JOURNAL OF VIROLOGY, Jan. 2007, p. 558–567
Vol. 81, No. 2
Hijacking Components of the Cellular Secretory Pathway for
Replication of Poliovirus RNA?
George A. Belov,1Nihal Altan-Bonnet,2† Gennadiy Kovtunovych,2Catherine L. Jackson,2
Jennifer Lippincott-Schwartz,2and Ellie Ehrenfeld1*
National Institute of Allergy and Infectious Diseases1and National Institute of Child Health and
Human Development,2National Institutes of Health, Bethesda, Maryland 20892
Received 7 August 2006/Accepted 17 October 2006
Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-
like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains un-
known, although some observations have implicated components of the cellular secretory and/or autophagy
pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory
trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with
newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific
target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from
its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins
independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus
replication. Intracellular Arf-GTP levels increase ?4-fold during poliovirus infection. The requirement for
these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression
of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important
clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.
All known positive-strand RNA viruses replicate their ge-
nomes in association with membranous structures that are
formed after the synthesis of viral proteins in infected cells by
remodeling membranes from existing intracellular organelles.
Different viruses target different organelle membranes (e.g.,
endoplasmic reticulum [ER], Golgi, endosomes, and mito-
chondria) and generate different morphological structures on
which the replication complexes assemble (37).
Among the Picornavirus family members, RNA replication
complexes from poliovirus-infected HeLa cells have been the
best studied. Heterogeneously sized, vesicle-like structures
that cluster in the perinuclear space in infected cells were
observed by electron microscopy and described more than 40
years ago. Viral RNA replication occurs on the cytosolic sur-
faces of the vesicles, which aggregate into clusters (7–9). Little
is known about how these structures are formed, although
some recent observations have suggested that components of
the cellular secretory and/or autophagy pathways are involved
(24, 39, 41).
The process of membrane traffic begins at the ER, where
polio proteins appear to be synthesized. Rust et al. (36) have
visualized, by three-dimensional reconstruction of serial con-
focal microscope sections, poliovirus protein 2B sequences,
and presumably other viral proteins, budding from multiple
sites on the ER and showed extensive colocalization of the 2B
sequences with COPII coatamer protein. Although it has not
been determined which polio protein sequences are directly
responsible for initiating the COPII-dependent budding pro-
cess, it is generally believed that the P2 proteins 2C, 2BC, or
precursors thereof, are required (2, 9, 12, 41). Normally, the
COPII machinery establishes a membrane flow from the ER to
the Golgi complex. However, poliovirus replication vesicles do
not fuse with the Golgi complex; indeed, the Golgi complex
appears to disassemble in poliovirus-infected cells, presumably
because retrograde membrane traffic from the Golgi continues
while anterograde traffic is rerouted to form the viral replica-
A complicating observation is that polio RNA replication is
inhibited by the fungal antimetabolite, brefeldin A (BFA) (18,
22, 27), whereas neither COPII-dependent traffic nor autoph-
agous vesicle formation is known to be sensitive to this drug.
Instead, BFA is known to inhibit the activation and function of
some members of the small GTPase family, Arf, by interacting
with specific guanine nucleotide exchange factors (GEFs) that
recycle Arf from its inactive GDP-bound form to its active,
GTP-bound form (30, 34). When activated, Arf-GTP recruits
effectors such as coat complexes and lipid-modifying enzymes
to specific membrane sites, creating domains competent for
cargo transport (29, 31). Thus, the Arf GTPases play a central
role in regulating membrane dynamics and protein transport.
The recruitment of Arfs to their target membranes is mediated
by a diverse group of GEFs, all of which share a Sec7 domain
necessary for guanine nucleotide exchange (23). All Arf GEFs
are peripherally associated membrane proteins either tethered
to membranes through a pleckstrin homology domain that
interacts with specific phosphoinositides or through interac-
tions with specific membrane-bound proteins that are not well
characterized. The latter group includes the high-molecular-
weight GEFs GBF1, BIG1, and BIG2, which are sensitive to
BFA (14). The binding of specific GEFs to various membrane
* Corresponding author. Mailing address: NIAID. NIH, 50 South
Dr., Rm. 6120, Bethesda, MD 20892-8011. Phone: (301) 594-1654. Fax:
(301) 435-6021. E-mail: firstname.lastname@example.org.
† Present address: Department of Biological Sciences, Rutgers Uni-
versity, Newark, NJ 07102.
?Published ahead of print on 1 November 2006.
sites confers specificity to the recruitment and activation of
specific Arfs, serving to regulate different steps in the membrane
trafficking pathway (4).
The sensitivity of poliovirus RNA replication to BFA sug-
gested that, in addition (or subsequent) to the COPII-depen-
dent budding of vesicles from the ER, an Arf-dependent mem-
brane trafficking step may be required for polio replication
complex formation. For example, Gazina et al. (21) reported
that ?-COP, a component of the coatamer COPI, whose re-
cruitment to membranes is regulated by Arf1, localizes to the
membranous replication complexes in cells infected by echo-
virus 11, a member of the Enterovirus genus whose growth, like
that of poliovirus, is sensitive to BFA. Arf1 was not present on
vesicles associated with the replication complexes of picorna-
viruses such as encephalomyocarditis virus, whose growth is
resistant to BFA.
