JOURNAL OF VIROLOGY, Mar. 2011, p. 2386–2396
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 5
A PDI Family Network Acts Distinctly and Coordinately with ERp29
To Facilitate Polyomavirus Infection?
Christopher P. Walczak and Billy Tsai*
Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place,
Room 3043, Ann Arbor, Michigan 48109
Received 1 September 2010/Accepted 3 December 2010
Endoplasmic reticulum (ER)-to-cytosol membrane transport is a decisive infection step for the murine
polyomavirus (Py). We previously determined that ERp29, a protein disulfide isomerase (PDI) member,
extrudes the Py VP1 C-terminal arm to initiate ER membrane penetration. This reaction requires disruption
of Py’s disulfide bonds. Here, we found that the PDI family members ERp57, PDI, and ERp72 facilitate virus
infection. However, while all three proteins disrupt Py’s disulfide bonds in vitro, only ERp57 and PDI operate
in concert with ERp29 to unfold the VP1 C-terminal arm. An alkylated Py cannot stimulate infection, implying
a pivotal role of viral free cysteines during infection. Consistent with this, we found that although PDI and
ERp72 reduce Py, ERp57 principally isomerizes the virus in vitro, a reaction that requires viral free cysteines.
Our mutagenesis study subsequently identified VP1 C11 and C15 as important for infection, suggesting a role
for these residues during isomerization. C11 and C15 also act together to stabilize interpentamer interactions
for a subset of the virus pentamers, likely because some of these residues form interpentamer disulfide bonds.
This study reveals how a PDI family functions coordinately and distinctly to promote Py infection and
pinpoints a role of viral cysteines in this process.
Penetration of the host membrane represents a decisive step
in virus infection. For enveloped viruses that are surrounded
by a lipid bilayer, such as HIV and influenza virus, this process
requires fusion of viral and host membranes, resulting in the de
facto delivery of the viral particle across the limiting membrane
(14). In contrast, the mechanism by which nonenveloped
viruses penetrate biological membranes is less clear (29).
Despite this uncertainty, certain common principles have
emerged from studies of their penetration mechanisms.
One of these principles is that conformational changes are
imparted to the nonenveloped virus at the membrane penetration
site (29). This remodeling event may generate a hydrophobic viral
particle that binds and disrupts the limiting membrane, leading to
the subsequent transfer of a subviral particle across the mem-
brane. Alternatively, the conformational change can release an
internal virus peptide harboring intrinsic lytic activity (often
called a lytic peptide) buried inside the native virus. Disruption
of the limiting membrane by this peptide enables virus trans-
port across the membrane. In both instances, the critical trig-
ger for virus transport is the conformational change the viral
particle experiences. These structural alterations occur as a
result of the concerted actions of numerous cellular factors
acting on the virus. How the distinct functions of each contrib-
uting cellular factor are coordinated to produce the final pen-
etration-competent capsid conformation remains poorly un-
Previous studies describing the intracellular trafficking and
structure of nonenveloped viruses frame our understanding of
how these viruses cause infection. For example, to infect cells,
the nonenveloped murine polyomavirus (Py) binds to glyco-
lipid receptors called ganglioside GD1a or GT1b (5, 21, 28)
and is transported in a retrograde manner to the endoplasmic
reticulum (ER), where the virus penetrates the ER membrane
to access the cytosol (30). From the cytosol, Py is transferred
into the nucleus, with the ensuing transcription and replication
of the viral genome leading to lytic infection or cell transfor-
mation. How Py is transported across the ER membrane from
the ER lumen into the cytosol is a complicated process that
recent studies have begun to unravel (6, 10, 11, 17–19).
Structurally, Py is composed of 72 pentamers of the major
coat protein VP1, which encloses its DNA genome (9, 23, 24).
Twelve of the pentamers are surrounded by five other pentam-
ers (i.e., five coordinated), while the remaining 60 pentamers
are surrounded by six other pentamers (i.e., six coordinated)
(9). Each pentamer associates with a single copy of the minor
protein VP2 or VP3 (2).
Three major forces stabilize the architecture of the viral
capsid. First, the C terminus of VP1 invades a neighboring VP1
pentamer that stabilizes interpentamer interactions (9, 23).
