Three-Dimensional Analysis of a Viral
RNA Replication Complex Reveals a
Benjamin G. Kopek1, Guy Perkins2,3, David J. Miller4,5, Mark H. Ellisman2,3, Paul Ahlquist1,6*
1 Institute for Molecular Virology, University of Wisconsin–Madison, Madison, Wisconsin, United States of America, 2 National Center for Microscopy and Imaging Research,
University of California San Diego, La Jolla, California, United States of America, 3 Department of Neurosciences, University of California San Diego, La Jolla, California, United
States of America, 4 Department of Medicine, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 5 Department of Microbiology and
Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 6 Howard Hughes Medical Institute, University of Wisconsin–Madison,
Madison, Wisconsin, United States of America,
Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All
positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such
as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships
between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We
applied electron microscope tomography and complementary approaches to flock house virus (FHV)–infected
Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor,
protein A, and FHV-specific 5-bromouridine 5’-triphosphate incorporation localized between inner and outer
mitochondrial membranes inside ;50-nm vesicles (spherules), which thus are FHV-induced compartments for viral
RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to
the cytoplasm by a necked channel of ;10-nm diameter, which is sufficient for ribonucleotide import and product RNA
export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA
replication intermediates and an interior shell of ;100 membrane-spanning, self-interacting protein As. The results
identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions,
and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication
complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently
proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.
Citation: Kopek BG, Perkins G, Miller DJ, Ellisman MH, Ahlquist P (2007) Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.
PLoS Biol 5(9): e220. doi:10.1371/journal.pbio.0050220
Positive-strand RNA [(þ)RNA] viruses contain messenger-
sense, single-stranded RNA in their virions; they represent
over a third of known virus genera; and they include many
important human, animal, and plant pathogens . A
common, if not universal, feature of (þ)RNA virus replication
is the association of their RNA replication complexes with
infection-specific host intracellular membrane rearrange-
ments [2–19]. Characterizing the features of these mem-
brane-associated RNA replication complexes should identify
general principles and mechanisms of (þ)RNA virus repli-
cation and could lead to broadly applicable control strategies
for (þ)RNA viruses including, e.g., hepatitis C virus and the
For many (þ)RNA viruses—including alphaviruses , other
members of the alphavirus-like superfamily , rubiviruses
[7,20], flaviviruses , tombusviruses , and others [4,23–
25] —RNA replication occurs in association with ;50–70-nm
diameter membranous vesicles or spherules that form in the
lumen of specific secretory compartments or organelles. The
similarity of these structures suggests that RNA replication by
such otherwise distinct viruses involves important conserved
features related to membranes. For some viruses, the local-
ization of viral replicase proteins [11,17,23,26–28] or viral
RNA synthesis [5,15,29] suggest that such spherules may
contain or comprise the viral RNA replication complex. For
brome mosaic virus (BMV) and some other viruses, two-
dimensional (2-D) electron microscopy (EM) reveals that a
fraction of such spherules have interiors that appear to be
connected to the cytoplasm by membranous necks [15,25,28].
However, limitations inherent in random sectioning and 2-D
analysis prevent standard EM from resolving many issues
crucial to understanding spherule structure and function,
such as the range of spherule diameter and volume, and
whether all spherule interiors are connected to the cytoplasm
or if some bud free from their adjacent bounding mem-
To resolve these and other issues central to the mechanism
Academic Editor: Skip Virgin, Washington University School of Medicine, United
States of America
Received March 30, 2007; Accepted June 15, 2007; Published August 14, 2007
Copyright: ? 2007 Kopek et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: (?)RNA, negative-strand RNA; (þ)RNA, positive-strand RNA; BMV,
brome mosaic virus; BrUTP, 5-bromouridine 5’-triphosphate; dsRNA, double-
stranded RNA; EM, electron microscopy; EMT, electron microscope tomography;
FHV, flock house virus; hpi, h post infection;
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
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P PL Lo oS S BIOLOGY
of RNA replication, we used EM tomography (EMT) to
provide the first, to our knowledge, three-dimensional (3-D)
ultrastructural study of the membrane-bound RNA replica-
tion complexes of a (þ)RNA virus. EMT generates high-
resolution, 3-D images or tomograms by digitally processing a
series of 50–100 electron micrographs collected as a specimen
is tilted in 1–2 8 increments on an axis perpendicular to the
electron beam . Similar 3-D EMT analyses have been
crucial to reveal many important features of complex cellular
organelles such as the Golgi apparatus [31–34], endoplasmic
reticulum [33,34], and mitochondria [35–37].
We chose flock house virus (FHV), the best characterized
member of the Nodaviridae, as a (þ)RNA virus with advanta-
geous features for such studies. FHV has been used as a model
to study RNA replication [8,9,38–40], virion structure and
assembly [41,42], and genomic packaging [42–46]. FHV has a
4.5-kb bipartite RNA genome in which RNA2 (1.4 kb) encodes
the capsid precursor  whereas RNA1 (3.1 kb) encodes an
RNA silencing inhibitor [48,49] and a multifunctional RNA
replication factor, protein A [40,50,51]. Protein A, the only
FHV protein needed for RNA replication, is directed by an N-
terminal targeting and transmembrane sequence to outer
mitochondrial membranes, where it colocalizes by immuno-
fluorescence with the sites of viral RNA synthesis [8,38].
Gradient flotation and dissociation assays showed that
protein A behaves as an integral transmembrane protein
. Additionally, protease digestion and selective permeabi-
lization after differential epitope tagging demonstrated that
protein A is inserted into the outer mitochondrial membrane
with the N terminus in the inner membrane space or matrix,
while the majority of the protein A sequence is exposed to the
cytoplasm . Protein A also self-interacts in vivo in ways
that are important for RNA replication . Like many other
(þ)RNA viruses [4,5,7,15,20–25], FHV infection induces the
formation of ;50-nm membranous vesicles or spherules,
which, for the case of FHV, are found between the
mitochondrial outer and inner membranes .
Here we use EMT and multiple complementary approaches
to provide 3-D visualization of a (þ)RNA virus replication
complex. Among other findings, the results show that FHV
spherules are compartments or mini-organelles for viral RNA
synthesis, which form by invagination of the outer mitochon-
drial membrane and communicate with the cytoplasm
through ;10-nm diameter necks. The results further indicate
that each spherule contains, on average, ;100 membrane-
spanning, self-interacting protein A molecules and that FHV-
infected cells contain 2–4 genomic RNA replication inter-
mediates per spherule. These observations define a new level
of understanding of the nature, structure, and organization
of a viral RNA replication complex, including principles that
are likely relevant to many other (þ)RNA viruses.
