and infection procedure
All experiments were conducted with Spodoptera frugiperda
LPLB-Sf-21 cells grown at 28°C in BML-TC/10 medium
(Gardiner and Stockdale, 1970) with 10% FCS. The source of
virus was a second passage E2 strain of AcMNPV extracellular virus (Smith and Summers, 1978) harvested from culture
medium at 48 h post-infection (p.i.). Cells were infected at a
multiplicity of infection (m.o.i.) of 20 plaque forming units
(p.f.u.) per cell. Infection with an m.o.i. of 20 should ensure
that 99.99% of the cells receive at least 1 infectious particle
(particle number per cell is thought to occur according to a
Poisson distribution), and thus essentially all cells become
infected by the input inoculum. Nevertheless, infection
cannot be regarded as synchronous, because different cells
will receive different numbers of particles, and infection
kinetics vary with dosage. In addition, lepidopteran cell lines
are notorously heteroploid, thus even in recently cloned lines
cells cannot be regarded as genetically identical (Ennis and
Sohi, 1976). Furthermore, the cells were not synchronized
with regard to cell cycle for these experiments. Time zero was
defined as the time at which viral inoculum was removed
following a 1-h adsorption period.
Drug treatments
Treatment of infected cells with CD (Sigma Chemical
Company, St. Louis, MO) was performed as previously
described (Talhouk and Volkman, 1991). A stock solution of
CD was prepared at a concentration of 5 mg/ml in
dimethylsulfoxide (DMSO) and diluted into culture medium
immediately before use. After a 1-h adsorption period, the
viral inoculum was removed and cells were rinsed once with
fresh medium containing 5 /ig/ml CD. Controls were treated
with 0.1% DMSO. CH (Sigma) was prepared in BML-TC/10
medium and used at 200 ^g/ml.
lnfectivity assay
Titers of progeny virus were determined by the immunoplaque technique (Volkman and Goldsmith, 1982). Briefly,
2X104 log phase cells in 10 fA medium were placed in each 5mm well of a 12-well printed glass slide. After cell attachment
(30 min), the medium was drawn off and replaced with 10 fA of
appropriately diluted inoculum and incubated for 2 h at 27°C.
At the end of the 2 h adsorption period the inoculum was
removed and replaced by a drop of medium containing 0.6%
methylcellulose (methylcellulose overlay) to confine progeny
virions to cells immediately adjacent to initially infected cells.
The slides were incubated in a humidity chamber for 40 h at
27°C, after which the overlay was removed and the cells fixed
with formyl-buffered acetone. Following fixation, foci of cells
containing viral antigens were stained using a peroxidase/antiperoxidase procedure and rabbit antiserum to AcMNPV
purified from polyhedra.
Antibodies and fluorescent probes
Antiserum to AcMNPV purified from polyhedra and prepared in rabbits was described previously (Volkman, 1983).
Monoclonal antibody 39P10 (mAb 39P10), reactive with the
AcMNPV major capsid protein, was originally obtained from
Sharon Roberts and Jarue Manning, University of California,
Davis (Whitt and Manning, 1988). Fluorescein isothiocyanate-conjugated, affinity-purified goat anti-mouse immunoglobulin G (FITC-anti-IgG), FITC-conjugated phalloidin,
tetramethylrhodamine isothiocyanate (TRlTC)-conjugated
phalloidin and propidium iodide (PI) were purchased from
Sigma. BODIPY™ phallacidin (ex 581/em591) was purchased from Molecular Probes, Eugene, Oregon.
