Membrane-shaping host reticulon proteins play
crucial roles in viral RNA replication compartment
formation and function
Arturo Diaza, Xiaofeng Wanga,1, and Paul Ahlquista,b,2
aInstitute for Molecular Virology andbHoward Hughes Medical Institute, University of Wisconsin, Madison, WI 53706
Contributed by Paul Ahlquist, July 29, 2010 (sent for review May 14, 2010)
Positive-strand RNA viruses replicate their genomes on membranes
with virus-induced rearrangements such as single- or double-
membrane vesicles, but the mechanisms of such rearrangements,
including the role of host proteins, are poorly understood. Brome
mosaic virus (BMV) RNA synthesis occurs in ≈70 nm, negatively
curved endoplasmic reticulum (ER) membrane invaginations in-
duced by multifunctional BMV protein 1a. We show that BMV
RNA replication is inhibited 80–90% by deleting the reticulon ho-
mology proteins (RHPs), a family of membrane-shaping proteins
that normally induce and stabilize positively curved peripheral ER
membrane tubules. In RHP-depleted cells, 1a localized normally to
However, 1a failed to induce ER replication compartments or to
recruit viral RNA templates. Partial RHP depletion allowed forma-
tion of functional replication vesicles but reduced their diameter by
30–50%. RHPs coimmunoprecipitated with 1a and 1a expression
redirected >50% of RHPs from peripheral ER tubules to the interior
of BMV-induced RNA replication compartments on perinuclear ER.
Moreover, RHP-GFP fusions retained 1a interaction but shifted 1a-
induced membrane rearrangements from normal vesicles to double
membrane layers,a phenotypealso inducedbyexcess1a-interacting
2apol. Thus, RHPs interact with 1a, are incorporated into RNA replica-
lication compartment formation and function. The results suggest
possible RHP roles in the bodies and necks of replication vesicles.
positive-strand RNA virus|reticulons|genomic RNA replication complex|
endoplasmic reticulum vesicles
tion with vesiculation or other membrane rearrangements (1–3).
As a highly conserved feature of (+)RNA virus life cycles, such
membrane involvement is a potentially valuable target for broad-
spectrum antiviral treatments.
Brome mosaic virus (BMV) is a member of the alphavirus-like
superfamily of human, animal, and plant viruses. BMV encodes
two RNA replication proteins. BMV 1a contains an N-proximal
domain with m7G methyltransferase and covalent m7GMP-
binding activities required for viral RNA capping (4–6), and a C-
terminal NTPase/RNA helicase-like domain (7). BMV 2apolhas
a central RNA-dependent RNA polymerase-like domain and an
N-terminal domain that interacts with the 1a helicase domain (8,
9). In both the yeast Saccharomyces cerevisiae and its natural plant
hosts, BMV RNA is selectively encapsidated into progeny virions
(10), and BMV RNA replication depends on 1a, 2apoland specific
cis-acting RNA signals (11), localizes to ER membranes (12, 13),
generates a dramatic excess of positive- to negative-strand RNA
(14), and efficiently directs subgenomic mRNA synthesis (14).
BMV 1a localizes to perinuclear ER membranes and induces
60- to 75-nm vesicular invaginations or spherules. BMV 1a’s high
multiplicity in spherules (15), strong membrane association (16),
and self-interaction (17) imply that 1a may induce spherule in-
vagination away from the cytoplasm (negative membrane cur-
vature) by forming a capsid-like shell on the spherule interior
universal feature of (+)RNA viruses is that they replicate
their RNA on intracellular membranes, usually in associa-
(15). In the presence of low levels of 2apoland a viral RNA
template, spherules serve as compartments or mini-organelles
for RNA replication (15). Many other (+)RNA viruses, in-
cluding flaviviruses (18), nodaviruses (3) and alphaviruses (19),
also replicate in association with ≈50- to 80-nm-diameter
spherules. However, for BMV, increasing 2apollevels shift the
spherular membrane rearrangements to large multilayer stacks
of appressed double-membrane layers that, like spherules, are
the sites of 1a and 2apolaccumulation, protect RNA templates
from nucleases, and support RNA replication (20).
The mechanisms by which (+)RNA viruses induce and
maintain such membrane rearranged RNA replication com-
partments and the role of host factors in this process are not
resolved. Here, we reveal and dissect the critical roles of the
reticulon homology domain proteins (RHPs) in BMV RNA
replication compartment formation and function. The RHPs are
a family of membrane-shaping proteins conserved from yeast to
humans and plants (21, 22). RHPs share similar ≈200-aa
domains with two long hydrophobic segments that insert in the
cytoplasmic face of membranes to induce positive membrane
curvature (protrusion toward the cytoplasm). RHPs primarily
partition to and stabilize positively curved peripheral ER mem-
brane tubules while avoiding the low-curvature ER domains of
the nuclear envelope and peripheral ER sheets (23–28). Ge-
nome-wide deletion analysis in yeast, which encodes three RHPs
(Rtn1p, Rtn2p, and Yop1p), previously revealed only a 2-fold
reduction in BMV replication on deleting Rtn1p and just a ≈10–
20% reduction on deleting Rtn2p or Yop1p, respectively (29).