Second, intrapentamer disulfide bonds between cysteine 19 of
one monomer and cysteine 114 of another monomer further
stabilize the VP1 capsid (24, 26). While the X-ray structure of
Py indicates that C273 and C282 do not form disulfide bonds,
this structure does not provide information on the nature of
the remaining C11 and C15 residues located at the VP1 N
terminus (24, 26). Third, calcium ions that bind to the virus
provide additional structural support (25). Thus, local unfold-
ing of the VP1 C-terminal arm, disulfide bond disruption, and
removal of calcium ions are reactions that destabilize Py struc-
ture, initiating the uncoating process that prepares the virus for
ER membrane penetration. The host activities responsible for
these structural disruptions, however, have not been fully de-
fined. In the case of the related simian polyomavirus simian
* Corresponding author. Mailing address: Department of Cell and
Developmental Biology, University of Michigan Medical School, 109
Zina Pitcher Place, Room 3043, Ann Arbor, MI 48109. Phone: (734)
764-4167. Fax: (734) 764-5155. E-mail: email@example.com.
?Published ahead of print on 15 December 2010.
virus 40 (SV40), ER-resident protein disulfide isomerase
(PDI) family members have been shown to disrupt the viral
disulfide bonds to facilitate infection (20).
We showed previously that ERp29, a PDI family member,
extrudes Py VP1 in a reaction that requires disruption of the
virus disulfide bonds (11). ERp29, however, does not appear to
act on SV40 (20). Here, using a combination of cell infection
studies and biochemistry, we identify a network of ER-resident
PDI proteins called ERp57, PDI, and ERp72 that act on Py’s
disulfide bonds to facilitate infection and reveal an important
role of the previously uncharacterized viral C11 and C15 res-
idues in mediating this process.
MATERIALS AND METHODS
Reagents. Crude and purified murine Py, NIH 3T3 cells, and M1 VP1 antibody
were generously provided by T. Benjamin (Harvard Medical School, Boston,
MA). Polyclonal I-58 VP1 antibody was provided by R. Garcea (University of
Colorado, Boulder, CO), the polyclonal antibody against ERp29 was a gift from
S. Mkrtchian (Karolinska Institutet, Stockholm, Sweden), the polyclonal anti-
body against ERp57 was a gift from S. High (University of Manchester,
Manchester, England), and the polyclonal antibody against Derlin-1 was a gift
from T. Rapoport (Harvard Medical School, Boston, MA). The monoclonal
antibody against BiP was purchased from BD Biosciences (San Jose, CA).
The polyclonal antibody against PDI was purchased from Santa Cruz Bio-
technology (Santa Cruz, CA). The polyclonal antibody against ERp72 was
purchased from Assay Designs (Ann Arbor, MI). Dulbecco’s modified Eagle’s
medium (DMEM), Optimem, Lipofectamine 2000, and 0.05% trypsin-EDTA
were purchased from Invitrogen (Carlsbad, CA). Fetalclone III (FC) was pur-
chased from HyClone (Logan, UT). Complete Mini EDTA-free protease inhib-
itor cocktail tablets were purchased from Roche (Indianapolis, IN). The cross-
linking reagent dithiobis succinimidylpropionate (DSP) was purchased from
Pierce Biotechnology (Rockford, IL). Reduced and oxidized glutathione, pro-
teinase K, trypsin, dithiothreitol (DTT), N-ethylmaleimide (NEM), and anti-
FLAG M2-agarose were purchased from Sigma (St. Louis, MO). Calmodulin
was purchased from Calbiochem. Micro Bio-Spin P-30 Tris chromatography
columns were purchased from Bio-Rad.
siRNA knockdown. Stealth RNA interference (RNAi) negative-control du-
plexes (low or medium GC% duplex) were purchased from Invitrogen. Duplex
small interfering RNAs (siRNAs) corresponding to a segment of mouse PDI
(siRNA 1, 5?-GCA ACA ACU UUG AGG GUG AUU-3? and 5?-UCA CCC
UCA AAG UUG UUG CUU-3?; siRNA 2, 5?-GCA ACA ACU UUG AGG
GUG AUU-3? and 5?-UCA CCC UCA AAG UUG UUG CUU-3?), mouse
ERp57 (siRNA 1, 5?-CCA GCA ACU UGA GAG AUA ATT-3? and 5?-UUA
UCU CUC AAG UUG CUG GCT-3?; siRNA 2, 5?-GCC AGC AAC UUG
AGA GAU AUU-3? and 5?-UAU CUC UCA AGU UGC UGG CUU-3?), and
mouse ERp72 (siRNA 1, 5?-GCA GUU UGC UCC AGA AUA UTT-3? and
5?-AUA UUC UGG AGC AAA CUG CTT-3?; siRNA 2, 5?-UGA CAA AGA
UAC AGU GCU AUU-3? and 5?-UAG CAC UGU AUC UUU GUC AUU-3?)