Immunofluorescence microscopy
Cells were prepared for immunofluorescence microscopy
basically as reported previously (Charlton and Volkman,
1991). Briefly, specially cleaned, sterile coverslips were placed
in tissue culture dishes and suspensions of 5 x 105 cells in 500
fA samples of media were applied to them. Cells were allowed
to adhere for 1 h or overnight before being infected and/or
treated with CD as described above. At 23 h post-infection,
cells were rinsed with fresh media containing CH, CD or
DMSO and allowed to incubate for one hour. At 24 h p.i.,
coverslips were processed by removing media, fixing for 15
minutes in 2% paraformaldehyde in PHEM buffer (60 mM
PIPES (piperazine-A'-A''-bis (2-ethanesulfonic acid)), 25 mM
HEPES
(A'-2-hydroxyethylpiperazine-A''-2-ethanesulfonic
acid), 10 mM EGTA (ethylene glycol-bisQS-aminoethyl
ether)-A',A',A'',A''-tetraacetic acid), and 2 mM MgCI2, pH
6.9), and solubilizing for 15 min in 0.15% Triton X-100 in
PHEM buffer. Cells to be labeled with PI were incubated for
15 min in 30 jig/ml RNase A in PHEM and rinsed twice with
PHEM. Coverslips were then floated for 45 min at 27°C on 25
^1 of 3 x 10"7 M FITC-phalloidin, BODIPY™ phallacidin or
TRJTC-phalloidin in the presence or absence of monoclonal
antibody 39P10 (1:100 in PHEM) (as specified in figure
legends), rinsed three times in PHEM, and floated for 45 min
on 25 jul FITC-anti-IgG (1:100 in PHEM) if the coverslip had
been treated with the primary antibody. Cells were rinsed
three times in PHEM and either directly mounted for viewing,
or treated for 30 s with PI (1.0 f<g/ml in PHEM), rinsed once
more with PHEM and then mounted for viewing in a drop of
nonbleach mountant. Coverslips were viewed with a Sarastro
confocal laser scanning microscope (CLSM 1000) set for dual
detection with a xlO ocular and a xlOO (1.4 NA) objective.
Confocal images were visualized as single channel images on a
Silicon Graphics 4D/35TG workstation and merged using
Sarastro software. Images were transferred to a Macintosh IIx
for lettering and measurement bars and then photographed
with a Lasergraphics LFR Plus Film Recorder. All images
except those shown in Fig. 4 were 0.4 fim step sections
through the cell with a 0.1 fim pixel size. Images for Fig. 4
were 0.1 fan step sections with a 0.1 fim pixel size.
Results
When 24-h infected cells were stained with fluorescent
Fig. 1. Laser confocal microscopic mid-nuclear optical sections showing DNA (PI) and F-actin (BODIPY™ phallacidin or
FITC-phalloidin), or p39 (mAb 39P10 and FTTC-anti-IgG) of 24 h infected cells. (A) DNA, mid-nuclear section. (B) Factin (FITC-phalloidin), same section as A. (C) A and B merged. (D) DNA, mid-nuclear section. (E) p39, same section as
D. (F) D and E merged. (G) F-actin (BODIPY™ phallacidin), mid-nuclear section. (H) p39, same section as G. (I) G and
H merged. Colocalization of red and green fluorescent dyes appears as yellow in merged images, vs, virogenic stroma; rz,
ring zone. Bar, 5 fim.
AcMNP\'-induced nuclear F-actin
probes for both DNA and F-actin and examined by
laser confocal microscopy, F-actin was found to be in
the nucleus in 47.5% of the cells (Fig. 1A through C).
Detailed analyses of mid-nuclear optical sections of
these cells indicated that the F-actin was specifically
located in the "ring zone", a nuclear region surrounding
the virogenic stroma, which normally forms late in
infection during nucleocapsid morphogenesis (Xeros,
1956). When similarly infected cells were probed for
p39 (the major capsid protein) and DNA, p39 was
17
found to be distributed throughout the nucleus, but
most highly concentrated in the ring zone (Fig. ID
through F). F-actin in the ring zone appeared to be colocalized with p39 (Fig. 1G through I).
Similar analyses were conducted on 24-h infected
cells treated with CD throughout the infection period.
In these cells, the ring zone appeared to be much
narrower (Fig. 2A and C), and p39 was restricted
almost entirely to it (Fig. 2D and F). Nuclear F-actin
was observed in 18% of these cells, but no co-
Fig. 2. Laser confocal
microscopic mid-nuclear
optical sections showing
DNA (PI) and F-actin
(BODIPY™ phallacidin,
FITC-phalloidin or TRTTCphalloidin), or p39 (mAb
39P10 and FTTC-anti-IgG) of
24 h infected cells treated
with with CD. (A) DNA,
mid-nuclear section.
(B) F-actin (FITCphalloidin), same section as
A. (C) DNA, mid-nuclear
section. (D) p39, same
section as C. (E) F-actin
(TRITC-phalloidin), midnuclear section. (F) p39,
same section as E. rz, ring
zone. Bar, 5 JOTI.