However, we show that 1a interacts with RHPs and relocalizes
them from peripheral ER tubules to the interior of the spherular
BMV RNA replication compartments. Moreover, consistent
with the RHPs’ redundant roles in the cell (25), simultaneously
deleting Rtn1p and Yop1p or all three RHPs severely inhibited
BMV RNA replication, abolished 1a-induced spherule forma-
tion and altered the number and morphology of 1a-induced
double-membrane layers, whereas GFP-tagged versions of the
RHPs prevented spherule formation. These and other results
show that the RHPs play important roles in the formation and
function of BMV-induced replication compartments.
Deleting RHP Genes Inhibits BMV RNA Replication. BMV 1a by itself
or with low levels of 2apol, expressed from the ADH1 promoter,
induces spherular ER invaginations, whereas 1a plus high 2apol
Author contributions: A.D., X.W.,andP.A. designedresearch; A.D. performedresearch; A.D.
contributed new reagents/analytic tools; A.D., X.W., and P.A. analyzed data; and A.D. and
P.A. wrote the paper.
The authors declare no conflict of interest.
1Present address: Texas AgriLife Research and Department of Plant Pathology and Micro-
biology, Texas A&M University System, Weslaco, TX 78596.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 14, 2010
| vol. 107
| no. 37
levels, driven by the GAL1 promoter, induce stacked layers of
double-membrane ER (15, 20). To assess the role of RHPs in
subgenomic RNA4 accumulation in wt, single- (rtn1Δ, rtn2Δ, or
yop1Δ), and double-knockout (rtn2Δyop1Δ or rtn1Δyop1Δ) yeast
expressing 1a, RNA3, and ADH1 2apolor GAL1 2apol. Wt yeast
cells supported high levels of positive-strand RNA3 and sub-
genomic RNA4 in both spherules and layers (Fig. 1, lane 1). rtn2Δ,
yop1Δ, and rtn2Δyop1Δ yeast also supported RNA4 synthesis to wt
levels (Fig. 1, lanes 3–5). rtn1Δ yeast supported RNA4 synthesis to
1, lane 2). Moreover, in rtn1Δyop1Δ yeast, RNA4 synthesis was
inhibited by 92% and 67% in spherules and layers, respectively
(Fig. 1, lane 6). A similar trend was observed for negative-strand
the wt rate,general transcription and translation were not affected
RHP gene deletions on BMV mirror their relative expression
levels of Rtn1p > Yop1p > Rtn2p (30).
The above yeast deletion mutants were made in S. cerevisiae
(12, 15, 20, 31). Despite extensive efforts, we could not generate
YPH500 rtn1Δrtn2Δ or rtn1Δrtn2Δyop1Δ strains, suggesting that
these deletions inhibit cell growth in the YPH500 background.
However, for widely used strain BY4741, which also has been
used to study many aspects of BMV replication (29, 32), an
rtn1Δrtn2Δyop1Δ triple knockout strain has been generated (25).
This mutant has no growth defect, and ER morphology changes
caused by RHP deletion do not block vesicular trafficking out of
the ER (25). In rtn1Δrtn2Δyop1Δ yeast, positive-strand RNA4 ac-
cumulation decreased by 80% and 90% in spherules and layers
compared with their levels in wt BY4741 yeast (Fig. 1, lanes 7 and
8). Hereafter, results described for single- and double-knockouts
were based on YPH500 derivatives, whereas rtn1Δrtn2Δyop1Δ
yeast were on the BY4741 background.
Moreover, 1a and 2apollevels were not altered in the absence
of RHPs, ruling out the possibility that the decrease in RNA
replication might be due to a destabilizing effect on the viral rep-
licase proteins (Fig. S1 B and C). Additionally, 1a and 2apollo-
calized normally to the perinuclear ER in most strains (Fig. S2).
However, in rtn1Δrtn2Δyop1Δ yeast-expressing layers, only a por-
tion of 1a was associated with the perinuclear ER, whereas the
majority of 1a colocalized with Sec63 in aberrant regions of ER
that extended away from the nucleus (Fig. S2C).
Deleting RHPs Reduces Spherule Numbers and Diameter. To de-
termine whether spherule formation or structure was affected by
RHP deletion, we measured the abundance and diameter of
spherules in the subset of cells that were sectioned through their
nuclei among a total of 200 cells for each strain. In wt and rtn2Δ
yeast, the average spherule diameter was 58–74 nm, whereas in
yop1Δ, rtn1Δ, and rtn2Δyop1Δ yeast, spherules averaged 48, 35,
and 27 nm in diameter, respectively (Fig. 2 A–C and Fig. S3). For
rtn2Δyop1Δ yeast in particular, the >2-fold decrease in spherule
diameter was paralleled by a >2-fold increase in spherule fre-
quency (Fig. 2C), possibly reflecting the unchanged level of
spherule-lining 1a protein (Fig. S1). Finally, in rtn1Δyop1Δ and
rtn1Δrtn2Δyop1Δ yeast, spherules were not detected among
thousands of cells examined (Fig. 2C).