FHV RNA Replication Protein A and RNA Synthesis Are
Located within Membrane-Bound Spherules
Protein A is the only FHV protein needed for RNA
replication and so must co-localize with viral RNA replication
complexes. Prior immunofluorescence and immunogold
labeling EM localized protein A to the outer mitochondrial
membrane in FHV-infected cells . However, in those prior
attempts at immunogold labeling, fixation conditions needed
to preserve spherule ultrastructure abolished protein A
antigenicity for the polyclonal antibody used, hence blocking
protein A localization relative to spherules. To overcome this,
we identified a monoclonal antibody against protein A 
that was able to detect protein A under fixation conditions
that sufficiently retained spherule ultrastructure. Immuno-
gold EM with this protein A monoclonal antibody revealed
that nearly all protein A was in or on mitochondrial spherules
in FHV-infected cells (Figure 1). Over 900 gold particles in 25
different electron micrographs were counted and 88% 6 5%
of the specific gold labeling density above background (see
Materials and Methods) was associated with spherules.
Cytoplasmic labeling, presumably including protein A being
translated and/or trafficked in the cytoplasm, was just 2% 6
7% above background labeling levels. The remaining 10% 6
5% of immunogold label was associated with mitochondria
but not discernable spherules, including gold particles on the
cytoplasmic face of the outer mitochondrial membrane
where some protein A might have been localized that was
not, or not yet, internalized into spherules. The clustering
pattern of immunogold particles in a subset of spherules may
be due to nonuniform epitope exposure or signal amplifica-
tion by secondary antibodies. To avoid over-weighting the
calculations due to such clustering effects, we also analyzed
the same 25 micrographs counting each cluster as one event.
The resulting count of clusters gave a very similar pattern to
the one described above (94% spherule associated).
By immunofluorescence microscopy, we found that 5-
bromouridine (BrU)-labeled FHV RNA synthesis occurs
exclusively at outer mitochondrial membranes in infected
Drosophila cells . To localize more precisely FHV RNA
synthesis in relation to spherules, we incubated mitochondria
isolated from uninfected and FHV-infected Drosophila cells
with a nucleotide mix including 5-bromouridine 5’-triphos-
phate (BrUTP) and performed immunogold labeling EM with
an antibody recognizing BrU incorporated into RNA, but not
For mitochondrial preparations from FHV-infected cells,
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RNA Replication Complex Structure and Organization
Whereas cells store and replicate their genomes as DNA, most
viruses have RNA genomes that replicate by using virus-specific
pathways in the host cell. The largest class of RNA viruses, the
positive-strand RNA viruses, replicate their genomes on intracellular
membranes. However, little is understood about how and why these
viruses use membranes in RNA replication. The well-studied flock
house virus (FHV) replicates its RNA on mitochondrial membranes.
We found that the single FHV RNA replication factor and newly
synthesized FHV RNA localized predominantly in numerous infec-
tion-specific membrane vesicles inside the outer mitochondrial
membrane. We used electron microscope tomography to image
these membranes in three dimensions and found that the interior of
each vesicle was connected to the cytoplasm by a single necked
channel large enough to import ribonucleotide substrates and to
export product RNA. The results suggest that FHV uses these
vesicles as replication compartments, which may also protect
replicating RNA from competing processes and host defenses.
These findings complement results from other viruses to support
possible parallels between genome replication by positive-strand
RNA viruses and two distinct virus classes, double-stranded RNA and
spherules were the major site of immunogold labeling (Figure
2A and 2B). Of 221 gold particles examined, 70% 6 18%
were on spherules. The remaining 30% of gold particles that
fell outside of spherules may include mature RNA products
released from spherules and nonspecific background labeling.
For mitochondria from uninfected cells, background labeling
levels were independent of the addition or omission of
BrUTP and averaged 15% of the total immunogold labeling
of BrUTP-treated mitochondria from FHV-infected cells. We
found that using isolated mitochondria was advantageous for
the BrUTP-labeling experiments because of low transfection
efficiencies of BrUTP into whole Drosophila cells. Never-
theless, we were able to obtain some immunogold labeling
results using intact Drosophila cells, which also showed that
spherules were the major sites of BrUTP-labeling (Figure 2C).
Gold particles in the intermembrane space of the mitochond-
rion in the lower right are well within the distance (20 nm)
from spherules that may be spanned by the primary and
secondary antibodies linking the immunogold particles to
their target epitopes .
All Replication Spherules Retain an Open Connection to
Having shown that spherules were the sites of protein A
accumulation and FHV RNA synthesis, we applied 3-D EMT
to provide a new level of analysis of spherule morphology and
topology. As noted in the Introduction, the 3-D nature of
EMT overcomes many serious limitations of 2-D EM analysis
to reveal possible connections to surrounding membranes
and compartments, complete dimensions, and other funda-
mental characteristics not accessible from conventional
transmission EM analyses of random sections. For example,
along the z-axis parallel to the electron beam, standard
transmission EM projects a 50–70-nm section into a single
view, whereas EMT allows computationally dissecting an
entire ;250-nm-thick sample volume into successively view-
able planes spaced with a resolution of just a few nanometers
To produce 3-D reconstructions of FHV-infected cells
including modified mitochondria, Drosophila S2 cells were
harvested 12 h post infection (hpi) and fixed, embedded, and
sectioned as described under Materials and Methods. For
each reconstruction, a tilt series of 60 images was collected by
rotating a 250-nm-thick section of resin-embedded sample in
2 8 increments between ?60 8 to þ60 8 relative to the plane
perpendicular to the beam, and was digitally processed to
produce a tomographic reconstruction. Using Drosophila cells
from three independent FHV infection experiments, five
independent reconstructions were generated using a single-
tilt series technique (Figure 3C–3D and additional unpub-
lished data) and one reconstruction was performed using a
double tilt technique (Figure 3A and 3B) to improve tomo-
graphic resolution further . Representative results are
shown in the figures.