18
L. E. Volkman and others
Fig. 3. One hour after
removal of CD from a 24-h
infected culture treated with
CD. (A) F-actin
(BOD1PY™ phallacidin),
mid-nuclear section.
(B) p39 (mAb 39P10 and
FITC-anti-IgG), same
section as A. Bar, 5 fan.
localization with p39 was evident. In the majority of
these cells (82%), F-actin occurred in the cytoplasm,
frequently forming rings juxtaposed to and surrounding
the nucleus (Fig. 2B and E).
When the CD was rinsed away from such 24-h
infected, CD-treated cells and analyzed for F-actin and
p39 location, F-actin was observed to be co-localized
with the narrow ring of p39 in the nucleus within an
hour (Fig. 3). The co-localization of F-actin and p39 in
these cells was remarkable. The same result was
obtained when CH was added just before and during
the removal of CD, indicating that no new protein
synthesis was needed for the re-organization of F-actin
to occur (data not shown).
An unexpected result was noted when we examined
uninfected cells treated with CD for 24 h. In contrast to
infected cells given this treatment, 66% of uninfected
cells had actin cables coursing through the nucleus as
determined by a "through" focus series (Fig. 4D
through H). DNA staining of the same series showed
that the F-actin occurred in the same regions as the
DNA, indicating that the F-actin was within the nucleus
and was not an artifact attributable to nuclear folding
(Fig. 4C and I). F-actin was not detected to any
significant extent (0.2%) in nuclei of uninfected cells
cultured in drug-free medium (Fig. 4A-B).
To determine the biological relevance of these
results, we designed experiments to examine the effect
of CD on virus replication at different stages of
infection. When CD was added at the onset of
infection, or at 16 h p.i. (the time when infectious
progeny virions normally begin to appear in the
medium in our system), an increase in extracellular
virus titer did not occur (Fig. 5). (Titers of 1 x 104
p.f.u./ml and below can be regarded as background in
this system due to technical difficulties in removing
residual inoculum.) Additionally, when CD that had
been added at the onset of infection was removed at 16
h p.i., the viral growth curves closely resembled those
of the untreated controls. These results indicated that
viral replicative events occuring during the first 16 h of
infection were not involved in the CD-sensitive step.
When CD was added at 24 h after the onset of
infection (a time of active extracellular virus production), the infectious particle concentration continued to increase for approximately 8 h (although at a
rate somewhat lower than in the control cultures) and
then reached a plateau while control values increased
further (Fig. 6). When CD was removed from infected
cells treated with the drug for 24 h, infectious virus
production resumed (apparently without delay) and a
steep production rate was observed from 24 through 38
h p.i. (Fig. 6). The production rate was significantly
decreased from 38 through 72 h p.i., but nevertheless,
by 72 h p.i., the titer in the cultures treated with CD for
the first 24 h was the same as in control cultures. Virus
titers from infected cells continually exposed to CD did
not increase above background levels.
The complete parity of extracellular virus titer after a
24 h delay was unexpected for several reasons, among
them the knowledge that many events in viral replication are temporally regulated by the stoichiometry of
both host and viral factors, and also the knowledge that
genome synthesis and genome packaging are usually
tightly coupled. We were curious, therefore, to determine whether recovery was possible throughout the
course of infection. Accordingly, we set up parallel
cultures of infected cells treated or not treated with CD.
Starting at 14 h p.i., and continuing at 16 h intervals, we
removed the medium from both treated and untreated
cultures, rinsed them, and incubated them for another
16 h when samples were taken and analyzed for the
number of infectious progeny produced in each 16 h
period. The results shown in Fig. 7 demonstrate that
partial recovery was possible even after infected cells
had been incubated with CD for 78 h, and that the
patterns of relative amounts produced in each interval
were similar for experimental and control cultures.
To determine whether recovery of infectious virus
production upon removal of CD was dependent upon
protein synthesis, we pretreated cells with CH for 2 h
prior to the removal of CD, and thereafter for another
18 h before removing samples for titer determination.
We found that infected cells rinsed free of CD and
resuspended in drug-free medium produced 32-fold
more infectious progeny than similar cells resuspended
AcMNPV'-induced nuclear F-actin
Fig. 4. Laser confocal microscopic mid-nuclear optical sections showing DNA (PI) and F-actin (FITC-phalloidin) in
uninfected, untreated cells (A,B) or uninfected cells treated with CD for 24 h (C-T). (A) DNA, mid-nuclear section.