In yeast cells, the half-life of RNA3 increases from 5 to 7 min in
a marked increase in RNA3 accumulation (33). This 1a-induced,
the 1a-induced spherules (15). In wt, single-deletion, and rtn2-
Δyop1Δ yeast, 1a increased RNA3 accumulation 8- to 11-fold over
that in cells lacking 1a (Fig. S4). However, in rtn1Δyop1Δ yeast,
RNA3 accumulation did not increase in the presence of 1a (Fig.
S4). Thus, Rtn1p and Yop1p in particular modulate the for-
mation and size of 1a-induced membrane spherules.
Deleting RHPs Alters Number and Morphology of Double-Membrane
Layers. Electron micrographs of wt yeast expressing 1a plus GAL1
ER surrounding the nucleus (Fig. 3A). Likewise, multiple double-
membrane layers were found in rtn1Δ (Fig. 3B), rtn2Δ, yop1Δ, and
rtn2Δyop1Δ yeast (Fig. S3). rtn1Δyop1Δ and rtn1Δrtn2Δyop1Δ
yeast, in which RNA replication was inhibited by 70–90% (Fig. 1),
predominantly had only a single double-membrane layer sur-
rounding the nucleus (Fig. 3 C and D). Moreover, simultaneously
deleting the three RHPs disrupted the normal close pairing of
adjacent double-membrane layers, leading to dramatic bulges in
the normally regular, narrow interlayer space (Fig. 3D), reminis-
1 2 3 4 5 6 7 8
% of WT
strains.TotalRNAextracts were obtained from wtYPH500(lane 1), rtn1Δ (lane
2), rtn2Δ (lane 3), yop1Δ (lane 4), rtn2Δyop1Δ (lane 5), rtn1Δyop1Δ (lane 6), wt
BY4741 (lane7),orrtn1Δrtn2Δyop1Δ (lane8)yeastcoexpressing 1a,RNA3, and
either ADH1 2apol(Spherules) or GAL1 2apol(Layers), and accumulation of
was verified by probing for 18S ribosomal RNA. Values represent the mean of
three independent experiments.
BMV RNA replication is inhibited in specific reticulon deletion yeast
74 ± 16 nm
34 ± 9 nm
48 ± 9 nm
27 ± 4 nm
58 ± 9 nm
N N N
C C C
N N N
C C C
RHPs. Representative EM images of wt (A) and rtn1Δ (B) yeast cells
expressing BMV-induced spherules. White arrows point out individual
spherular structures. N, nucleus; C, cytoplasm. (Scale bars: 200 nm.) (C) Effect
of RHP deletion on spherule diameter and number of spherules per cell.
BMV-induced spherule formation is abolished in the absence of
| www.pnas.org/cgi/doi/10.1073/pnas.1011105107 Diaz et al.
cent of the aberrant regions of ER seen by using confocal mi-
croscopy (Fig. S2C).
Because many lipid synthesis steps occur on smooth tubular
ER (34) shaped by RHPs (25, 26, 35), it seemed possible that
RHP depletion might affect these steps, perhaps explaining why
spherule and normal double-membrane layer formation were
suppressed in rtn1Δyop1Δ and rtn1Δrtn2Δyop1Δ yeast. However,
total lipid levels and the major fatty acid species in S. cerevisiae,
palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1),
and oleic acid (18:1) in wt and rtn1Δrtn2Δyop1Δ yeast expressing
either spherules or layers were not significantly changed (Fig.
S5). Therefore, the decreased number of double-membrane
layers in rtn1Δyop1Δ and rtn1Δrtn2Δyop1Δ yeast was not due to
a change in total lipid levels or lipid composition.
BMV 1a Interacts with and Relocalizes RHPs to BMV RNA Replication
Sites. Because the ER membrane-shaping RHPs were required
to form BMV-induced spherules and normal double-membrane
layers, we examined the localization of FLAG-tagged Rtn1p and
Yop1p expressed from their endogenous promoters. Consistent
with previous reports, RHPs were almost absent from the nu-
clear envelope but were abundant in the peripheral ER (Fig. 4A)
(25), which in yeast is located primarily beneath the plasma
membrane (36). In contrast, in yeast expressing 1a alone or with
2apol(Fig. 4B), the majority of RHPs colocalized with 1a in the
perinuclear ER (12, 13).
Next, we used immunoprecipitation (IP) to determine whether
with anti-FLAG antibody, 1a was detected in yeast coexpressing
FLAG-tagged RHPs and 1a or 1a plus 2apol, but not in yeast
lacking 1a (Fig. 4C Lower) or expressing 1a but not FLAG-tagged
RHPs. Similarly, after IP with anti-1a antibody, Rtn1p-, Rtn2p-,
and Yop1p-FLAG were strongly detected (Fig. 4D Upper). IP with
anti-FLAG antibody and immunoblotting for FLAG confirmed
that the RHP-FLAG proteins were expressed (Fig. 4D Lower).