For one such tomogram, Figure 3A shows the image of a
computationally dissected, 2.2-nm-thick virtual section, re-
vealing an FHV-modified mitochondrion containing spher-
ules in the mitochondrial intermembrane space. This 2-D
image shows a typical view of randomly sectioned, FHV-
Figure 1. FHV Protein A Is Localized in Virus-Induced Mitochondrial Spherules
(A and B) Examples of anti-protein A immunogold labeling of mitochondrial spherules in FHV-infected cells. Cells were fixed in 4% paraformaldehyde
and 0.2% glutaraldehyde, postfixed in 0.1% osmium, dehydrated and embedded in LR Gold resin, sectioned and placed on nickel-grids, immunostained
with protein A antisera and a secondary antibody conjugated to ultrasmall gold particles, and silver-enhanced. The mitochondrial matrix, cytoplasm
(Cyt) and examples of the many spherules (S) in the mitochondrial intermembrane space (IMS) are labeled for reference. White arrowheads indicate gold
particles at a mitochondrial membrane but not directly over a spherule. Black arrowheads indicate gold particles in the cytoplasm. As illustrated in panel
A, islands of cytoplasm surrounded by a mitochondrial ring are seen frequently in EM sections of FHV-infected mitochondria.
(C) A schematic, based on 3-D tomographic analysis shown below, of how such cytoplasmic islands are generated by EM sectioning of the frequently
cup-shaped, FHV-modified mitochondria [see panel (B)]. The bottom image in (C) further shows how some planes of sectioning give rise to ‘‘vesicle
packet’’ structures seen in Figures 2C and 3D below. White, cytoplasm; blue, space between outer and inner mitochondrial membranes; yellow, matrix.
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RNA Replication Complex Structure and Organization
modified mitochondria, in which some spherules appear to be
light bulb–shaped invaginations attached to the outer
membrane by small diameter necks (white arrowheads),
whereas others appear to be free vesicles in the intermem-
brane space (asterisks). Figure 3B shows another virtual
section from the same tomogram, displaced down the
perpendicular z-axis by ;15 nm to a point where those
spherules that appeared to be free vesicles in Figure 3A
(asterisks) now show necked attachments to the outer
membrane. To determine if all spherules were attached to
the outer mitochondrial membrane, or if a population of
spherules budded free of this membrane, we followed
individual spherules through dozens of successive 2.2-nm-
spaced adjacent planes perpendicular to the electron beam (a
‘‘z-series’’ of sections). When all six reconstructions were
examined in this way, all ;500 spherules in all ;8 FHV-
modified mitochondria examined were found to be connected
to the outer mitochondrial membrane by a membranous neck
observable in some plane of the sample. The red arrowhead in
Figure 3A points to a channel through the spherule neck that
connects the interior of a spherule to the cytoplasm.
Thus, all spherules are necked invaginations of the outer
mitochondrial membrane whose interiors remain connected
to the cytoplasm, and sections in which a given spherule
appears to be a free vesicle simply represent planes that did
not pass through the smaller diameter neck linking the
spherule membrane to the mitochondrial outer membrane.
This is illustrated more dynamically in Video S1, which
animates the progression through a z-series of sections of the
tomogram of Figure 3A and 3B.
Figure 3C–3D shows two virtual sections from another
tomogram, which are displaced ;150 nm down the perpen-
dicular z-axis from each other. As shown in a video through
this z-series (Video S2), mitochondrion 1 curves significantly
in the space between these two sections, such that the plane
of Figure 3C sections mitochondrion 1 spherules parallel to
an axis through the spherule necks, whereas the parallel plane
of Figure 3D sections the spherules on another part of the
Figure 2. Association of Viral RNA Synthesis with FHV Spherules
(A and B) Examples of incorporated BrUTP immunogold labeling of mitochondrial spherules from cells infected with FHV. Isolated mitochondria were
incubated for 1 h at 28 8C with a transcription mix that included BrUTP.
(C) Localization of RNA synthesis to spherules in FHV-infected cells into which BrUTP was introduced by transfection. Length of labeling period was 15
min. Samples were prepared for EM and immunogold labeling as in Figure 1, except that anti-BrU antibody was used instead of anti-protein A. See
Figure 1C for an explanation of the nonstandard mitochondrial morphologies seen in the upper left and lower right of (C). The arrowheads and labeling
of mitochondria, spherules, and cytoplasm are as in Figure 1. In interpreting the immunogold localization, note that the primary-secondary antibody
complex linking the gold particles to their target epitopes may span up to 20 nm.
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RNA Replication Complex Structure and Organization
mitochondrion 1 surface tangential to the axes through their
necks. These two perpendicular views of similar spherules on
the same mitochondrion are notable because Figure 3C
strongly resembles images of spherules induced by alphavi-
ruses, nodaviruses, etc. [8,28], whereas Figure 3D resembles
images of apparently distinct ‘‘vesicle packets’’ described for
flaviviruses . Thus, some apparently distinct membrane
rearrangements and vesicle structures observed in connec-
tion with RNA replication by different (þ)RNA viruses may
represent related structures distinguished in part by the
perspective from which they were viewed.
3-D Mapping of Spherule Membranes
To generate 3-D surface maps of the virus-induced
membrane rearrangements associated with FHV RNA repli-
cation, we manually traced the inner and outer mitochondrial
membranes (including spherules) over ;100 adjacent, 2.2-
nm-spaced virtual sections of selected tomographic recon-
structions, and we used a computer-generated mesh overlay
to join these tracings into continuous surfaces (Figure 4).
Figure 4A shows part of the relationship between the electron
density of the mitochondrion in Figure 3A and its 3-D map,
and Video S3 provides a much more dynamic visualization of
this relationship and the complete 3-D map. For clarity, the
cytoplasmic faces of outer mitochondrial membranes are
colored blue, spherule membranes are white, and inner
mitochondrial membranes are yellow.
Figure 4B shows a close-up view of a portion of the 3-D
map in Figure 4A that demonstrates the connection of the
spherules to the outer mitochondrial membrane. This and
other similar maps confirmed as noted above that the
spherule membranes (white) are continuous with the outer
mitochondrial membrane (blue). Figure 4C is a 90 8 rotation
of Figure 4B that shows a view looking down on the surface of
an FHV-modified mitochondrion, with the outer membrane
(blue) rendered translucent to reveal the spherules beneath
(Video S4). The necked channels connecting the interior of
each spherule to the cytoplasm (red arrowhead) are clearly
visible as circular openings in the outer membrane. For 150
individual spherules in four mitochondria from four cells and
three experiments, we measured the interior diameters of
these neck channels as the distance between the two lipid
bilayers, from inner leaflet to inner leaflet, at the point where
the tomographic plane sliced through the center of the neck.