Previously, we demonstrated that Arf1 proteins colocalize
with the newly formed poliovirus replication complexes in in-
fected cells and that some members of the Arf family are
recruited to cellular membranes by poliovirus proteins synthe-
sized in HeLa cell extracts that support the complete transla-
tion and/or replication of viral RNA in vitro (5). These extracts
have been used extensively to investigate many aspects of viral
protein synthesis and processing, RNA replication, and assem-
bly of infectious virions (3, 28), and we have shown previously
that viral RNA synthesis, but not translation, is dependent
upon the presence of intact membranes in the extracts (19).
Recruitment of Arf to the membranes was induced indepen-
dently by two viral proteins from the P3, but not the P2, region:
3A and 3CD.
We show here that the two viral proteins, 3A and 3CD,
independently recruit different Arf GEFs (GBF1 and BIG1/2,
respectively) to membranes. These GEFs then are responsible
for Arf activation and translocation to these membranes. The
requirement for these specific BFA-inhibited GEFs explains
the sensitivity of virus growth to BFA, which can be rescued by
overexpression of GBF1.
MATERIALS AND METHODS
Cells and viruses. Monolayer cultures of HeLa or Vero cells, grown either in
multiwell chambers for time-lapse microscopy or on 18-mm coverslips in 12-well
plates for immunofluorescence, were infected with the Mahoney strain of polio-
virus type 1 in Dulbecco modified Eagle medium containing ?5 ? 107PFU of
Plasmids. pXpA-SH contains full-length cDNA of poliovirus type 1 under
control of the T7 promoter, with the hammerhead ribozyme coding sequence at
the 5?end of the poliovirus sequence and additional unique restriction sites
within the viral cDNA (5). pXpA-RenR plasmid encodes a poliovirus replicon
with the Renilla luciferase gene from phRL-CMV (Promega) substituting for the
capsid coding sequence in pXpA-SH. Plasmids used for transcription of RNAs
coding for individual poliovirus proteins have been previously described (5).
pArf1-GFP for Arf-GFP fusion expression, pHA-GBF1-Yc for expression of
GBF1, and pHA-BIG2 have been previously described (32, 40, 43).
Transfection. DNA transfections were performed with Fugene 6 reagent
(Roche), and RNA transfections utilized the Trans-It mRNA transfection kit
(Mirus) according to the manufacturer’s instructions.
Cell-free translation assays. In vitro transcription and translation of viral
RNAs and Western blotting of the membrane fractions were performed essen-
tially as described previously (5). Briefly, HeLa cell S10 extracts were pro-
grammed with poliovirus-specific RNAs and incubated for 3.5 h at 34°C, and the
membrane material was collected by centrifugation at 15,700 ? g for 20 min at
4°C prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis (SDS-PAGE) and immunoblotting.
Antibodies. Antibodies to BIG1 and BIG2 were provided by M. Vaughan and
J. Moss (NHLBI). Anti-GTPase activating protein (GAP) and anti-GBF1 anti-
bodies were from P. Randazzo (National Cancer Institute) and J. Bonifacino
(National Institute of Child Health and Human Development). Monoclonal
anti-poliovirus antibodies to 2C and 3A were a gift from K. Bienz, University of
Basel, Basel, Switzerland. Secondary antibody conjugates used in immunofluo-
rescence were from Molecular Probes; those used in Western blots were from
Microscopy. Live cell microscopy was performed on a Zeiss LSM510 line
scanning confocal microscope. HeLa cells were grown in coverglass-bottom
chambers (Lab-Tek). The cells were maintained at 37°C on the microscope stage
for the duration of the experiments. High-spatial-resolution images were ob-
tained with a ?63/1.4 numerical aperture plan Apochromat oil immersion ob-
jective lens with a small (1.2 Airy unit) pinhole aperture, whereas images for
purposes of quantitation (e.g., fluorescence recovery after photobleaching
[FRAP]) were acquired with a ?40/1.3 N.A. oil immersion objective lens with a
fully open pinhole setting (?14 Airy units). All images were acquired on 12-bit
photomultiplier tubes. No saturated images were analyzed for quantitation.
For FRAP experiments, a region of interest (ROI) was drawn around the
Arf1-GFP-labeled structures, and this ROI was photobleached for 5 s with a
high-intensity 488-nm laser beam. Images of the samples were then taken with
low-intensity light for 3 min. Input from lateral diffusion effects was excluded by
photobleaching areas that did not have apparent contacts with other intracellular
structures associated with Arf1-GFP. The FRAP data were analyzed with image
analysis software which allowed us to measure the mean fluorescence per pixel in
the defined ROIs. To obtain the total fluorescence associated with Arf1-GFP
either at the Golgi apparatus or at cytoplasmic foci in an infected cell, we
multiplied the background-subtracted mean fluorescence per pixel in the ROI
with the area of that ROI. The total fluorescence at each time point was cor-
rected for photobleaching (which typically occurs after a time series) by dividing
it by the total fluorescence of the cell at that time.