A control IP confirmed that 2apol-GFP, which like wt 2apol
interacts with 1a (8, 9), immunoprecipitated with 1a, whereas
free GFP and Sec63-GFP, a transmembrane ER protein, did not
(Fig. S6B). Moreover, RHPs did not co-IP with 2apolin the ab-
sence of 1a (Fig. S6A). Together, the results indicate that RHPs
are recruited to or retained at BMV RNA replication sites
through a specific interaction with 1a.
RHPs Localize to Interior of Spherules and Double-Membrane Layers.
To more precisely localize RHPs in relation to BMV-induced
replication compartments, we performed immunogold EM with
an antibody recognizing FLAG-tagged RHPs and a secondary
treatment to preserve antigenicity reduced direct membrane
staining but, as in prior studies (15, 20), spherules remained
readily recognizable by the electron lucence of their bounding
membranes and high electron density of their interior contents
relative to general cytoplasm (Fig. 5 A and B). Of >100 gold
were on or near the perinuclear ER (Fig. 5E). In cells expressing
1a, ≈55% of RHP-targeted gold particles were inside the ≈70 nm
spherules (Fig. 5 B and E) or well within the ≈20 nm distance
spanned by the primary and secondary antibodies (37). Likewise,
in cells expressing 1a plus GAL1 2apol, ≈57% of gold particles
connected spaces between layers (Fig. 5 D and E). Notably, as
integral membrane proteins inserted in the ER membrane cyto-
plasmic face (25), RHPs cannot be in the ER lumen. Thus, RHPs
localize to the interior of spherules and double-membrane layers,
where 1a also localizes (15, 20).
Fusing GFP to RHPs Shifts Spherules to Double-Membrane Layers.
The small 8-aa FLAG tag used in previous experiments did not
interfere with the normal localization of RHPs (Fig. 4A) and had
no adverse effect on the formation and morphology of BMV-
induced spherules and double-membrane layers (Fig. 5). How-
ever, we found that RHP-GFP fusions prevented spherule for-
mationand instead formed a layer-like structure under conditions
that normally produce spherules (Fig. S7B). The GFP-tagged
RHPs relocalized from peripheral to perinuclear ER in the
presence of 1a or 1a plus GAL1 2apol(Fig. S7 C and D) and co-IP
N N N
C C C
N N N
C C C
N N N
C C C
N N N
C C C
triple and onedoubledeletion strain. EM images of yeastcells expressing BMV
induced layers in wt (A), rtn1Δ (B), rtn1Δyop1Δ (C), or rtn1Δrtn2Δyop1Δ (D)
yeast. Black arrows point out double-membrane layers, whereas white arrows
Morphology and number of double-membrane layers is altered in
Empty 1a1a + 2apol
R1FR1F R2F R2FY1F Y1FR1FR2F Y1F
Empty 1a + 2apol
replication. Localization of FLAG-tagged RHPs in yeast transformed with
empty plasmids (A) or 1a alone (B). DNA was stained with DAPI (blue). Images
were cropped just outside the yeast cell wall to include only one cell. (Scale
plasmids, 1a alone, or 1a plus GAL1 2apolwere lysed, and the cleared lysates
were subjected to immunoprecipitation by using anti-1a or anti-FLAG anti-
bodies (IP 1a and IP FLAG, respectively). The resulting immunoprecipitates
were analyzed on 4–15% SDS/PAGE and immunoblotted using anti-1a (C) or
anti-FLAG (D) antibodies. The positions of 1a, Rtn1p-FLAG (R1F), Rtn2p-FLAG
(R2F), and Yop1p-FLAG (Y1F) are shown on the right.
RHPs interact with BMV 1a protein and relocalize to site of BMV
Diaz et al. PNAS
| September 14, 2010
| vol. 107
| no. 37
with 1a (Fig. S6C). Thus, RHPs are necessary to form spherular
compartments as a GFP tag prevented formation of spherules
while not interfering with RHP–1a interaction.
To replicate their genomic RNAs, (+)RNA viruses induce
membrane rearrangements that create membrane-linked RNA
replication compartments, but how such rearrangements are ac-
complished remains largely unknown. Our results show that the
membrane-shaping host RHPs localize to the sites of BMV RNA
synthesis through an interaction with viral replication factor 1a,
play crucial roles in BMV RNA synthesis, and are essential for
forming the membrane-bounded replication compartments in
which RNA synthesis occurs.