were synthesized by Invitrogen. One hundred nanomolar (ERp57 siRNA 2 and
ERp72 siRNA 2) or 200 nM (ERp57 siRNA 1, ERp72 siRNA 1, and PDI siRNA
1 and 2) duplexed siRNAs were transfected into 15 to 30% confluent NIH 3T3
cells using Lipofectamine 2000 according to the manufacturer’s protocol. Neg-
ative-control duplexes were transfected similarly at 100 nM or 200 nM concen-
trations. Protein expression was assessed by SDS-PAGE and immunoblot anal-
ysis at 72 h posttransfection. Infection assays were initiated 72 h posttransfection.
XBP1 splicing assay. Detection of XBP1 splicing was performed as described
previously (31). The forward and reverse primers used were 5? GAA CCA GGA
GTT AAG AAC ACG 3? and 5? AGG CAA CAG TGT CAG AGT CC 3?,
Infection assay. Cells treated with the indicated siRNA were plated on glass
coverslips 48 h posttransfection at a density of 3 ? 104cells/well in a 6-well plate.
At 72 h posttransfection, crude Py (100 PFU/cell) was added to the cells in fresh
DMEM plus 10% FC. The infected cells were incubated at 37°C for 24 h, washed
with phosphate-buffered saline (PBS), provided with fresh DMEM plus 10% FC,
and allowed to incubate for an additional 24 h. The cells were then fixed and
stained with a rat monoclonal antibody against murine Py large T antigen and a
rhodamine-conjugated donkey anti-rat IgG (Jackson ImmunoResearch, West
Grove, PA). Each condition depicted in Fig. 1C and F (also see Fig. 3B and 5A)
represents the average value of at least three independent experiments. In each
experiment, at least 500 cells per condition were scored for the absence or
presence of nuclear large T antigen expression using standard immunofluores-
cence microscopy as described previously (19). A Nikon epifluoresence micro-
scope (model Eclipse TS100; Nikon, Melville, NY) equipped with a Texas Red
emission filter and a 40? objective was used.
Production of recombinant PDI, ERp57, and ERp72. PDI proteins were ex-
pressed and purified as described previously (4). Full-length mouse PDI, human
ERp57, and mouse ERp72 containing N-terminal His6tags were expressed from
pQE30 (Qiagen) constructs in Escherichia coli strain BL21-pro (Clontech) for 2
to 4 h at 37°C upon induction with isopropyl thio-?-galactoside (1 mM; Invitro-
gen). Cells were lysed by incubation in buffer containing 1% Triton X-100, 300
mM potassium acetate (KOAc), 250 mM sucrose, 2 mM magnesium acetate
[Mg(OAc)2], 50 mM HEPES (pH 7.5), and protease inhibitors, followed by
sonication. The lysates were centrifuged, and the resulting supernatant fractions
were applied to a nickel nitrilotriacetic acid-agarose column (Qiagen) in the
presence of imidazole (20 mM, Sigma). The His-tagged proteins were eluted
from the column with imidazole (100, 300, or 500 mM). Eluates containing
purified proteins were dialyzed extensively overnight in PBS, frozen in liquid
nitrogen, and stored at ?80°C.
Py reduction and isomerization assay. Reaction mixtures containing the indi-
cated components were incubated for 1 h at 37°C. Each reaction mixture was
subjected to nonreducing SDS-PAGE, followed by immunoblotting with an an-
tibody against VP1. As shown in Fig. 2B to D, untreated purifed Py (100 ng) was
incubated in the presence or absence of ERp57 (8 ?M), ERp72 (6 ?M), or PDI
(5 ?M). ERp57 (heat treated) was heated for 1 h at 95°C prior to incubation with
Py. NEM-treated ERp57, ERp72, and PDI were reacted with NEM (10 mM) for
2 h at 37°C, followed by overnight dialysis against PBS to remove excess NEM.
For the reactions shown in Fig. 3C to D, the reaction mixtures were treated as for
Fig. 2B to D, except that the virus used was either mock treated or NEM treated
as indicated. Where indicated, DTT (5 mM) was added to the reaction mixtures.
Acid pretreatement of Py. Wild-type (WT) Py was pretreated under acidic
conditions (pH 5) or neutral conditions (pH 7) as described previously (16).