For immunofluorescence, cells were grown on coverslips, fixed with 4% para-
formaldehyde–phosphate-buffered saline (PBS) for 20 min, and permeabilized
with 0.2% Triton X-100 in PBS for 5 min. The cells were then incubated in 3%
nonfat dry milk solution for 1 h to block nonspecific binding sites. This solution
also was used for dilution of primary and secondary antibodies in which cells
were sequentially incubated for 1 h. Images were taken with Leica DMIRE
microscope. Digital images were processed with Adobe Photoshop software.
Arf-GEF assay. Detection of Arf GEF activity was performed as described
previously (35). Briefly, cytoplasmic extract from ?107HeLa cells was incubated
with recombinant myristoylated Arf1 in the presence of liposomes and
[?-35S]GTP. At various times aliquots of the reaction mix were put into stop
solution and kept on ice. When all of the samples were collected, they were
precipitated with 10% trichloroacetic acid on filter paper and washed twice with
5% trichloroacetic acid, and the precipitated radioactivity was measured by
Arf-GTP pull-down assay. HeLa cells were grown overnight on 10-cm petri
dishes. The cells were then infected with 10 PFU of poliovirus/cell. At various
times the cells were lysed on ice for 15 min in 1 ml of lysis buffer (50 mM Tris-Cl
[pH 7.5], 100 mM NaCl, 2 mM MgCl2, 0.5% sodium deoxycholate, 1% Triton
X-100, 10% glycerol) containing 100 ?M phenylmethylsulfonyl fluoride and a
1/100 of volume of protease inhibitor cocktail (Sigma-Aldrich). Lysates were
clarified by centrifugation at maximum speed for 20 min in an Eppendorf mini-
fuge at 4°C. The supernatants were collected and stored at ?80°C. The Arf-GTP
pull-down assay was performed as described in reference 25. Briefly, lysates
were precleared with 50 ?l of a 50% slurry of glutathione-Sepharose 4B beads
(Amersham-Biosciences) for 1 h and centrifuged. Supernatants were incubated
with 50 ?l of beads that contained 40 ?g of prebound GST-GGA3-GAT fusion
protein (expression construct kindly provided by Julie Donaldson, NHLBI,
NIH). Beads were collected by centrifugation and washed three times with 250
?l (10 times the bed volume of beads) washing buffer. Proteins were eluted from
beads by adding 1? SDS-PAGE sample buffer and heating the samples at 70°C
for 15 min. All manipulations with lysates were carried out at 4°C.
Viral RNA replication assays. HeLa cells grown in 96-well plates were trans-
fected with specified plasmids and the next day were transfected with poliovirus
replicon RNA (0.8 ng/well) transcribed from pXpA-RenR. After 1 h, normal
growth medium containing 60 ?M EnduRen Renilla luciferase substrate (Pro-
mega) and 1 ?g of BFA/ml or solvent dimethyl sulfoxide was provided. Light
from Renilla luciferase activity was measured at hourly intervals with a Molecular
Devices MV microplate reader. After the 7-h time point, cells were lysed with
DualGlo (Promega) firefly luciferase assay buffer, and the firefly luciferase ac-
tivity was measured to assess DNA transfection efficiency.
VOL. 81, 2007FORMATION OF POLIOVIRUS REPLICATION COMPLEXES559
Dynamics of Arf1-EGFP redistribution upon poliovirus in-
fection. The Arf GTPases regulate vesicular traffic and or-
ganelle structure by recruiting coat proteins and regulating
phospholipid metabolism (4). The sensitivity of poliovirus rep-
lication to BFA, which is known to inhibit the activation and
function of Arf, previously prompted us to determine whether
Arf was associated with the viral RNA replication complexes
that formed in poliovirus-infected cells (5). The results showed
a clear redistribution of Arf1-EGFP from its predominant con-
centration at the Golgi complex in uninfected cells to perinuclear
structures that colocalized with viral replication proteins used to
mark the replication complexes in infected cells. In order to mon-
itor the kinetics and pathway of Arf translocation during the
course of infection, HeLa cells transiently expressing Arf1-GFP
fusion protein were infected with poliovirus and imaged by using
time-lapse confocal microscopy at 6-min intervals throughout the
course of infection (Fig. 1). The conditions were optimized to
maintain cell viability during the entire virus replication cycle,
allowing the molecular relocalization of Arf1-GFP to be moni-
tored in individual live, infected cells. At the start of the infection,
Arf1-GFP was primarily concentrated on the surface of Golgi
membranes, with a soluble pool distributed diffusely throughout
the cytoplasm. In most cells Arf1-GFP began to dissociate from
the Golgi region between 2 and 3 h postinfection and, simulta-
neously, multiple new foci of fluorescence emerged in the cyto-
FIG. 1. Relocalization of Arf1-GFP in infected cells. (A) HeLa cells transfected with pArf1-GFP for 18 h were infected with poliovirus and
monitored every 6 min for 16 h. The figure shows selected images taken at the indicated times postinfection. (For the complete sequence, see the
movie [and corresponding Web site] referred to in Results.) (B) HeLa cells were cotransfected with pGalT-YFP and pArf-CFP. After 18 h of
incubation, the cells were infected with poliovirus and monitored for fluorescence of both of the expressed proteins.