The resulting distribution of neck diameters is shown in
Figure 5A. The average diameter of the neck channel was 10.5
6 1.8 nm (Figure 5A), which is more than large enough to
allow import of ribonucleotides and export of RNA products
(diameter , 2 nm).
Surface-rendered, 3-D maps of the two mitochondria from
Figure 3C are shown in Figure 4D, illustrating also the inner
mitochondrial membrane (yellow). Using such surface-ren-
dered maps (Figure 4 and other unpublished data), we also
measured the interior volume and membrane surface area of
175 spherules. As illustrated in Figure 5C, the spherule
volumes spanned a range of ;15,000 to 50,000 nm3. A range
of spherule sizes is seen in Figure 4E, which is a rotated view
Figure 3. EMT Reconstructions of Mitochondria from FHV-Infected
Labels denote outer mitochondrial membrane (OM) and inner mitochon-
drial membrane (IM). (A and B) Slices through a tomographic
reconstruction showing FHV-induced spherule rearrangements of a
mitochondrion (see Video S1 to view the entire z-series). (B) is a slice that
is ;15 nm down the z-axis (perpendicular to the plane of the image)
from the slice in (A). White arrowheads indicate the necks that connect
spherules to the OM. Asterisks mark two spherules that appear to be free
vesicles in (A) but are shown to have necked connections to the outer
membrane in (B). A red arrow marks the ;10-nm channel connecting a
representative spherule interior to the cytoplasm. This red arrow also
corresponds to the same spherule and connection as the red arrow in
(C and D) Images from another tomogram that are displaced from each
other in the z-axis by ;150 nm. Note the change in morphology of
mitochondrion 1 where spherules that appear to be connected to the
outer mitochondrial membrane in (C) appear as a vesicle packet in (D).
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RNA Replication Complex Structure and Organization
of mitochondrion 2 in Figure 4D, with the outer membrane
removed. The average spherule interior volume was ;33,000
nm3(Figure 5C), and the average interior spherule membrane
surface area was ;6,000 nm2(Figure 5B).
Stoichiometry of FHV RNA Replication Components
Reveals High Protein A Copy Number
Since both protein A (Figure 1) and nascent FHV RNA
(Figure 2) localized predominantly or exclusively to spherules,
the relative numbers of protein A, RNA replication templates,
and spherules could provide important insights into the
structure and organization of FHV RNA replication com-
plexes. Accordingly, we measured the number of molecules of
protein A and FHV RNAs per cell in Drosophila S2 cells at 4, 8,
12, and 24 hpi with FHV. The numbers of positive- and
negative-strand genomic RNAs per cell were measured by
quantitative Northern blotting calibrated with known amounts
of in vitro transcripts (Figure 6A and 6B). The number of
Figure 4. 3-D Maps of FHV-Modified Mitochondria
Blue indicates outer mitochondrial membrane, white indicates FHV spherules, yellow indicates inner mitochondrial membrane.
(A) Merged image of a 3-D map of the outer membrane and spherules of the mitochondrion from Figure 3A and 3B and a slice of the tomogram
showing the electron density map from which it was derived.
(B) A portion of the map in (A) showing a close-up view of the connections between the outer mitochondrial membrane and the spherules. The red
arrow marks that same spherule as the one depicted by the red arrow in Figure 3A.
(C) A 90 8 rotation of (B) showing the channels that connect the spherule interiors to the cytoplasm. The outer membrane has been made translucent to
show the spherules behind it. Again, the red arrow corresponds to the red arrows in Figures 3A and 4B.
(D) 3-D maps of the mitochondria shown in Figure 3C.
(E) A view of mitochondrion 2 from (D) that has been rotated and has had the outer membrane removed to show the range of spherule sizes.
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RNA Replication Complex Structure and Organization
protein A molecules per cell was measured by quantitative
Western blotting calibrated with known amounts of co-
electrophoresed, purified protein A standards (Figure 6E).
Starting before 4 hpi and continuing thereafter in FHV-
infected Drosophila cells, the primary mode of viral RNA
synthesis is (þ)RNA synthesis from negative-strand RNA
[(?)RNA] templates (Figure 6A–6D). The number of
(þ)RNA1 and (þ)RNA2 per cell increased from ;40,000
molecules of each RNA species at 4 hpi to ;2–3 million each
by 24 hpi (Figure 6C). Such (þ)RNA products primarily
accumulate in the cytoplasm for translation and encapsida-
tion, and only a minor fraction of (þ)RNAs fractionate with
the membrane-associated RNA replication complex (P. Van
Wynsberghe, P. Ahlquist, unpublished data).
By contrast to positive-strand export and accumulation in
the cytoplasm, FHV (?)RNAs appear to function only as RNA
replication intermediates and are completely membrane-
associated (P. Van Wynsberghe, P. Ahlquist, unpublished
data). (?)RNA thus is a key measure of a minimal RNA
replication complex, because every mature RNA replication
complex, active in (þ)RNA synthesis, must contain at least one
(?)RNA template. Therefore, the number of (?)RNAs gives an
estimate of the maximal number of replication complexes per
cell. (?)RNA1 accumulation plateaued by 8 hpi at ;16,000
copies per cell (Figure 6D). (?)RNA2 accumulation increased
throughout the first 24 hpi, although more slowly after 12 hpi,
reaching ;50,000 molecules per cell by 24 hpi (Figure 6D).
The number of protein A molecules plateaued by 8 hpi
(Figure 6F), which is consistent with prior results that protein
A synthesis occurs early in infection and then declines .
Intriguingly, the peak level of protein A was ;2 million
molecules per cell (Figure 6F). Protein A was thus present at
dramatically higher levels than (?) RNA templates were. The
ratio of protein A to (?)RNAs was relatively consistent over all
time points examined, with averages throughout infection of
118 6 23 and 64 6 20 protein A copies per (?)RNA1 and
(?)RNA2, respectively (Figure 6G).
To understand the organization of the replication complex
in relation to the spherules better, we compared the number
of spherules per cell with the number of protein A and (?)RNA
molecules per cell. To measure the number of spherules per
cell, we collected FHV-infected Drosophila S2 cells at 12 hpi,
processed them for transmission EM, and imaged 25 randomly
sectioned cell profiles. All spherules in each imaged cell
section were counted and divided by the cell section volume,
which was calculated by measuring the cell area using ImageJ
(National Institutes of Health) and multiplying by the effective
section thickness (see Materials and Methods). The number of
spherules per cell was calculated by multiplying the resulting
density of spherules by the average volume of the almost
perfectly round, 10 lm-diameter Drosophila S2 cells  (and
our independent, matching measurements).