Alkylation of Py. Crude or purified Py was alkylated with NEM (10 mM) for
2 h at 37°C or mock treated. Excess NEM was removed efficiently with subse-
quent buffer exchange using a spin column (Bio-Rad) according to the manu-
Trypsin digestion assay. An ER lumenal extract was produced from dog
pancreatic microsomes by three freeze-thaw cycles, followed by centrifugation of
the microsomes at 50,000 ? g for 30 min to remove the membrane material. The
contents of the supernatant represented soluble proteins in the ER lumen,
referred to as an ER lumenal extract. Trypsin digestion assays were performed
similarly to those described previously (11). For experiments using crude virus,
virus was pretreated with DTT (3 mM) and EGTA (10 mM) for 20 min at 37°C,
followed by the addition of the ER lumenal extract or bovine serum albumin
(BSA) (1 mg/ml) and continued incubation for 1 h at 37°C. The reaction mixtures
were then treated with trypsin (0.25 mg/ml) for 30 min at 4°C or left untreated.
The reaction was stopped by the addition of TLCK (N?-p-tosyl-L-lysine chlo-
romethyl ketone) (1 mM) for 10 min at 4°C. Samples were analyzed by reducing
SDS-PAGE, followed by immunoblotting with a VP1 antibody.
For experiments using purified Py, an ER lumenal extract enriched for ERp29
was used as described previously (11). This extract was pretreated with DTT (1
mM) for 1 h at 37°C, followed by 3 to 6 h of dialysis against PBS to remove excess
DTT. Calmodulin was also dialyzed against PBS to remove trace amounts of
protease inhibitors. The purified Py (50 ng) was first incubated with the indicated
protein (10 ?M) and EGTA (10 mM) for 1 h at 37°C. An ER lumenal extract (1
mg/ml) was then added to the reaction mixtures and incubated for 30 min at
37°C. Trypsin (0.25 mg/ml) was added next, and the incubation proceeded for 30
min at 4°C. The reaction was stopped by the addition of TLCK (1 mM) for 10
min at 4°C. Samples were analyzed by reducing SDS-PAGE, followed by immu-
noblotting with a VP1 antibody.
Construction of N-terminally FLAG-tagged rat ERp29. The cDNA of ERp29
was amplified by PCR using a 5? primer containing the FLAG tag sequence with
pcDNA3.1(?)-rat ERp29 as a template (18). The fragment was inserted into
modified pcDNA3.1 (Invitrogen) that contained the ERp29 signal sequence.
Chemical cross-linking and coimmunoprecipitation analyses. NIH 3T3 cells
were grown to 80 to 90% confluence on a 10-cm dish before being transfected
with constructs expressing rat ERp29 or N-terminally FLAG-tagged rat ERp29
using Lipofectamine 2000 according to the manufacturer’s protocol. At 48 h
posttransfection, the cells were harvested, pelleted, and washed twice with PBS.
Where indicated, the cells were treated with the DSP cross-linker or mock
treated. DSP dissolved in dimethyl sulfoxide (DMSO) (25 mM) was diluted to a
concentration of 1 mM in 1.5 ml of PBS, which was used to resuspend the cells,
followed by incubation for 1 h at 4°C. The cells were pelleted, and the DSP was
VOL. 85, 2011A PDI FAMILY NETWORK MEDIATES POLYOMAVIRUS INFECTION2387
removed. After being washed with PBS, the cells were lysed for 30 min at 4°C in
a buffer containing 1% Triton X-100 for DSP-treated cells or 1% deoxyBigChap
(Calbiochem) for mock-treated cells. The cells were centrifuged at 16,000 ? g for
15 min, and 10% of the supernatant was taken as input. A 30-?l slurry of an
anti-FLAG M2 agarose was equilibrated, added to the remaining supernatant,
and incubated overnight at 4°C. The agarose was pelleted, and the supernatant
was removed before extensive washing. Samples were subjected to SDS-PAGE,
followed by immunoblotting with the appropriate antibody.
Mutagenesis of Py and analyses of WT and mutant viruses. The WT Py
genome (RA strain) cloned into a PBS vector was generously provided by T.
FIG. 1. ERp57, ERp72, and PDI facilitate Py infection. (A) Knockdown of PDI family members. NIH 3T3 cells were transfected with the
indicated siRNA, and lysates derived from the cells were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (B) Induction
of XBP1 splicing. Shown is RT-PCR analysis of the unspliced (u) and spliced (s) forms of the XBP1 mRNA from cells treated with DTT or
tunicamycin or transfected with the indicated siRNA. (C) Py infection in knockdown cells. Cells transfected with scrambled siRNA or one of two
independent siRNAs against ERp57, ERp72, and PDI (siRNAs 1 and 2) were challenged with Py (100 PFU/cell), and the large T antigen
expression was analyzed by standard immunofluorescence microscopy. The values were normalized to scrambled siRNA. The data represent the
means and standard deviations (SD) of at least three independent experiments. (D) As for panel A, except the indicated siRNAs were used. (E) As
for panel B, except the indicated siRNAs were used. (F) As for panel C, except the indicated siRNAs were used. (G) Average infection of single-
and double-knockdown cells. A two-tailed t test was used.