560 BELOV ET AL.J. VIROL.
plasm (Fig. 1A). The foci began to coalesce and concentrate in
the perinuclear region within approximately 30 min after they
began to form. This pattern has been shown previously to corre-
spond to the pattern of poliovirus replication complexes (9),
which expands and remains perinuclear until cell death (Fig. 1;
the imaging sequence can be viewed in movie form [http://www3
/ehrenfeld.htm]). In some cells all of the Golgi-associated Arf1-GFP
distributed to the cytoplasm before relocalizing to the new
structures, whereas in others some residual fluorescence
remained in the original location.
The loss of Arf1 from the Golgi region in poliovirus-infected
cells coincided with the disassembly of the Golgi apparatus
based on experiments in which Arf1-CFP was coexpressed with
the Golgi enzyme marker, galactosyltransferase tagged with
YFP (GalT-YFP). In these cells the two proteins initially com-
pletely colocalized in the Golgi area (Fig. 1B, 0 h postinfec-
tion). As infection proceeded, they both lost their juxtanuclear,
Golgi localization and redistributed in different patterns.
Based on these results, we concluded that Arf1 relocates from
Golgi to non-Golgi membrane foci between 2 and 3 h postin-
To determine whether Arf relocalization occurred as a gen-
eral response of infected cells to ER stress or induction of
apoptosis, known to occur early in poliovirus infection (42),
HeLa cells were treated with tunicamycin, which induces ER
stress by interference with protein glycosylation, or with tumor
necrosis factor, a potent inducer of receptor-mediated apop-
tosis. No significant change in the fluorescence pattern of Arf1-
GFP was observed, and cells treated with these substances
underwent apoptotic death with Arf1-GFP still associated with
Golgi structures (not shown). Infection of cells in the presence
of a caspase inhibitor, zVAD-fmk, previously shown to prevent
apoptosis induction by poliovirus (1), also did not affect the
translocation of Arf fluorescence. Thus, Arf1 redistribution in
infected cells does not represent a response to general stress
conditions but is specifically induced by poliovirus. Infection of
Vero cells expressing Arf1-GFP resulted in a similar redistri-
bution of Arf1-GFP as observed in HeLa cells, indicating that
the cellular response to poliovirus infection is not host cell
specific (not shown).
FRAP analysis of Arf1 on poliovirus replication complexes.
Normal function of Arf involves rapid exchange between mem-
brane-associated and cytoplasmic Arf pools. To compare the
rate of Arf cycling on and off membranes in polio-infected and
mock-infected cells we performed FRAP experiments in cells
expressing Arf1-GFP fusion protein. The FRAP experiments
were performed in cells approximately 3 to 4 h postinfection,
when Arf1-GFP containing foci were abundant in the cyto-
plasm. Regions of membrane-associated Arf1-EGFP fusion
were selectively photobleached with laser irradiation, and the
recovery of fluorescence, which occurs if there is an exchange
of Arf between the membranes and the cytoplasmic pool, was
monitored. Input from lateral diffusion was excluded by photo-
bleaching areas that did not have apparent contacts with other
intracellular membrane structures with associated Arf1-GFP.
The rates of recovery of fluorescence in poliovirus- and mock-
infected cells were similar (t1/2? 22.2 ? 5.2 s in mock-infected
cells and 19.5 ? 3.1 s in infected cells; however, infected cells
contained a higher proportion [35% ? 13%] of noncycling Arf
compared to 10% ? 7% in mock-infected cells [Fig. 2]). These
results suggest that Arf1 undergoes less exchange between
membrane and cytoplasmic pools in infected cells compared to
uninfected cells. Alternatively, the cytoplasmic Arf1-GFP may
be prevented from diffusing into and out of the photobleached
regions due to the high concentration of membranes in this
Poliovirus infection stimulates production of Arf-GTP in
vivo. Inactive, GDP-bound Arf is located in the cytosol, and
active GTP-bound Arf is associated with membranes. Arf ac-
tivation then leads to recruitment of coat proteins and other
factors that in turn regulate the protein sorting and membrane
deformation events that direct intracellular trafficking. To con-
firm that the Arf associated with polio replication complexes
FIG. 2. Arf1-GFP dynamics in poliovirus-infected HeLa cells. (A) Regions of interest (dotted lines) were selected for photobleaching in cells
?3 h postinfection. In mock-infected cells the Arf1-GFP-labeled Golgi apparatus was selected. A laser pulse was directed at the selected regions
to bleach the fluorescence. Recovery of fluorescence was monitored with low intensity laser light every 5 s. (B) The recovery of fluorescence into
the selected regions was quantified and plotted as described in Materials and Methods. Photobleaching was performed on 9 virus-infected and 12
mock-infected cells in two separate experiments. The mean rate of recovery (?) for control cells was 32 ? 7.5 s?1, and for virus-infected cells it
was 28 ? 4 s?1. The mean immobile fraction for control cells was 10% ? 7%, whereas for poliovirus-infected cells it was 35% ? 13%.
Representative recovery curves from infected and mock-infected cells are plotted.