These calculations revealed the average number of spher-
ules per cell at 12 hpi to be ;20,000 6 11,000 (Table 1). The
ratio of protein A per cell to spherules per cell revealed that
on average, there are ;100 copies of protein A per spherule
(Table 1). Further comparison to the Figure 6 data shows that,
on average there are ;1 (?)RNA1 and ;2 (?)RNA2 molecules
per spherule (Table 1). The implications of these results for
the organization of replication complexes are considered
further in the Discussion.
To advance understanding of the crucial relationship
between (þ)RNA viruses and the intracellular membranes
Figure 5. Spherule Dimensions
Distribution of spherule neck channel diameters (A), interior surface areas
(B), and interior volumes (C). The data shown represents measurements
of 150 (A) and 175 (B and C) individual spherules.
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RNA Replication Complex Structure and Organization
Figure 6. Measurements of the number of molecules of FHV (þ)RNAs and (?)RNAs and protein A per cell in FHV-infected Drosophila cells
(A and B) Cells were harvested and counted at the time points indicated above the figure, and total RNA was extracted. An amount of RNA
corresponding to 1.0 3 105, 1.0 3 104, or 1.0 3 103cell equivalents was loaded as indicated above each lane and subjected to Northern blot
hybridization with radio-labeled probes specific for the detection of (þ)RNA1 (top panel of A), (?)RNA1 (top panel of B), (þ)RNA2 (bottom panel of A), or
(?)RNA2 (bottom panel of B). Specific signals are indicated by the arrowheads.
(C) Graph of the number of molecules of (þ)RNA1 (diamond) and (þ)RNA2 (squares) per cell over a 24-h time course.
(D) Graph of the number of molecules of (?)RNA1 and (?)RNA2 per cell over a 24-h time course.
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RNA Replication Complex Structure and Organization
on which they replicate their RNA genomes, we combined 3-
D ultrastructural imaging with quantitative biochemical data
and other results to model the architecture and organization
of a nodavirus RNA replication complex. Immunogold
labeling identified virus-induced membranous spherules as
the sites of accumulation of the sole FHV RNA replication
protein, protein A, and of FHV RNA synthesis (Figures 1 and
2). EMT revealed that all FHV spherules maintain an open
connection with the cytoplasm with a diameter of ;10 nm,
which is wide enough to allow the exchange of ribonucleo-
tides and RNA products (Figures 4C and 5A). Our stoichi-
ometry measurements further revealed the presence of, on
average, 100 copies of the viral replicase protein A and 2–4
RNA replication intermediates per spherule (Table 1). As
discussed further below, these findings have substantial
implications for the structure, assembly, and function of the
FHV RNA replication complex and likely also for the
organization of many similar membrane-associated viral
RNA replication complexes. In addition to advancing under-
standing of viral replication mechanisms, such insights also
should prove valuable for developing additional antiviral
strategies or agents.
Organization of the FHV RNA Replication Complex
Protein A is a transmembrane protein in outer mitochon-
drial membranes  and is ;90% localized within spherules
(Figure 1). Therefore, protein A must line the interior
membrane surface of spherules. If protein A is similar to
typical globular proteins, its volume would be ;183 nm3,
based on the protein A molecular weight of 112 kDa  and
the average partial specific volume of typical proteins . If
globular, protein A then would have a diameter of ;7 nm and
cover a surface area of ;40 nm2. Thus, the average spherule
interior membrane surface area of 6,000 nm2(Figure 5B)
provides enough space to accommodate at most ;150
protein A molecules, under a perfect close-packing arrange-
ment. Therefore, the measured value of ;100 protein A
molecules per spherule (Table 1) is near saturation for the
spherule interior membrane surface area. We modeled 50 7-
nm-diameter spheres representing protein A adjacent to the
membrane surface within a tomographic model of half a
typical spherule (Figure 7) to demonstrate how protein A may
pack into the spherules.
The resulting near-full occupancy of the interior mem-
brane surface area by protein A (Figure 7) and the nature of
protein A as a transmembrane protein whose self-interaction
is required for RNA replication [38,52] imply that the ;100
copies of protein A form an inner network or shell within the
spherule (Figure 7B). Such a shell would explain the
formation and maintenance of the high-energy membrane
deformation of spherules. A shell of these dimensions appears
reasonable, given that the main shell of a reovirus core is 60
nm in diameter and is composed of 120 copies of a slightly
larger protein k1 (142 kDa) . The distribution of FHV
spherule size spans a defined range of ;30–45-nm intra-
membrane diameter, suggesting some flexibility in the
assembly of the protein A shell. Other examples of high-
density protein shells of flexible size and shape include the
capsids of retroviruses, influenza, and retrotransposons [60–
62]. For example, one species of Ty retrotransposon forms
virus-like capsids that have a 30–50-nm range of diameters,
similar to FHV spherules, and contain on average 300 copies
of a 381–amino acid protein subunit , a protein content
very close to the FHV spherule average of 100 copies of 998–
amino acid protein A. Endocytic vesicles, secretory transport
vesicles, and synaptic vesicles are further examples of
protein-induced membrane vesicles that each have a range
of variable sizes (50–100-nm diameter), despite being formed
by regular arrays of uniform proteins [63,64].
Because ;90% of protein A, the FHV RNA polymerase
(Figure 1), and ;70% of newly synthesized FHV RNA (Figure
2) are spherule-associated, and essentially all FHV (?)RNA
templates are membrane associated (P. Van Wynsberghe, P.
Ahlquist, unpublished data), it appears likely that (?)RNAs,
and thus any double-stranded RNAs (dsRNAs) are within
spherules. Sequestration of dsRNA within such a compart-
ment may allow the virus to avoid, minimize, or delay dsRNA-
induced host–cell defense responses such as protein kinase,
RNA activated (PKR) and RNase L  or RNA interference
(RNAi) . Such dsRNA localization is consistent with earlier
observations of virus-induced membrane spherules contain-
ing fibrils with salt-dependent nuclease sensitivity [25,67].