RHP Roles in Replication Vesicle Formation and Function. Previous
work demonstrated that 1a membrane association and 1a–2apolin-
(7, 31). Here, our data show that RHP deletion did not reduce 1a
2apol(Fig. S2D). However, deleting all three RHP genes (rtn1Δ-
rtn2Δyop1Δ) or the two most highly expressed RHP genes
(rtn1Δyop1Δ) inhibited RNA replication 10-fold (Fig. 1) and abol-
ished 1a-induced spherule formation (Fig. 2C) and viral RNA tem-
plate protection (Fig. S4). This failure toprotect viral RNAs may be
interiors are the protected site in which 1a sequesters viral RNAs
(7, 15) and, upon 1a mutation, increases and decreases in spherule
BMV 1a strikingly localized more than one-half of RHPs to
the perinuclear ER and incorporated them inside the replication
vesicles (Figs. 4 and 5). A challenge in understanding how RHPs
contribute to spherule formation is that although 1a-induced
spherules are invaginated away from the cytoplasm, RHPs pull
ER membranes to protrude into the cytoplasm. Thus, superfi-
cially, it appears that RPH and 1a action should be antagonistic,
not cooperative. However, RHPs also are involved in forming
nuclear pores (38), which are topologically equivalent to spher-
ule necks: both are positively curved channels from the peri-
nuclear ER into the cytoplasm. Thus, one important role for
RHPs may be stabilizing spherule necks (Fig. 6A). Because no
membrane breakage is involved, every degree of negative cur-
vature induced by 1a must be compensated by corresponding
positive curvature at the neck. Consistent with such a role, RHPs
polymerize into short arcs and need to occupy only ≈10% of
a membrane surface to form tubules (35). Without RHPs to
stabilize neck formation, 1a may not be able to progress beyond
initial membrane deformation to make a full vesicle.
RHP expression levels and density on membranes vary the rate
of bending and, thus, the diameter of ER tubules (35). Similar
considerations may explain why partial RHP depletion in rtn1Δ,
yop1Δ, and rtn2Δyop1Δ yeast reduced spherule diameter ≈2-fold
suggest that RHPs also may be incorporated with 1ainto the main
vesicle body (Fig. 6A). There, local positive curvature induced by
membrane insertion of wedge-shaped RHPs should partially
the membrane to bend toward closure more slowly and increasing
spherule diameter relative to a pure 1a shell (Fig. 6B).
Notably, the smaller spherules supported substantial levels of
RNA replication (Fig. 1). Thus, as long as large or small 1a-
induced spherules formed, viral RNA templates were stabilized
(Fig. S4) and replicated (Fig. 1), suggesting that a major RNA
replication defect in rtn1Δyop1Δ and rtn1Δrtn2Δyop1Δ yeast was
the failure to form replication compartments.
N N N
N N N
C C C
N N N
N N N
C C C
C C C
C C C
% of peri-
7.5 ± 4.5
55.0 ± 7
57.0 ± 8
osmium fixed yeast cells containing spherules (A) or layers (C). Anti-FLAG
immunogold EM labeling in yeast cells expressing Rtn1p-FLAG and 1a (B)
or 1a plus GAL1 2apol(D). Insets in B show individual spherules. To preserve
the antigenicity of the proteins, osmium fixation and staining were re-
duced, resulting in electron lucent regions where the lipid membranes
used to be due to lipid extraction during the dehydration steps. N, nu-
cleus; C, cytoplasm. (E) Percentage of total gold particles that localized
to perinuclear ER in yeast expressing no BMV components, spherules, or
RHPs localize to interior of spherules and layers. EM images of
tenance of BMV-induced spherules. (A) Schematic of BMV spherules showing
vesicle body, whereas arced RHP multimers (red) might stabilize the positively
curved neck region and also be incorporated in the main vesicle body. (B)
Schematics replacing 1a and RHP clusters in the main vesicle body with blue
and red arcs, respectively, to show how expansion of the vesicle body in RHP-
containing spherule A is primarily due to the action of RHPs in partially neu-
tralizing the intrinsic rate of membrane curvature by 1a alone.
Models for the potential role of RHPs in the formation and/or main-
| www.pnas.org/cgi/doi/10.1073/pnas.1011105107 Diaz et al.
RHP Roles in Double Membrane Layers. RHPs also were crucial for
RNA replication under conditions of high 2apolexpression,
which shift 1a-induced replication compartments from vesicles to
stacked double-membrane layers (20). RHP depletion in rtn1Δ,
rtn1Δyop1Δ, and rtn1Δrtn2Δyop1Δ yeast progressively inhibited
layer formation and deranged layer morphology in parallel with
inhibiting RNA replication (Figs. 1 and 4). Just as they stabilize
ER tubules, RHPs might help to initiate each new layer by
forming arc-like scaffolds to stabilize the positively curved ends
(half tubules) where each perinuclear ER layer folds over to
begin the next outer layer (Fig. S8A) (20). Moreover, because
RHPs were recruited to the intermembrane spaces (Fig. 5D), the
self-interacting 1a and RHP proteins (38) might cluster in the
concave and convex membrane regions, respectively, neutralizing
each other’s induced membrane curvature to yield relatively flat
sheets (Fig. S8A). Consistent with this possibility, absence of
RHPs produced aberrant single layers with prominent, nega-
tively curved bulges consistent with unbridled 1a-induced cur-
vature (Fig. S8B).