VOL. 81, 2007FORMATION OF POLIOVIRUS REPLICATION COMPLEXES 561
was in an active, GTP-bound form and to determine whether
the amount of activated Arf increased during the course of
infection, we performed a pull-down assay using a GST-GGA3
fusion protein to monitor the amount of Arf-GTP protein in
lysates of cells during the course of infection (Fig. 3) (25, 32,
38). GGA3 is an effector of Arf, and binds with high affinity
specifically to Arf-GTP (10). Infected cells showed a three- to
fourfold increase in Arf-GTP that accumulated steadily at least
up to 5 h postinfection. Similar experiments were performed
with antibodies specific for different members of the Arf family
as probes. GTP-bound Arfs 1 and 3 (class I) showed the same
pattern of increase during infection as the total Arf-GTP
shown in Fig. 3; Arf5-GTP increased less dramatically, whereas
Arf6-GTP showed no change during the virus growth cycle
(data not shown).
Arf GAPs do not accumulate on poliovirus replication com-
plexes. The hydrolysis of GTP bound to Arf proteins requires
the activity of GAPs to enhance the weak intrinsic GTPase
activity of Arf. The levels of cellular Arf-GTP result from a
balance between GAP activity and GEF activity, which cata-
lyzes the formation of Arf-GTP by exchanging GDP bound to
Arf with GTP. To determine whether poliovirus protein-in-
duced translocation of Arf to membranes was accompanied by
changes in GAPs on the membranes, we translated full-length
poliovirus RNA and RNAs coding for individual viral proteins
in HeLa cell extracts, routinely used to study poliovirus trans-
lation-replication in vitro (3, 28). After the translation of po-
liovirus-specific RNAs we collected the membranes, with their
associated proteins, from the extract by centrifugation and
examined this material by immunoblotting with GAP-specific
antibodies. None of the GAPs tested (ArfGAP1, ARAP1,
ARAP2, ASAP1, ASAP2, AGAP1, AGAP2, and GIT1) man-
ifested significant alterations in membrane association (not
shown). These data suggest that the larger fraction of Arf1-
GFP that is stably associated with membranes in poliovirus-
infected cells (Fig. 2B) is due to limited GAP activity at new
recruitment sites. This could explain the reduced exchange
rate of Arf1-GFP on and off membranes as well as the accu-
mulation of Arf-GTP during the course of infection. We there-
fore examined the distribution of ArfGAP1 tagged with GFP
(ArfGAP1-GFP) (26) in poliovirus-infected cells. In agree-
ment with the in vitro data, no accumulation of ArfGAP1-GFP
on viral replication complexes was observed during infection
(Fig. 4). In some fields, the fluorescence in virus-infected cells
FIG. 4. ArfGAP1 in poliovirus-infected cells. HeLa cells trans-
fected with an ArfGAP1-EGFP expression plasmid for 18 h were
infected with poliovirus. Cells were fixed at 5 h postinfection and
stained with anti-poliovirus 3A antibodies to visualize the replication
FIG. 3. GTP-bound Arf in poliovirus-infected cells. HeLa cells
were mock infected or infected with poliovirus. (A) At the indicated
times postinfection, cells were lysed, and the Arf-GTP pull-down assay
was performed as described in Materials and Methods. The upper
panel shows the amount of GTP-bound Arf recovered from each cell
lysates, and the middle panel shows the total amount of Arf in each
lysates. Anti-polio-2C staining (bottom panel) was performed on the
same membrane used for Arf-GTP blot after stripping it with Chemi-
con Re-Blot Plus Mild solution. (B) Quantitation was performed by
using Total Lab 1D gel image analysis software.
562 BELOV ET AL.J. VIROL.
appeared to be more intense than in control cells, most likely
because the shape and morphology of the cells change upon
infection and the components appear more concentrated. In
mock-infected cells, ArfGAP1-GFP was associated with ER,
as previously described (26). After poliovirus infection, the
ArfGAP1-GFP became more diffusely distributed but showed
no colocalization with the viral replication complexes defined
by the perinuclear localization of viral protein 3A (Fig. 4,
Viral proteins show no GEF activity. Cyclic regeneration of
Arf from the GDP-bound to the active GTP-bound form re-
quires the activity of a GEF. This activity could be provided by
either preexisting cellular GEFs or by some viral protein(s).
We therefore sought to identify which GEF(s) may be respon-
sible for recruiting Arf. Initial experiments were performed to
test whether any poliovirus proteins contributed a unique GEF
activity for Arf activation during infection. Lysate from in-
fected cells was incubated with recombinant Arf1 and the non-
hydrolyzable GTP analog [?-35S]GTP, and the production of
radiolabeled Arf1 was measured in a standard GEF assay (35).
As positive and negative controls we used lysates from HeLa
cells transiently expressing the GEF, ARNO, or its mutated
inactive version ARNO E156K (6). Although lysate from cells
overexpressing ARNO demonstrated high levels of GEF ac-
tivity, lysate from infected cells did not show any increase in
GTP binding compared to the negative control (Fig. 5). Thus,
we found no indication of significant GEF activity provided by
any viral proteins in this assay.