2388WALCZAK AND TSAIJ. VIROL.
Benjamin (Harvard Medical School, Boston, MA) and was used as a template for
PCR-based site-directed mutagenesis with a QuikChange II site-directed mu-
tagenesis kit from Stratagene (La Jolla, CA). The desired mutations were con-
firmed by sequencing. The viral genomes were removed from the PBS construct
by restriction digestion and religated in a dilute reaction. Purified WT or mutant
genomes were transfected into 80 to 90% confluent NIH 3T3 cells using Lipo-
fectamine 2000 according to the manufacturer’s protocol. After 24 h, the cells
were washed and provided with fresh medium containing penicillin-streptomycin
(Invitrogen). Medium containing viral particles was collected 5 to 7 days post-
transfection and used for subsequent infection and in vitro experiments. The
medium containing viral particles was subjected to reducing or nonreducing
SDS-PAGE and immunoblotting with an antibody against VP1. Infection assays
were performed essentially as described above, and cells were treated with equal
amounts of crude WT or mutant virus as determined by VP1 signal in immuno-
Proteolytic analyses of WT, alkylated, and mutant Py. Crude WT, mutant,
alkylated, or mock-treated Py was incubated for 30 min at 4°C with various
concentrations of proteinase K as indicated. Samples were subjected to reducing
SDS-PAGE, followed by immunoblotting with an antibody against VP1.
Native agarose electrophoresis of WT and mutant Py. Crude WT or mutant
virus was mixed with sample-loading buffer without reducing agent or SDS and
loaded onto a 0.4% agarose gel. Electrophoresis in 50 mM Tris-acetate (pH 8.1)
was carried out at 4°C for at least 3.5 h, with the running buffer replaced
FIG. 2. ERp57, PDI, and ERp72 disrupt Py’s disulfide bonds in vitro. (A) Expression and purification of ERp57, PDI, and ERp72. N-terminally
His-tagged ERp57, PDI, and ERp72 constructs were expressed in bacteria, purified to homogeneity, and subjected to Coomassie staining.
(B) ERp57 acts on Py directly. Where indicated, purified Py was incubated with ERp57, heat-treated ERp57, or NEM-treated ERp57. The samples
were subjected to nonreducing SDS-PAGE and immunoblotted with an I-58 VP1 antibody. The asterisk indicates a nonspecific band recognized
by the VP1 antibody. (C) As for panel B, except Py was pretreated at pH 5. (D) PDI acts on Py directly. PDI or NEM-treated PDI was incubated
with or without purified Py and analyzed as for panel B. (E) As for panel D, except Py was pretreated at pH 5. (F) ERp72 acts on Py directly.
ERp72 or NEM-treated ERp72 was incubated with or without purified Py and analyzed as for panel B. (G) As for panel F, except Py was
pretreated at pH 5.
VOL. 85, 2011 A PDI FAMILY NETWORK MEDIATES POLYOMAVIRUS INFECTION2389
ERp57, ERp72, and PDI facilitate Py infection. Using an in
vitro system, we showed previously that the PDI-like protein
ERp29 extrudes the C-terminal arm of VP1 in a remodeling
reaction that depends on the reductant DTT (11). This finding
suggests that disulfide bond reduction or isomerization of Py is
required for extrusion of the VP1 C-terminal arm and impli-
cates ER-resident reductases and/or isomerases in this reac-
tion. While stable knockdown of PDI in the heterologous hu-
man HeLa cell line blocks Py infection (6), it is not known
whether PDI or other PDI family members reduce and/or
isomerize Py directly.