Like excess 1a-interacting 2aPol, GFP fusion to 1a-interacting
RHPs inhibited spherule formation and induced membrane layer
formation (Fig. S7). This phenotype is consistent with our in-
dependent findings that RHPs interact with 1a and localize to
spherule and layer interiors (Figs. 4 and 5). As with 2apol(20),
these effects may be due to steric issues or interference with
competing 1a interactions, such as certain 1a–1a interactions.
RHPs also might contribute to RNA synthesis in ways beyond
membrane shaping. By interacting with 1a, RHPs might mod-
ulate 1a conformation, multimerization, or membrane in-
teraction to activate one or more 1a functions required for RNA
synthesis, similar to membrane activation of Semliki Forest vi-
rus nsP1 capping functions (39). BMV RNA replication also
requires high, balanced levels of lipid synthesis (40), many steps
of which occur on RHP-stabilized tubular ER (25, 26, 34).
However, deleting all three RHPs did not alter fatty acid levels
or composition (Fig. S5). Moreover, although RHPs affect
vesicular trafficking (41), deleting all three RHPs did not alter
ER membrane localization of BMV RNA replication proteins
1a or 2aPol(Fig. S2). It remains possible that RHP deletion
might alter the distribution of a host factor required for
Relation to Other (+)RNA Viruses. Several other membrane-asso-
ciated host factors recently have been implicated in (+)RNA
virus replication. BMV and poliovirus RNA replication depend
on ER-localized Δ9 fatty acid desaturase Ole1p and secretory
regulating factor GBF1, which is recruited to poliovirus RNA
replication sites, although neither factor is required for replica-
tion-associated membrane rearrangements (40). Poliovirus also
subverts membrane-rearranging autophagosomal pathways, with
implications for RNA replication and virion release (42). Addi-
tionally, dominant-negative mutants of ESCRT-III proteins re-
duced tombusvirus RNA replication and the frequency of
spherules in infected cells (43). Moreover, enterovirus 71 and
poliovirus 2C proteins interact with human reticulon 3 (RTN3),
and RTN3 knockdown decreased enterovirus 71 double-
stranded RNA synthesis and protein expression (44). Although
possible effects on membrane rearrangements were not de-
termined, these results suggest that the BMV-RHP findings here
may have implications for other (+)RNA viruses.
Overall, our data show that RHPs interact with BMV 1a and
are essential for inducing and/or stabilizing the membrane
rearrangements linked to BMV RNA replication. Further un-
derstanding of RPH roles in RNA replication by BMV and other
viruses should provide additional insights into replication path-
ways and facilitate use of such host genes as potential targets for
S. cerevisiae Strains and Plasmids. The yeast strains used were as follows:
YPH500 (MATα ura3–52 lys2–801 ade2–101 trp1-Δ63 his3-Δ200 leu2-Δ1),
BY4741 (MATα his3Δ1 leu2Δ met15Δ ura3Δ), and NDY257 (BY471 rtn1::
kanMX4 rtn2::kanMX4 yop1::kanMX4) (25). The following strains were
generated for this work: RM1 (YPH500 rtn1::kanMX4), RM2 (YPH500 rtn2::
kanMX4), RM3 (YPH500 yop1::kanMX4), RM4 (YPH500 rtn1::kanMX4 yop1::
natMX4), and RM5 (YPH500 rtn2::trp1 yop1::KanMX4). Genomic insertions
were made by using amplified KanMX4, NatMX4, or trp1 cassettes flanked
by 5′ and 3′ homologous recombination regions. Strains expressing the
chromosomal alleles of RTN1, RTN2, and YOP1 as GFP fusions were obtained
from Invitrogen. For endogenous expression of the RHPs, the coding region
of the full length protein plus the 300-bp upstream sequences were PCR-
amplified from wt yeast DNA with appropriate primers and inserted into
a CEN plasmid. A FLAG-tag was added at the C terminus of the RHPs. BMV 1a
was expressed from the GAL1 promoter by using pB1YT3 (5), 2apolwas
expressed from pB2CT15 (ADH1 promoter) (14) or pB2YT5 (GAL1 promoter)
(5), and BMV RNA3 was expressed from a CUP1 promoter using
pB3VG128His, a pB3MS82 derivative (5). Plasmids encoding Sec63-GFP
(pJK59) (20), free GFP, and 2apol-GFP (8) have been described. Plasmid
transformation and yeast cultivation were performed as described (14).