Arf GEFs GBF1 and BIG1/2 are recruited to membranes by
viral proteins 3A and 3CD. We examined the possible involve-
ment of specific cellular GEFs in virus-induced membrane
reorganization associated with Arf recruitment. RNAs coding
for individual poliovirus proteins were translated in HeLa cell
extracts (Fig. 6B), and the membranous pellets were collected
by centrifugation and assayed by immunoblotting with GEF-
specific antibodies. The synthesis of two viral proteins that
were previously shown to induce translocation of Arf to the
membranes (5) also demonstrated an increased association of
specific GEFs with membranes. BIG1 and BIG2 were greatly
enriched in the membrane fraction after the translation of
RNA coding for protein 3CD (Fig. 6A). Translation of 3A
RNA, on the other hand, did not recruit BIG1/2 but resulted in
significant translocation to membranes of another GEF, GBF1
(Fig. 6A). Other GEFs tested (cytohesine 1, ARNO, and
GRP1) showed no response to the synthesis of poliovirus pro-
FIG. 5. GEF activity in poliovirus-infected cells. Cytoplasmic ex-
tracts from poliovirus-infected cells were assayed for guanine nucleo-
tide exchange activity on purified recombinant Arf1 protein in vitro as
described in Materials and Methods. GEF activity in infected cells was
compared to that in mock-infected cells or cells expressing ARNO or
an inactive, mutant protein, ARNO E156K.
FIG. 6. Recruitment of GEFs to membranes by poliovirus proteins in vitro. (A) Poliovirus proteins were synthesized in HeLa cell extracts, and
the membrane fractions were collected and assayed by immunoblotting with antibodies to BIG1, BIG2, GBF1, and GRP1. (B) Translation of each
poliovirus-specific RNA was monitored by labeling an aliquot with [35S]methionine and analyzing the translation products by SDS-PAGE.
VOL. 81, 2007 FORMATION OF POLIOVIRUS REPLICATION COMPLEXES563
teins (see, for example, GRP1 in Fig. 6A, which serves also as
a loading control).
To confirm that the recruitment of specific GEFs to mem-
branes after the synthesis of poliovirus proteins in vitro re-
flected processes that also occurred during poliovirus infection
in vivo, we monitored the translocation of GEFs in poliovirus-
infected cells. Staining of mock-infected cells with anti-BIG1
antibodies clearly showed the protein localized to the Golgi
area (Fig. 7A), whereas in infected cells it was redistributed to
the virus-induced perinuclear ring of vesicles, where the pro-
tein showed extensive colocalization with poliovirus replication
protein 2C (Fig. 7B to D). Staining of GBF1 similarly demon-
strated its relocalization to the perinuclear ring of poliovirus
replication complexes (Fig. 7E to H). This pattern is in clear
contrast to the nonspecific distribution of ArfGAP1-GFP in
infected cells (cf. Figure 4). BIG2 antibodies suitable for im-
munofluorescence analysis were not available. The data in Fig.
6 and 7 together show a highly specific translocation of two Arf
GEFs to the viral replication complex membranes and suggest
that two different viral proteins may independently induce re-
cruitment of GBF1 and BIG1/2.
GBF1 rescues poliovirus sensitivity to BFA. The specificity
of GEF recruitment by poliovirus proteins likely explains the
previously reported sensitivity of poliovirus replication to BFA
(15, 18, 27). BIG1, BIG2, and GBF1 belong to the family of
high-molecular-weight GEFs whose members are sensitive to
BFA (14). Specific amino acid residues within the Sec7 domain
of these proteins bind BFA and trap a GEF/Arf-GDP complex
in an inactive state, thus blocking nucleotide exchange. Re-
cruitment of Arf to the membranes of the increasing number
of RNA replication complexes that continue to form during
the viral growth cycle is indicated by the sensitivity of virus
production to BFA even when the drug is added in the middle
or at later times in the growth cycle (27) (our unpublished
To test the functional significance of GEF involvement in
poliovirus replication complex formation, we studied viral
RNA replication in cells ectopically expressing GBF1 and
BIG2. (Unfortunately, there is currently no reliable expression
system for BIG1.) Initial attempts to lower endogenous levels
of these GEFs with siRNAs resulted in a significant cytotoxic
effect, making it difficult to reliably attribute a reduction in
virus replication to the downregulation of a particular protein
(not shown). Overexpression of GBF1 and BIG2 has been
shown previously to rescue certain steps of intracellular mem-
brane traffic from inhibition by BFA (25, 40). We tested
whether those proteins can rescue BFA-induced inhibition of
poliovirus replication. HeLa cells were transfected with (i) a
plasmid encoding GBF1 fused to the C-terminal portion of
YFP (HA-GBF1-Yc) that was shown previously to manifest
the GEF activity of intact GBF1 (32); (ii) a BIG2 expression
plasmid, pHA-BIG2 (40); or (iii) both plasmids together. To
monitor the transfection efficiencies, 1% of a firefly luciferase
expression plasmid pGL4.13 (Promega) was included in the
transfection mix (Fig. 8B). The expression of GBF1 and BIG2
was confirmed by Western blotting (Fig. 8C). Reduced expres-
sion of both proteins in the sample transfected with both plas-
mids was likely due to promoter competition. After 18 h, the
cells were transfected with a poliovirus replicon RNA contain-
ing the Renilla luciferase gene in place of the structural pro-
tein-coding region. Renilla luciferase activity was monitored in
intact cells as a measure of viral RNA replication. Figure 8A
shows similar replicon amplification in cells expressing GBF1,
BIG2, or both GEFs together, and this level of RNA replica-
tion was generally the same as in control cells that were trans-
fected with an empty vector (not shown). In the presence of
BFA, however, GBF1 partially rescued RNA replication. It
should be noted that rescue could only occur in the subpopu-
lation of cells that had been transfected with GBF1-expressing
plasmids, which we estimate to be 30 to 50% of total; thus, the
actual rescue is significantly greater than that represented by
the measured increase in replicon replication in the total cell
FIG. 7. Relocalization of BIG1 and GBF1 in HeLa cells upon poliovirus infection. (A) Mock-infected HeLa cells stained with anti-BIG1
antibodies. (B and C) Poliovirus-infected cells stained 3 h postinfection with antibodies against BIG1 and poliovirus nonstructural protein 2C,
respectively. (D) Merged image of panels B and C. (E) Mock-infected cells stained with anti-GBF1 antibodies. (F and G) Poliovirus-infected cells
stained 3 h postinfection with antibodies to GBF1 and poliovirus nonstructural protein 3A, respectively. (H) Merged image of panels F and G.