(B) F-actin, same section as A. (C) DNA. (D) F-actin, same section as C. (D-H) F-actin, sequential sections. (I) DNA,
last section in sequence. Numbers indicate section number in image series. Bar, 5 /im.
in CH-containing medium. Cells resuspended in CHcontaining medium, however, produced over 100-fold
more infectious progeny than cells resuspended in CDcontaining medium (Fig. 8). These results suggested
that while the ability to synthesize proteins provided a
distinct advantage in resuming progeny production
after removal of CD, it was not essential. The
effectiveness of the CH treatment was confirmed by
assessing the degree of incorporation of 35S-methionine
into 24 h-infected cells in the presence and absence of
CH (Fig. 8).
Discussion
Like adenoviruses and herpesviruses, baculoviruses
replicate within the nucleus and bind detergentinsoluble elements of the nuclear matrix during assembly (Wilson and Price, 1988). Electron microscopic
studies of baculovirus-infected cells indicate that during
nucleocapsid morphogenesis empty capsids appear to
form in association with basal structures attached to the
nuclear matrix. Capsids are oriented toward the DNAcontaining virogenic stroma and, in this- position,
20
L. E. Volkman and others
10 7 ,
I
10»,
d
103,
102
10'
10
20
30
40
50
60
70
80
Hours post-infection
Fig. 5. The effect of adding or removing CD on production
of extracellular virus (p.f.u./ml) at 16 h p.i. Infectedcontrol, (D); infected with the continuous presence of CD,
(A); CD treatment started at 16 h p.i., ( • ) ; CD treatment
removed at 16 h p.i., (A). Bars represent one standard
deviation of the mean.
107,
I
10".
10
Control
+CD 24 h p.i.
CO control
-CD24hp.L
10'
20
30
40
50
60
70
80
Hours post-infection
Fig. 6. The effect of adding or removing CD on production
of extracellular virus (p.f.u./ml) at 24 h p.i. Infectedcontrol ( • ) ; infected with the continuous presence of CD
(A); CD treatment started at 24 h p.i. ( • ) ; CD treatment
removed at 24 h p.i. (A). Bars represent one standard
deviation of the mean.
appear to be filled with DNA (Fraser, 1986). As has
been shown here, however, AcMNPV is different from
other nuclear-replicating viruses because it induces the
polymerization of F-actin within the nucleus during
replication. Our hypothesis is that nuclear F-actin
functions as a scaffold during nucleocapsid morphogenesis.
The addition of CD after 16 h of infection blocked the
appearance of infectious progeny as effectively as the
addition of CD at at the beginning of infection,
indicating the inhibitory effect of CD is not dependent
on a process or event that occurred during the first 16 h
of infection. The immediate recovery of p.f.u. pro-
14-30
30-46
46-62
62-78
78-94
Hours post-infection (h)
Fig. 7. Profile of p.f.u./ml released from control infected
cells, or from infected cells rinsed free of CD during 16 h
intervals throughout the course of infection. Cells were
infected in the presence or absence of CD. At 16 h
intervals, beginning at 14 h p.i. the medium was removed
from three dishes each of CD " + " and " - " cells, the cells
were rinsed, and fresh drug-free medium was added.
Media samples were taken 16 h later (30, 46, 62, 78, and
94 h p.i.) and p.f.u./ml determined. Extracellular virus
concentration for the continuous presence of CD was less
than 5 x 104 p.f.u./ml in these experiments. Bars represent
one standard deviation of the mean.
duction upon removal of CD at 16 h p.i. further
supports this contention.
When CD was added at 24 h p.i., the inhibitory effect
was immediate, but not absolute until 8 h later. This
observation is consistent with the occurrence of an
intracellular pool of nucleocapsids, sufficiently completed so as to be beyond the CD-sensitive step, which
continues to bud while new assembly is prohibited by
missing or dysfunctional scaffolding. The rapid recovery observed in p.f.u. production when 24 h-infected
cells were rinsed free of CD, along with the observed
rapid reappearance of F-actin co-localized with p39
within the nucleus, is consistent with the view that Factin is the CD-sensitive factor important to viral
morphogenesis. Also, the recovery of infectious
progeny production from infected cells rinsed free of
CD at any time during the period when viral morphogenesis is taking place, i.e. from 14 to 94 h p.i., is
consistent with our scaffolding hypothesis. Finally, the
recovery of infectious virus from infected cells rinsed
free of CD in the presence of CH indicates that all
proteins required for the synthesis of infectious virus
are synthesized in the presence of CD, and that they can
somehow reconstitute appropriate structures to form
infectious virus when CD is removed. These results
indicate that CD interferes with a structural, or possibly
a post-translational biosynthetic event, or both.