The ;100 protein A molecules per spherule (Table 1)
would consume ;18,300 nm3of interior volume, leaving
;14,000 nm3within an average spherule to accommodate
FHV RNA. Based on 0.655 nm3per hydrated nucleotide for
the crystal structure of duplex RNA [43,68,69], the volumes of
FHV RNA1, RNA2, and RNA3 would be 2035, 917, and 254
nm3, respectively. Thus, in addition to ;100 protein A
molecules, a spherule of average size has enough interior
space to contain at most four single-stranded RNA (ssRNA) or
two dsRNA copies of all three FHV RNA species. Given this
maximal occupancy, the estimate from biochemical data of
an average of one (?)RNA1 and two (?)RNA2 templates per
Table 1. Ratio of Spherules per Cell to Protein A and (?)RNA at
spherules per cell
20,000 6 11,000
101 6 51
0.9 6 0.4
2.2 6 1.0
(E) An aliquot of the same cells harvested for the RNA analysis at the indicated time points was lysed in Laemmli sample buffer and subjected to
immunoblot analysis with polyclonal antisera for protein A. Protein A levels were measured by comparison with the signal intensities derived from
known amounts of purified protein A.
(F) Graph of the number of molecules of protein A per cell over a 24-h time course.
(G) Graph of the ratios of protein A versus (?)RNA1 (diamonds) and (?)RNA2 during the course of infection. The 24 hpi data shown represent the mean
and standard deviation for three independent experiments. The data shown for 4, 8, and 12 hpi represent the mean and standard deviation for two
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RNA Replication Complex Structure and Organization
spherule (Table 1), together with at least one nascent (þ)RNA
progeny strand for each, appears fully reasonable.
Currently, it is not known if FHV RNA1 and RNA2 are
replicated in separate or common spherules. If RNA1 and
RNA2 were in separate spherules (i.e., 50% of spherules
containing RNA1 and 50% containing RNA2), then the ratios
of (?)RNA1 and (?)RNA2 to total spherules (Table 1) imply
each RNA1-containing spherule would have two replication
intermediates, and each RNA2-containing spherule would
have approximately four replication intermediates. Because
RNA1 (3.1 kb) is twice as long as RNA2 (1.4 kb), the total RNA
content in both cases then would be nearly equal. If RNA1
and RNA2 were together in the same spherule, then each
spherule would hold on average three replication intermedi-
ates (one RNA1 and two RNA2). The possibility of spherules
containing both species of RNAs is intriguing, considering
the interactions of FHV RNAs required for replication: FHV
subgenomic RNA3, which is templated from RNA1, trans-
activates RNA2 replication and, in turn, RNA3 replication is
suppressed by the resulting progeny RNA2 . RNA3, and
not its protein product, is responsible for transactivating
RNA2 . However, it is also possible that RNA3 is produced
in one spherule during RNA1 replication and then exported
to the cytoplasm prior to transactivating RNA2.
Parallels with Other Viral RNA Replication Complexes
Membrane spherules similar to those of FHV are induced
by many other (þ)RNA viruses including alphaviruses ,
other members of the alphavirus-like superfamily ,
rubiviruses [7,20], flaviviruses , tombusviruses , and
others [4,23–25]. Among these, one of the best-studied with
regard to the localization and stoichiometry of RNA
replication complex components is BMV. BMV and FHV
differ in many important respects including that BMV
encodes a much larger complement of RNA replication
proteins [15,19]. Nevertheless, although the understanding
that we present here for FHV RNA replication complexes is
more advanced in many ways, the known characteristics of
BMV RNA replication complexes are strikingly similar to
those for FHV. Both BMV and FHV induce spherules of
similar dimensions where viral RNA synthesis and viral
replication proteins are localized . BMV replication
protein 1a, which is sufficient to induce spherules , is
also a strongly membrane-associated , self-interacting
[72,73] protein that is present at high copy number per
spherule . Similarly, whereas the ultrastructural organ-
ization of hepatitis C virus RNA replication complexes has
not been defined, recent results suggest that these may also
involve a dramatic excess of nonstructural protein copies per
In addition to the many (þ)RNA viruses whose RNA
replication is associated with spherules, other (þ)RNA viruses
induce various, apparently distinct membrane rearrange-
ments [4,17,21,26,27,75,76]. Although some or most of this
variability reflects real ultrastructural differences, at least
some of the perceived differences may be due to differences
in perspective under conventional 2-D imaging. Our tomog-
raphy results demonstrated that equivalent FHV spherules
appeared to vary in morphology and topological relation to
adjacent membranes when viewed in two dimensions from
different perspectives (Figures 3C and 3D). A greater under-
standing of the 3-D nature of membrane rearrangements
associated with RNA replication by other (þ)RNA viruses may
reveal shared features or common underlying principles.
Based on results with BMV, Schwartz et al. identified
potential parallels between the assembly, structure, and
function of membrane-associated RNA replication complexes
and the cores of reverse-transcribing and dsRNA virus
virions, including the sequestration of genomic RNA tem-
plates within a virus-induced compartment for replication
[15,19]. The results presented here for FHV validate and
extend these parallels by showing that all FHV spherules are
Figure 7. Modeling of FHV Transmembrane Protein A into a Spherule
(A) 3-D map of a single spherule where half of the membrane has been removed to show the interior. As in Figure 4, the spherule membrane is white
and the contiguous outer mitochondrial membrane is blue.
(B) Schematic of likely protein A organization within a spherule. Based on the average density of globular proteins, FHV protein A (112 kDa) is modeled
in as a green sphere of ;7 nm in diameter. Based on the average of ;100 protein A molecules per spherule (Table 1), the figure shows the potential
packing arrangement of 50 protein A molecules in half a spherule. The spheres are shown lining the interior surface area of the spherule membrane
because protein A is a transmembrane protein .
(C) Schematic representation of the structure and organization of the FHV RNA replication complex. Protein A (ptn A; green spheres) forms a shell within
the mitochondrial membrane spherule within which RNA synthesis occurs (N ¼ N terminus; C ¼ C terminus). Protein A is also shown as possibly
extending into the spherule neck, since it may be a determinant of the relatively constant 10-nm diameter neck. As noted in Figure 1 (white
arrowheads), a small fraction of protein A may reside on the outer mitochondrial membrane external to spherules. The diagram shows (þ)RNA synthesis
(red arrow) from (?)RNA templates (black segmented line), which is the predominant form of FHV RNA synthesis throughout all but the earliest phases
of FHV infection (Figure 6A–6D).