Three PDI family members that are highly expressed in the
ER are ERp57, PDI, and ERp72. To test whether any of these
factors plays a role in Py infection, we downregulated each
protein in the murine fibroblast NIH 3T3 cells using two dif-
ferent siRNA oligonucleotides (i.e., siRNA oligonucleotides 1
and 2) against ERp57, PDI, and ERp72. Cell lysates derived
from cells transfected with scrambled or ERp57-, ERp72-, and
PDI-specific siRNAs were subjected to SDS-PAGE and im-
munoblotting. In cells incubated with the ERp57-specific
siRNA 1, the ERp57 level decreased without significantly af-
fecting the levels of ERp72, PDI, and ERp29 (Fig. 1A, top four
rows, compare lane 2 to lane 1). Importantly, downregula-
tion of ERp57 did not markedly cause the upregulation of
the known unfolded protein response (UPR) markers BiP and
Derlin-1 (13) (Fig. 1A, 5th and 6th rows from top, compare
lane 2 to lane 1), indicating that the absence of ERp57 did not
cause profound ER stress. ERp72 (Fig. 1A, second row from
top, lane 3) and PDI (Fig. 1A, third row from top, lane 4) were
also downregulated efficiently and specifically when cells were
treated with the corresponding siRNA 1. Again, in these cases,
BiP and Derlin-1 expression was also not affected (Fig. 1A, 5th
and 6th rows from top, compare lanes 3 and 4 to lane 1).
To further verify that knockdown of the PDI proteins
(using siRNA 1) did not induce ER stress leading to UPR
stimulation, we asked whether splicing of the XBP1 tran-
scription factor mRNA was activated and found that it was not
(Fig. 1B, compare lanes 4 to 6 to lane 1), in contrast to incu-
bating cells with the known ER stress inducers DTT and tuni-
camycin (Fig. 1B, compare lanes 2 and 3 to lane 1). These
findings demonstrate that the three PDI family proteins can be
FIG. 3. ERp57 and PDI function coordinately with ERp29 to unfold Py in vitro. (A) VP1 digestion pattern. Crude Py was incubated with DTT,
EGTA, and either BSA or an ER lumenal extract, followed by trypsin addition where indicated. The samples were subjected to SDS-PAGE,
followed by immunoblotting with a VP1 antibody. (B) Purified Py was pretreated with either calmodulin, ERp57, ERp72, or PDI in the presence
of EGTA. The samples were then incubated with an ERp29-enriched ER lumenal extract, supplemented with trypsin, subjected to SDS-PAGE,
and immunoblotted with a VP1 antibody. A 10% input for the amounts of ERp57, PDI, and the ERp29-enriched ER lumenal extract used is shown.
(C) As for panel B, except NEM-treated ERp57 and PDI were used where indicated. (D) NIH 3T3 cells transfected with either a rat ERp29 or
an N-terminally FLAG-tagged rat ERp29 construct were treated with the DSP cross-linker or left untreated. The resulting cell lysates were
subjected to immunoprecipitation using an antibody directed against the FLAG epitope conjugated to agarose (IP:FLAG). The precipitates, as
well as the cell lysates (input), were subjected to SDS-PAGE and immunoblotted with antibodies against ERp57 and ERp29. (E) As for panel D,
except antibodies against ERp72 and PDI were used instead of antibodies against ERp57.
2390 WALCZAK AND TSAIJ. VIROL.
effectively and specifically downregulated without triggering
significant detectable ER stress.
Py infection, detected by expression of the virus-encoded
large T antigen, was decreased by approximately 40% when the
PDI family proteins were knocked down using siRNA 1 (Fig.
1C). When two PDI family members were knocked down si-
multaneously in all combinations using siRNA 1 (Fig. 1D, top
three rows), ER stress was not triggered in any of the combi-
nations (Fig. 1D, 4th, 5th, and 6th rows from top, and E).
Under these double-knockdown conditions, infection was re-
duced by at least 60% in all combinations (Fig. 1F). The av-
erage infection decrease in the single- and double-knockdown
experiments is shown in Fig. 1G. Cells were unhealthy when
simultaneous downregulation of all three PDI family proteins
The PDI family proteins were downregulated efficiently
when another siRNA (i.e., siRNA 2) was transfected in NIH
3T3 cells (data not shown). Under these conditions, Py infec-
tion also decreased, similar to the results using siRNA 1 (Fig.
1C). We conclude that ERp57, PDI, and ERp72 play impor-
tant roles in mediating Py infection.