Poliovirus proteins 2C and 3A both served as markers of viral RNA replication complexes.
564 BELOV ET AL.J. VIROL.
population. No replication occurred in the presence of BFA in
cells expressing BIG2 alone. This signal was indistinguishable
from that observed in cells transfected with an empty vector or
from a blank sample that was not transfected with the viral
replicon (not shown). The rescue of replication by GBF1 was
unaffected by simultaneous expression of BIG2. Overexpres-
sion of another GEF, ARNO, which is not BFA sensitive, had
no effect on poliovirus replication in the presence of BFA (not
shown), confirming the specificity of GBF1 action. The rescue
of poliovirus replication in the presence of BFA by ectopic
expression of GBF1 was not the result of decreasing the effec-
tive concentration of inhibitor because of binding BFA by extra
GBF1 molecules, since BIG2 binds BFA and showed no such
rescue. We also performed an additional experiment in cells
expressing a GBF1 mutant (M832L) believed to be a fully
functional GEF but unable to bind BFA (32). (Expression of
yeast GBF1 homolog Gea1p with the corresponding M699L
mutation made them resistant to BFA .) This mutant res-
cued viral RNA replication as well as the wild-type GBF1 did
(not shown), indicating that the specific effect of GBF1 was not
caused merely by the binding of BFA. Thus, it appears likely
that at least GBF1 activity is required for formation and func-
tion of viral replication complexes and that this requirement is
the cause of inhibition of virus growth by BFA.
Although it is generally accepted that poliovirus replication
requires the participation of cellular factors in various steps of
its growth cycle, very few specific proteins or their functions
have been identified. Exceptions are the cellular receptor,
CD155, for virus entry, and several proteins that stimulate
internal ribosome entry site utilization for initiation of trans-
lation. Viral RNA synthesis appears to require binding of
poly(rC) binding protein to the 5?-terminal cloverleaf structure
(20, 44) and perhaps hnRNP C binding to the 3? untranslated
region (11); however, the biochemical functions provided by
these proteins are not known. The results described in the
present study define a new class of cellular proteins (GEFs)
that are “hijacked” for the purposes of remodeling intracellu-
lar membranes to generate a membranous replication complex
scaffold and that are responsible for the sensitivity of virus
growth to BFA. Our data suggest that viral protein 3A recruits
GBF1, which in turn recruits Arf1 to induce changes in mem-
brane architecture and function, and that protein 3CD, via
BIG1/2/Arf recruitment, contributes to the formation of the
characteristic perinuclear replication complexes.
Arf proteins regulate membrane transport by interacting
with effectors that induce membrane deformation and budding
and may recruit other cellular factors required for viral RNA
replication. Poliovirus protein 3A is a transmembrane protein
(16) that blocks ER to Golgi traffic, resulting in inhibition of
cellular secretion during poliovirus infection (17, 18). While
this work was in progress, Wessels et al. (45) reported that the
3A protein of coxsackievirus B3, a member of the enterovirus
genus closely related to poliovirus, specifically binds to GBF1,
trapping it on the membrane and preventing Arf activation and
Arf1-dependent recruitment of COPI, a key player in mainte-
nance of the ER-Golgi cycle of secretory pathway traffic. They
concluded that when 3A was expressed in cells by itself, cox-
sackievirus 3A-GBF1 interaction inactivates GBF1, preventing
it from recruiting Arf1, whereas our data show increased levels
of activated Arf1 and recruitment of both GBF1 and Arf1 to
replication complexes in infected cells. This discrepancy could
be attributed to a difference in 3A behavior when expressed in
cells as a separate protein versus expressed as part of the virus
polyprotein, when other viral polypeptides can modulate its
interactions with cellular counterparts (see for example, the
dramatic change in epitope presentation of polio 3A expressed
alone and in infected cells ). However, synthesis of the 3A
protein alone in vitro caused Arf binding to membranes (5),
and this apparent contradiction with the results of Wessels
et al. is not understood.