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RNA Replication Complex Structure and Organization
membrane invaginations topologically equivalent to a bud-
ding, enveloped virion (Figure 4), and that self-interacting,
transmembrane protein A is present at levels sufficient to
coat the inner spherule membrane in a multi-subunit shell
similar to the capsids of retrovirus and dsRNA virus cores
(Figure 7). As with dsRNA viruses, hepadnaviruses, and
retroviruses, the high copy of protein A per spherule suggests
that there may be threshold effects in replication protein
expression to initiate replication. Further analysis of the
structure, interactions, and function of FHV RNA replication
complexes should provide additional insights into the basic
mechanisms of (þ)RNA virus replication and potentially
identify new approaches for antiviral interference.
Materials and Methods
Cells and infection protocol. Drosophila S2 cells were grown at 28 8C
in Gibco Drosophila serum-free media (SFM). Cells were dislodged by
gentle scraping, pelleted, and resuspended at 107cells/ml. FHV was
added at a multiplicity of infection of 10 for all experiments. The cells
and virus were incubated at 26 8C on a rotary shaker at 1,000
revolutions per minute (rpm) for 1 h to let the virus attach. After the
hour incubation, the cells were plated onto a tissue culture dish and
further incubated at 28 8C.
Mitochondria isolation. Mitochondria were isolated from Drosophi-
la cells as described by Echalier . Briefly, cells were recovered by
scraping and centrifugation and resuspended in a hypotonic buffer
that contained 20 mM N-2-hydroxyethylpiperazine-N’-2-ethanesul-
fonic acid (HEPES; pH 7.4), 1 mM EGTA, and a protease inhibitor
cocktail (1 mM phenylmethanesulphonylfluoride, 5 lg/ml pepstatin A,
1 lg/ml chymostatin, 10 mM benzamidine, 10 lg/ml leupeptin, and 0.5
lg/ml bestatin). After a 10 min incubation at room temperature, an
equal volume of double isotonic buffer was added that consisted of
the hypotonic buffer plus 0.5 M mannitol. The cells were lysed for 7
min using a pre-chilled Potter-Elvehjem homogenizer fitted with a
Teflon pestle (Kimble-Kontes; www.kimble-kontes.com/) and attached
to a stirrer motor spinning at 250 rpm. The lysate was transferred to a
Dounce homogenizer fitted with a type B glass pestle (Kimble-Kontes)
and disrupted manually for 100 strokes on ice. Unbroken cells and
nuclei were removed by two 10 min centrifugation steps at 500g at 4
8C. Mitochondria were pelleted by centrifugation at 3700g for 10 min
at 4 8C, resuspended in an isotonic buffer containing 0.25 M
mannitol, and washed by a second centrifugation at 7000g. BrUTP
incorporation on the isolated mitochondria was performed at 28 8C
for 1 h as described previously .
BrUTP transfection. Drosophila cells were infected with FHV as
above. At 8 hpi, cells were treated with 20 lg/ml actinomycin D for 30
min. FuGENE 6 (Roche; http://www.roche.com) was diluted 10-fold in
phosphate buffered saline pH 7.4 and mixed with BrUTP and
actinomycin D to final concentrations of 10 mM and 20 lg/ml,
respectively. The FuGENE/BrUTP/actinomycin D mix was incubated
for 15 min at room temperature then added to the cells and
incubated at 4 8C for 15 min. After the 4 8C incubation, the cells were
moved to 28 8C for a 15-min labeling period and then immediately
fixed and processed for EM.
Monoclonal antibody. Mouse monoclonal antibodies against FHV
protein A have been described previously . MAb clone 2–126.96.36.199.8,
which recognizes the protein A epitope between amino acids 230 and
399, was used for immunogold EM labeling.
Immunogold EM labeling . BrUTP immunolabeling fixation was
performed as described previously , except that samples were
embedded in LR Gold resin. Samples were sectioned and placed on
nickel grids. Sections were blocked with a goat-blocking solution
(Aurion; http://www.aurion.nl), and incubated for 1 h with an anti-
BrU antibody (PRB-1; Molecular Probes; http://probes.invitrogen.
com), diluted 1:100 in an incubation solution containing 100 mM
phosphate-buffered saline pH 7.4 and 0.1% BSA-c (Aurion). Grids
were washed six times in incubation solution without antibody, then
incubated for 2 h with a goat-anti-mouse antibody conjugated to an
ultrasmall gold particle (Aurion) that was diluted 1:100 in incubation
solution, and washed six times again with incubation solution. Silver
enhancement was performed for 30 min using R-GENT SE-EM
(Aurion). Protein A immunogold EM was performed in the same
manner using the mouse monoclonal antibody at a dilution of 1:100.
Background labeling was determined using uninfected control cells.
Labeling density was determined by calculating the surface area of
spherules, mitochondria, and cytoplasm using the point-hit method
. Specific labeling was determined by subtracting the background
Northern blot analysis and quantitation of FHV RNA. Northern
blotting was done as described previously . The number of
molecules of FHV RNAs was determined by comparison with a serial
dilution of a known amount of in vitro transcripts representing a
known amount of (þ)RNA or (?)RNA molecules. RNA levels were
quantitated with ImageQuant software (Molecular Dynamics; http://
Western blot analysis and quantitation of FHV protein A. Western
blotting was done as described previously . The number of
molecules of protein A was determined by comparison with a
purified protein A standard. To generate the standard for quantita-
tion, protein A was expressed in Escherichia coli as described previously
. To purify protein A, the hydrophobic transmembrane domain of
protein A was deleted (amino acids 8–89), replaced with a C-terminal
His6 tag, and purified by talon column (Clontech; http://www.
clontech.com) affinity chromatography. To further purify protein
A, we performed preparative electrophoresis using a BioRad mini-
prep cell. A 6%, 9.5-cm gel was run at 200 V for 9 h with an elution
speed of 150 ll/min. Fractions containing the purified, truncated
protein A standard were collected and quantitated based on
comparison with known standards of bovine serum albumin and b-
galactosidase. We quantitated protein levels with Lumi-imager
EM. For conventional transmission EM, cells were fixed and
embedded as previously described . For electron tomography, cells
were fixed in 2% parafomaldehyde and 2.5% glutaraldehyde in 0.1 M
sodium cacodylate, pH 7.4, post-fixed in 1% osmium tetroxide with
0.8% potassium ferrocyanide in sodium cacodylate buffer, stained
with 2% uranyl acetate, dehydrated in a graded series of ethanol, and
embedded in Durcupan ACM resin.