RNA and Protein Analysis. RNAisolation,NorthernandWesternblotanalysis,and
mouse polyclonal antibodies were purchased from Sigma and Molecular
Coimmunoprecipitation. Yeastcellswerelysedin RIPAbuffer (1%NonidetP-40,
glass beads and a bead beater, and the supernatant was collected after centri-
fugation. For immunoprecipitation, yeast lysates were mixed with Protein A
Sepharose beads (GE Healthcare) and anti-1a, anti-FLAG, oranti-GFP antibodies
overnight at 4 °C. Beads were pelleted and washed with RIPA buffer before
boiling in 1× SDS gel loading buffer and running samples in 4–15% SDS/
Confocal Laser Microscopy. To detect the subcellular localization of BMV pro-
teins in RHP deletion strains, yeast were transformed with plasmids expressing
1a and 2apoland Sec63-GFP. To detect the localization of RHPs, FLAG-tagged
RHPs or strains expressing GFP fusions to the chromosomal alleles of RTN1,
RTN2, and YOP1 were transformed with empty plasmids or plasmids encoding
1a and 2apol. Confocal microscopy was performed on a Bio-Rad 1024 double-
channel confocal microscope as described (12).
Electron Microscopy. Conventional and immunolabeling fixation EM was
done as described (15). For immunogold labeling, the percentage of relo-
calized RHPs was calculated by comparing the number of gold particles in or
near the perinuclear ER in the absence versus the presence of 1a. Back-
ground labeling was determined by using cells lacking FLAG-tagged RHPs.
Specific labeling was determined by subtracting the background labeling
density. The diameter of >50 spherules was measured with the imaging
program ITEM Analysis (Soft Imaging Systems). For additional information,
see SI Experimental Procedures.
ACKNOWLEDGMENTS. We thank Benjamin August and Randall Massey of
the University of Wisconsin Medical School Electron Microscopy Facility for
assistance with EM, Lance Rodenkirch of the W. M. Keck Laboratory for
Biological Imaging for assistance with confocal microscopy, and Dr. William
Prinz (the National Institutes of Health, Bethesda, MD) for providing the
triple-knockout strain. This project was supported by National Institutes of
Health Grant GM35072. P.A. is a Howard Hughes Medical Institute investi-
gator. A.D. was supported by National Institutes of Health Predoctoral Grant
1. Miller S, Krijnse-Locker J (2008) Modification of intracellular membrane structures for
virus replication. Nat Rev Microbiol 6:363–374.
2. Knoops K, et al. (2008) SARS-coronavirus replication is supported by a reticulovesicular
network of modified endoplasmic reticulum. PLoS Biol 6:e226.
Diaz et al.PNAS
| September 14, 2010
| vol. 107
| no. 37
3. KopekBG,PerkinsG,MillerDJ,EllismanMH,AhlquistP(2007)Three-dimensionalanalysis Download full-text
4. Ahola T, Ahlquist P (1999) Putative RNA capping activities encoded by brome mosaic
virus: Methylation and covalent binding of guanylate by replicase protein 1a. J Virol
5. Ahola T, den Boon JA, Ahlquist P (2000) Helicase and capping enzyme active site
mutations in brome mosaic virus protein 1a cause defects in template recruitment,
negative-strand RNA synthesis, and viral RNA capping. J Virol 74:8803–8811.
6. Kong F, Sivakumaran K, Kao C (1999) The N-terminal half of the brome mosaic virus 1a
7. Wang X, et al. (2005) Brome mosaic virus 1a nucleoside triphosphatase/helicase domain
plays crucial roles in recruiting RNA replication templates. J Virol 79:13747–13758.
8. Chen J, Ahlquist P (2000) Brome mosaic virus polymerase-like protein 2a is directed
to the endoplasmic reticulum by helicase-like viral protein 1a. J Virol 74:4310–4318.
9. Kao CC, Ahlquist P (1992) Identification of the domains required for direct interaction
of the helicase-like and polymerase-like RNA replication proteins of brome mosaic
virus. J Virol 66:7293–7302.
10. Krol MA, et al. (1999) RNA-controlled polymorphism in the in vivo assembly of 180-
subunit and 120-subunit virions from a single capsid protein. Proc Natl Acad Sci USA
11. Sullivan ML, Ahlquist P (1999) A brome mosaic virus intergenic RNA3 replication
signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo.
J Virol 73:2622–2632.
12. Restrepo-Hartwig M, Ahlquist P (1999) Brome mosaic virus RNA replication proteins
1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic
reticulum. J Virol 73:10303–10309.
13. Restrepo-Hartwig MA, Ahlquist P (1996) Brome mosaic virus helicase- and
polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA
synthesis. J Virol 70:8908–8916.
14. Janda M, Ahlquist P (1993) RNA-dependent replication, transcription, and persistence
of brome mosaic virus RNA replicons in S. cerevisiae. Cell 72:961–970.
15. Schwartz M, et al. (2002) A positive-strand RNA virus replication complex parallels
form and function of retrovirus capsids. Mol Cell 9:505–514.
16. den Boon JA, Chen J, Ahlquist P (2001) Identification of sequences in Brome mosaic
virus replicase protein 1a that mediate association with endoplasmic reticulum
membranes. J Virol 75:12370–12381.
17. O’Reilly EK, Kao CC (1998) Analysis of RNA-dependent RNA polymerase structure and
function as guided by known polymerase structures and computer predictions of
secondary structure. Virology 252:287–303.