We previously reported that actin synthesis is
stimulated by CD in IPLB-Sf-21 cells. In untreated
AcMNPV-induced nuclear F-actin
21
Fig. 8. Left: recovery of infectious
progeny virus from infected cells rinsed
free of CD in the presence of CH. Cells
were infected in the presence or
absence of CD. At 22 h p.i., one CDcontaining flask was additionally treated
with CH, and eventually designated
CD/CH. At 24 h p.i., cells were rinsed
and the medium replaced with either
drug-free (DF), CD-containing (CD) or
CH-containing (CH) medium. At 42 h
p.i., media samples were taken and
p.f.u./ml determined. Treatments for 024 h/24-42 h, respectively, are indicated
as DF/DF; CD/DF; CD/CD; and
DF/DF CD/DF CD/CD CD/CH
-CH +CH -CH +CH
CD/CH. Bars represent one standard
deviation of the mean. Right: effect of
Treatment
CH on protein synthesis in infected
cells. Cells were infected in drug-free medium. At 23 h p.i., CH was added to one of two flasks, and thereafter was
present continuously throughout the following manipulations. At 24 h p.i., the cells were rinsed and starved for 1 h in
methionine-free medium then the medium was replaced with complete medium containing 20 /iCi/ml [35S]methionine. The
cells were incubated for one additional hour before being harvested, subjected to SDS-PAGE, transferred to nitrocellulose,
stained with Ponceau red (lanes 3 and 4), and examined by autoradiography (lanes 1 and 2).
cells, actin represents approximately 3.5% of total
protein synthesis per unit time; in cells treated with CD
for 8 h or more, it represents approximately 13.5% of
total protein synthesis per unit time (Talhouk and
Volkman, 1991). The amount of F-actin also increases
in CD-treated cells, appearing as aggregates or
networks of thick cables. In the current study we noted
that CD-induced F-actin structures coursed through the
nuclei of numerous uninfected, and a few infected, cells
after 24 h of treatment. Possibly the F-actin polymerizes
in the nuclei due to abnormally high concentrations of
actin in the cells in the presence of CD.
We detected nuclear F-actin in 0.2% of uninfected,
untreated IPLB-Sf 21 cells. Whether actin occurs
normally in nuclei of a much higher percentage of these
cells in a form non-reactive with phalloidin is an
unresolved question at this time. Nevertheless, the fact
that we detected F-actin within nuclei of AcMNPVinfected cells indicates that actin must be transported to
the nucleus by either a host-specified or a viralmediated mechanism. Further experimentation will be
necessary to distinguish between the two.
Nuclear F-actin observed in AcMNPV-infected
IPLB-Sf-21 cells appears to be functional in nucleocapsid morphogenesis. F-actin has been reported to occur
within the nuclei of other organisms, such as the slime
mold Physarum polycephalum (Lachapelle and
Aldrich, 1988), and higher eucaryotic cells heat shocked
or treated with chemicals such as DMSO (Welch and
Suhan, 1985; Osborn and Weber, 1980), but evidence
for functions of nuclear F-actin in these systems is
lacking. Since actin polymerization within the nucleus
of AcMNPV-infected cells is controlled by a late viral
gene product(s) (Charlton and Volkman, 1991), genetic
approaches should be useful for gaining information on
viral factors mediating the event, and further elucidate
its significance in viral replication. Insight gained
through studying this system could be useful in sorting
out transport mechanisms and the significance of
nuclear actin in other systems.
The laser confocal microscope and the equipment used for
recording images was made available by the NSF Center of
Plant Developmental Biology. The authors thank Steven E.
Ruzin for his assistance with the confocal microscopy, and
W.T. Jackman, Sara L. Tobin and Zac Cande for their helpful
comments on the manuscript. The research was financially
supported by NSF grant DCB-8701725, U.S.D.A. Competitive Research grant 87-CRCR-2416, and by federal Hatch
funds.
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(Received 14 February 1992 - Accepted, in revised form,
20 May 1992)