ERp57, PDI, and ERp72 disrupt Py’s disulfide bonds in
vitro. We next asked whether the three PDI family members
disrupt Py’s disulfide bonds in vitro. Recombinant N-termi-
nally His-tagged mammalian PDI, ERp57, and ERp72 were
expressed in bacteria and purified to homogeneity (Fig. 2A,
lanes 1 to 3). We first examined the ERp57 activity. Py was
incubated with or without ERp57, and the samples were sub-
jected to nonreducing SDS-PAGE, followed by immunoblot-
ting with an antibody against VP1. In the absence of ERp57,
immunoblotting for VP1 did not detect any bands (Fig. 2B,
lane 1), indicating that the native virus was stable and unable
to migrate into the gel. In contrast, band patterns approxi-
mately corresponding to the sizes of the VP1 monomer (42
kDa), trimer (126 kDa), pentamer (210 kDa), and higher
oligomers were observed when Py was incubated with ERp57
(Fig. 2B, compare lane 2 to lane 1). No signals were detected
when ERp57 was incubated in the absence of virus (Fig. 2B,
lane 5), demonstrating that the bands observed in the presence
of virus were virus-derived products.
When ERp57 was heat treated prior to incubation with the
virus, the various Py-derived species were not seen (Fig. 2B,
lane 3). Moreover, when Py was incubated with ERp57 pre-
treated with the alkylating reagent NEM to block its free cys-
teines, no virus-derived products were generated (Fig. 2B, lane
4). We conclude that ERp57 uses its catalytic cysteines to
disrupt Py’s disulfide bonds directly to generate the virus-de-
rived species. As Py is transported to the low-pH endolysosome
system before reaching the ER (16), we asked whether pre-
treatment of Py at low pH (i.e., pH 5) affects ERp57’s ability to
generate the VP1-derived products and found that it did not
(Fig. 2C, compare lane 2 to lane 1).
The VP1-derived products were also generated when PDI,
but not NEM-treated PDI (Fig. 2D, compare lane 1 to lane 2),
was incubated with (but not without) Py. PDI also acted on
low-pH-treated virus with efficiency similar to that of the con-
trol virus (Fig. 2E, compare lane 1 to lane 2). Finally, the
VP1-derived products were produced when ERp72, but not
NEM-treated ERp72 (Fig. 2F, compare lane 1 to lane 2), was
incubated with (but not without) Py. Again, pretreating Py at
low pH did not significantly prevent ERp72 from acting on the
virus (Fig. 2G, compare lane 1 to lane 2). Thus, similar to
ERp57, PDI and ERp72 also use their cysteines to produce the
virus-derived products. When Py and the PDI family proteins
were incubated in a 1:1 molar concentration of oxidized glu-
tathione (GSSG) and reduced glutathione (GSH) that mim-
icked the ER redox condition (20), the VP1-derived products
were generated (data not shown). We conclude that the PDI
family proteins can disrupt Py’s disulfide bonds even under the
relatively oxidizing ER conditions.
ERp57 and PDI function coordinately with ERp29 to unfold
Py in vitro. Our previous in vitro system demonstrated that
ERp29 extrudes the VP1 C-terminal arm in a reaction that
requires disruption of Py’s disulfide bonds (11). The in vitro
assay assumes that an ER activity imparts a conformational
change to Py to expose a hidden proteolytic cleavage site bur-
ied in the native virus (11). Incubation of Py with an extract
containing ER lumenal proteins (referred to as an ER lumenal
extract) enables a general protease (such as trypsin) to digest
the viral capsid proteins, which can be detected when the
sample is subjected to SDS-PAGE, followed by immunoblot-
We found that when Py was incubated with a control
protein (BSA) in the presence of DTT and EGTA (to re-
move the virus-bound calcium), followed by trypsin addi-
tion, a VP1-derived fragment we previously termed VP1a was
generated (Fig. 3A, compare lane 2 to lane 1), similar to our
previous observation (11). However, when Py was incubated
with an ER lumenal extract (instead of BSA), DTT, and
EGTA, followed by trypsin addition, two VP1-derived tryptic
digestion products, VP1a and VP1b, appeared (Fig. 3A, com-
pare lane 4 to lane 3). Formation of VP1b (but not VP1a)
requires the presence of an ER lumenal extract, EGTA, and
DTT (11, 17, 18). Using mass spectrometry analysis, we
previously identified VP1b as a fragment that lacks the C
terminus of VP1, indicating that ER factors unfold VP1 to
expose its C terminus (11). Again, using mass spectrometry,
the ER factor required for VP1b generation was identified as
Can ERp57, PDI, or ERp72 functionally replace DTT? Py
was pretreated with either the cysteine-less calmodulin,
ERp57, ERp72, or PDI in the presence of EGTA. The samples
were then incubated with an ER lumenal extract enriched for
ERp29 (18), followed by trypsin addition. Our results show
that VP1b was generated only when Py was pretreated with
either ERp57 or PDI, but not with calmodulin or ERp72 (Fig.