18. Welsch S, et al. (2009) Composition and three-dimensional architecture of the dengue
virus replication and assembly sites. Cell Host Microbe 5:365–375.
19. Kujala P, et al. (2001) Biogenesis of the Semliki Forest virus RNA replication complex.
J Virol 75:3873–3884.
20. Schwartz M, Chen J, Lee WM, Janda M, Ahlquist P (2004) Alternate, virus-induced
membrane rearrangements support positive-strand RNA virus genome replication.
Proc Natl Acad Sci USA 101:11263–11268.
21. Yang YS, Strittmatter SM (2007) The reticulons: A family of proteins with diverse
functions. Genome Biol 8:234.
22. Nziengui H, Schoefs B (2009) Functions of reticulons in plants: What we can learn
from animals and yeasts. Cell Mol Life Sci 66:584–595.
23. Audhya A, Desai A, Oegema K (2007) A role for Rab5 in structuring the endoplasmic
reticulum. J Cell Biol 178:43–56.
24. Wakefield S, Tear G (2006) The Drosophila reticulon, Rtnl-1, has multiple differentially
expressed isoforms that are associated with a sub-compartment of the endoplasmic
reticulum. Cell Mol Life Sci 63:2027–2038.
25. Voeltz GK, Prinz WA, Shibata Y, Rist JM, Rapoport TA (2006) A class of membrane
proteins shaping the tubular endoplasmic reticulum. Cell 124:573–586.
26. De Craene JO, et al. (2006) Rtn1p is involved in structuring the cortical endoplasmic
reticulum. Mol Biol Cell 17:3009–3020.
27. Kiseleva E, Morozova KN, Voeltz GK, Allen TD, Goldberg MW (2007) Reticulon 4a/
NogoA locates to regions of high membrane curvature and may have a role in nuclear
envelope growth. J Struct Biol 160:224–235.
28. Tolley N, et al. (2008) Overexpression of a plant reticulon remodels the lumen of the
cortical endoplasmic reticulum but does not perturb protein transport. Traffic 9:
29. Kushner DB, et al. (2003) Systematic, genome-wide identification of host genes
affecting replication of a positive-strand RNA virus. Proc Natl Acad Sci USA 100:
30. Ghaemmaghami S, et al. (2003) Global analysis of protein expression in yeast. Nature
31. Liu L, et al. (2009) An amphipathic alpha-helix controls multiple roles of brome mosaic
virus protein 1a in RNA replication complex assembly and function. PLoS Pathog 5:
32. Noueiry AO, Diez J, Falk SP, Chen J, Ahlquist P (2003) Yeast Lsm1p-7p/Pat1p
deadenylation-dependent mRNA-decapping factors are required for brome mosaic
virus genomic RNA translation. Mol Cell Biol 23:4094–4106.
33. Janda M, Ahlquist P (1998) Brome mosaic virus RNA replication protein 1a dra-
matically increases in vivo stability but not translation of viral genomic RNA3. Proc
Natl Acad Sci USA 95:2227–2232.
34. Baumann O, Walz B (2001) Endoplasmic reticulum of animal cells and its organization
into structural and functional domains. Int Rev Cytol 205:149–214.
35. Hu J, et al. (2008) Membrane proteins of the endoplasmic reticulum induce high-
curvature tubules. Science 319:1247–1250.
36. Prinz WA, et al. (2000) Mutants affecting the structure of the cortical endoplasmic
reticulum in Saccharomyces cerevisiae. J Cell Biol 150:461–474.
37. Hayat MA (1991) Colloidal Gold (Academic, San Diego).
38. Shibata Y, et al. (2008) The reticulon and DP1/Yop1p proteins form immobile
oligomers in the tubular endoplasmic reticulum. J Biol Chem 283:18892–18904.
39. Ahola T, Lampio A, Auvinen P, Kääriäinen L (1999) Semliki Forest virus mRNA capping
enzyme requires association with anionic membrane phospholipids for activity. EMBO
40. Lee WM, Ahlquist P (2003) Membrane synthesis, specific lipid requirements, and
localized lipid composition changes associated with a positive-strand RNA virus RNA
replication protein. J Virol 77:12819–12828.
41. Steiner P, et al. (2004) Reticulon 1-C/neuroendocrine-specific protein-C interacts with
SNARE proteins. J Neurochem 89:569–580.
42. Taylor MP, Burgon TB, Kirkegaard K, Jackson WT (2009) Role of microtubules in
extracellular release of poliovirus. J Virol 83:6599–6609.
43. Barajas D, Jiang Y, Nagy PD (2009) A unique role for the host ESCRT proteins in
replication of Tomato bushy stunt virus. PLoS Pathog 5:e1000705.
44. Tang WF, et al. (2007) Reticulon 3 binds the 2C protein of enterovirus 71 and is
required for viral replication. J Biol Chem 282:5888–5898.
| www.pnas.org/cgi/doi/10.1073/pnas.1011105107Diaz et al.