To calculate the total number of FHV-induced mitochondrial
spherules per cell, FHV-infected Drosophila S2 cells were collected at
12 hpi, processed for transmission EM, and sectioned into 70-nm-
thick slices. For each of 25 randomly selected cells imaged in these
sections, we then counted all observable spherules with diameters
larger than 20 nm. The number of spherules counted for each cell
then was divided by the relevant section volume, which was calculated
by measuring the cell area using ImageJ (National Institutes of
Health) and multiplying by the effective section thickness. The
effective section thickness is a correction used to avoid overcounting
spherules with centers outside of the 70-nm physical section, which
would otherwise be counted twice if adjacent sections were analyzed.
As previously used to calculate synaptic vesicles per cell , this
effective section thickness is the thickness that would encompass the
centers of all counted spherules. In this case, the effective section
thickness was 116 nm, based on adding 23 nm (the distance from the
spherule center to a radius-perpendicular plane bisecting the
spherule to yield a 20-nm-diameter section) to each face of the 70-
nm section. The number of spherules per cell was calculated by
multiplying the resulting density of spherules by the average volume
of the almost perfectly round, 10 lm-diameter Drosophila S2 cells 
(and our independent, matching measurements).
EMT. Three separate FHV infections produced samples for six
independent tomograms. To survey the preservation quality and
FHV-infection efficiency of the Drosophila cells, thin-sectioned
material (;80 nm thick) was examined using a JEOL 1200FX electron
microscope. 3-D reconstructions of portions of the cell containing
FHV-infected mitochondria were generated using current techniques
of electron tomography . Sections were cut with a thickness of
;250 nm from blocks exhibiting well-preserved ultrastructure. These
sections were stained for 30 min in 2% aqueous uranyl acetate,
followed by 30 min in lead salts. Fiducial cues consisting of 20-nm
colloidal gold particles were deposited on both sides of the section.
For each reconstruction, a series of images was collected with a JEOL
4000EX intermediate-voltage electron microscope operated at 400
kV. The specimens were irradiated before initiating a tilt series in
order to limit anisotropic specimen thinning during image collection.
Pre-irradiation in this manner subjected the specimen to the steepest
portion of the nonlinear shrinkage profile before images were
collected. Six tilt series were collected: five single-tilt and one double-
tilt. ‘‘FHV2’’ was the highest resolution single-tilt reconstruction and
‘‘FHV6’’ was the high-resolution double-tilt reconstruction; the
majority of the analyses were conducted on these two reconstruc-
tions. The single-tilt series were recorded at 40,000 magnification
with an angular increment of 2 8 from ?60 8 to þ60 8 about an axis
perpendicular to the optical axis of the microscope using a
PLoS Biology | www.plosbiology.orgSeptember 2007 | Volume 5 | Issue 9 | e2202032
RNA Replication Complex Structure and Organization
computer-controlled goniometer to increment accurately the angular
steps. These single-axis tilt series were collected using a CCD camera
with pixel dimensions 1,960 3 2,560. The pixel resolution was 0.55
nm. The illumination was held to near parallel beam conditions and
optical density maintained constant by varying the exposure time.
The IMOD package was used for generating the reconstructions .
Double-tilt tomography was performed by first collecting two tilt
series of the same cellular region around orthogonal axes. After the
first tilt series was complete using an angular increment of 2 8 from
?66 8 toþ66 8, the specimen grid was rotated 90 8, and the second tilt
series was acquired from?60 8 toþ66 8. The IMOD software suite was
used for fiducial mark tracking and alignment. The positions of 30
gold particles were tracked in both tilt series. After alignment, the
tomographic reconstruction was generated by a projective algorithm
Volume segmentation was performed by manual tracing in the
planes of highest resolution with the program Xvoxtrace . The
mitochondrial reconstructions were visualized using Analyze (Mayo
Foundation, Rochester, MN, United States), ImageJ (National
Institutes of Health), or the surface-rendering graphics of Synu
(National Center for Microscopy and Imaging Research, San Diego,
CA, United States) as described by Perkins et al. 2001 . These
programs allow one to step through slices of the reconstruction in
any orientation and to track or model features of interest in three
dimensions. Measurements of structural features were made from
planes within the reconstructed volume with the program ImageJ
(National Institutes of Health) or within segmented volumes by the
programs Synuarea and Synuvolume (National Center for Micro-
scopy and Imaging Research). Some 3-D maps, images, and videos
were created using the software Amira (Mercury TGS; http://www.tgs.
Video S1. Animation through a z-Series of Slices of a Double-Tilt
Tomogram Showing FHV-Induced Spherule Rearrangements of a
Found at doi:10.1371/journal.pbio.0050220.sv001 (8.1 MB MOV).
Video S2. Z-Series Animation Illustrating the Varied Appearance of
Spherule Clusters when Sectioned Parallel and Perpendicular to the
Axes through Spherule Necks
Note how the morphology of the mitochondrion near the center
changes during the progression through the z-series.
Found at doi:10.1371/journal.pbio.0050220.sv002 (1.3 MB MOV).
Video S3. Relationship of a 3-D Map of FHV-Induced Spherule
Rearrangements of a Mitochondrion and the Electron Density from
which the Map was Derived
Blue indicates outer mitochondrial membrane, white indicates FHV
spherules, yellow indicates inner mitochondrial membrane. The red
arrow points along the y-axis and the green arrow points along the x-
axis of the tomogram. The boundary of the total tomographic volume
is outlined by the orange bounding box.
Found at doi:10.1371/journal.pbio.0050220.sv003 (5.8 MB MOV).
Video S4. A 90 8 Rotation of Figure 4B to Figure 4C
Found at doi:10.1371/journal.pbio.0050220.sv004 (1.0 MB MOV).
We thank Randall Massey and Benjamin August of the University of
Wisconsin Medical School Electron Microscopy Facility for assistance
with electron microscopy; Priscilla Van Wynsberghe and Billy Dye for
helpful discussions; Dan Lautenschlager, Steve Lamont, and Jean
Yves-Sgro for computer assistance; and Johan den Boon for critical
reading of the manuscript.
Author contributions. All authors conceived and designed the
experiments and analyzed the data. BGK, GP, and DJM performed the
experiments. BGK and PA wrote the paper.
Funding. This work was supported by NIH grants GM35072 to PA
and P41 RR04050 to MHE. PA is an Investigator of the Howard
Hughes Medical Institute.
Competing interests. The authors have declared that no competing
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PLoS Biology | www.plosbiology.org September 2007 | Volume 5 | Issue 9 | e2202034
RNA Replication Complex Structure and Organization