The interference by 3A with GBF1/Arf metabolism results in
the inhibition of protein transport in cells expressing 3A pro-
tein alone or in infected cells. Although this inhibition of
protein transport may play a role in the virus’s evasion of the
host’s immune responses, it is not absolutely required for rep-
FIG. 8. Rescue of poliovirus RNA replication in the presence of
BFA by GBF1. (A) Poliovirus replicon RNA containing the Renilla
luciferase gene was introduced into HeLa cells previously transfected
with expression plasmids for GBF1, BIG2, or both together. A total of
1 ?g of BFA/ml was added where indicated. Each point is an average
value from 16 wells of a 96-well plate. (B) Firefly luciferase activity was
measured in the cells shown in panel A to evaluate transfection effi-
ciencies. (C) Immunoblot showing expression of GBF1 and BIG2 in
the transfected cells.
VOL. 81, 2007 FORMATION OF POLIOVIRUS REPLICATION COMPLEXES565
lication in cultured cells, since mutations that fail to inhibit
protein secretion are not lethal to virus replication (18). The
rescue of poliovirus replication in the presence of BFA by
overexpression of GBF1, however, suggests that there is a
requirement for some activity of GBF1 for a successful infec-
Viral protein 3CD is a proteinase that generates individual
capsid proteins from a precursor polyprotein, and also serves
as an RNA-binding protein for at least two different steps in
viral RNA synthesis (33). It has no inherent membrane-bind-
ing properties, but it interacts with another polio membrane-
binding protein, 3AB (46). The mechanism by which 3CD
induces association of BIG1 and BIG2 with membranes in the
absence of other poliovirus proteins is not obvious and re-
quires further investigation. Although overexpression of GBF1
rescued replication of poliovirus from inhibition by BFA, over-
expression of BIG2 did not show this effect. The contribution
made by each of these GEFs in the formation of poliovirus
replication complexes is not yet understood. It is possible that
these GEFs share some common functions and thus may sub-
stitute to a certain extent for one another or that GBF1 acti-
vated by 3A is the major player in supporting virus replication,
whereas BIG1 and BIG2, brought to membranes by 3CD, may
participate in other, nonessential processes. These interactions
of virus and cellular proteins and their role in virus replication
require further investigation.
The extensive remodeling of intracellular membranes that
occurs after poliovirus and other plus-strand RNA virus infec-
tions of cultured cells was observed by electron microscopy
many years ago. Despite considerable recent progress in un-
derstanding the biochemistry and cell biology of intracellular
membrane trafficking and structural transformations, very little
has been learned about the biochemical processes and mor-
phological events induced by viruses to generate the functional
and structural scaffolds that support viral RNA synthesis. Ini-
tial studies designed to characterize the events following po-
liovirus infection suggested that some products of the P2 re-
gion of the poliovirus genome (protein 2BC and/or its cleavage
products) might initiate a COPII-dependent trafficking of viral
proteins and cellular membranes to begin the remodeling re-
quired for the formation of viral RNA replication complexes
(36). Our data suggest that cellular GEFs recruited by proteins
from the P3 region of the poliovirus genome to the sites of
poliovirus replication, GBF1 by 3A and BIG1/BIG2 by 3CD, in
turn recruit and activate Arf1 (and likely other Arf species).
This diverts these proteins from their normal activity of trans-
forming the COPII-initiated membrane protrusions into early
Golgi-like compartments and further transporting material
through the secretory pathway. Instead, they participate in
poliovirus-induced membrane remodeling, resulting in the
clusters of vesicle-like replication complexes. The recruitment
of GEFs to replication sites was not accompanied by increased
binding of GAPs, thus allowing the accumulation of Arf-GTP
at the replication complexes. Another series of studies pro-
vided both morphological and biochemical evidence that au-
tophagy mechanisms may contribute to the formation of mem-
branous vesicles that support poliovirus replication (24, 39,
41). The secretory pathway conducts traffic of cargo that is
destined for insertion into the plasma membrane or secretion
from the cell; autophagy is the major self-degradative process
in eukaryotic cells, carrying material targeted for destruction in
vesicles to the lysosomes. However, since all cellular mem-
brane is generally thought to derive from the ER and since the
points of divergence from cellular pathways to the new struc-
tures that form the viral replication complexes are not known,
it may be that both of these pathways are initiated and ulti-
mately utilized to support virus propagation.
The remodeling of cellular membrane structures to sites of
viral RNA replication is complex and still far from understood.
Multiple viral gene products and, no doubt, multiple cellular
factors contribute to the various steps in this pathway. The data
presented here suggest that viral proteins 3A and 3CD recruit
Arf via the BFA-sensitive GEFs, GBF1 and BIG1/2, respec-
tively, to participate in poliovirus-induced membrane remod-
eling. These activities, either sequentially or synergistically,
contribute to the formation of the characteristic viral replica-
tion complexes. Thus, it seems that several nonstructural pro-
teins from different regions of the poliovirus genome can in-
duce interactions with components of different steps of the
cellular secretory pathway in order to transform intracellular
membranes into replication structures.
We are grateful to P. Randazzo for supplying purified recombinant
Arf and for help with the assays for GEF activity.
This study was supported in part by the intramural research pro-
grams of the National Institute of Allergy and Infectious Disease and
National Institute of Child Health and Human Development, National
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