3B, compare lanes 2 and 4 to lanes 1 and 3). The ERp29-
enriched lumenal extract lacks ERp57 and PDI (Fig. 3B, com-
pare lane 6 to lane 5), thus explaining the requirement for
these proteins in the reaction. NEM-treated ERp57 and PDI
cannot assist the lumenal extract in generating VP1b (Fig.
3C, compare lane 2 to lane 1 and lane 4 to lane 3). Thus, the
catalytic activities of ERp57 and PDI replace DTT to produce
VP1b. We conclude that ERp57 and PDI act coordinately with
ERp29 to extrude the VP1 C-terminal arm. We note that the
lumenal extract used in this reaction was pretreated with DTT,
and the excess DTT in the extract was removed thoroughly by
In cells transfected with N-terminally FLAG-tagged ERp29,
immunoprecipitation of FLAG-ERp29 coprecipitated endog-
VOL. 85, 2011A PDI FAMILY NETWORK MEDIATES POLYOMAVIRUS INFECTION2391
enous ERp57 following treatment of cells with the cross-linker
DSP (Fig. 3D, top row, compare lane 3 to lane 2). Similar
results were found when the FLAG-ERp29 precipitate was
blotted for ERp72 (Fig. 3E, top, compare lane 3 to lane 2) or
PDI (Fig. 3E, second row from top, compare lane 3 to lane 2).
Thus, overexpressed ERp29 binds to endogenous ERp57,
ERp72, and PDI, suggesting that they may function as a net-
ERp57 principally isomerizes Py, while PDI and ERp72
reduce the virus in vitro. We next asked whether free cysteines
in Py are necessary for infection. To this end, Py was incubated
initially with NEM to alkylate these residues, and then the
sample was subjected to a spin column to remove the excess
NEM. We found that the infection efficiency of the NEM-
treated Py was severely attenuated compared to WT Py (Fig.
4A). Limited proteolysis experiments showed that the NEM-
treated Py exhibited sensitivity to proteinase K similar to that
of nonalkylated WT Py (Fig. 4B, compare lanes 5 to 8 to lanes
1 to 4), suggesting that NEM did not grossly disrupt the overall
structural integrity of the virus. We conclude that free cysteines
in Py are critical for infection.
In disulfide bond isomerization, a free cysteine in the sub-
strate is required to resolve the transient disulfide bond formed
between the enzyme and substrate that generates a new disul-
fide bond in the substrate. In disulfide bond reduction, a free
cysteine in the enzyme is used to resolve this transient disulfide
bond. The finding that free cysteines of Py are pivotal for
infection suggests that they may be involved in isomerization
To test whether free cysteines in Py are required for ERp57
to generate the VP1 species from intact virus, WT and NEM-
treated viruses were incubated with ERp57. We found that
generation of the VP1 higher oligomer, pentamer, trimer, and,
to a lesser extent, monomer was diminished markedly when
ERp57 was incubated with the NEM-treated Py compared to
WT Py (Fig. 4C, compare lane 4 to lane 2). DTT treatment of
WT and NEM-treated viruses produced similar VP1 monomer
levels (Fig. 4C, compare lane 5 to lane 6), demonstrating that
FIG. 4. ERp57 principally isomerizes Py, while PDI and ERp72 reduce the virus in vitro. (A) Free cysteines in Py are required for infection.
NIH 3T3 cells were incubated with either WT or NEM-treated Py (100 PFU/cell), and the infection efficiency was analyzed as for Fig. 1C.
(B) NEM-treated virus is not grossly misfolded. WT and NEM-treated Py were incubated with the indicated concentrations of proteinase K, and
the samples were subjected to SDS-PAGE and immunoblotted with a VP1 antibody. (C) ERp57 largely isomerizes Py. WT or NEM-treated Py
was incubated with ERp57 or DTT (where indicated), and the samples were analyzed by nonreducing SDS-PAGE, followed by immunoblotting
with a VP1 antibody. (D) PDI reduces Py directly. As for panel C, except PDI was used instead of ERp57. (E) ERp72 reduces Py directly. As for
panel C, except ERp72 was used instead of ERp57. The asterisk indicates a nonspecific band recognized by the M1 VP1 antibody.
2392 WALCZAK AND TSAI J. VIROL.
the same amount of WT and NEM-treated virus was used and
that both virus types display the same sensitivity to this general
reductant. We conclude that free cysteines in Py are necessary
for ERp57 to generate the smaller virus-derived products. The
results shown in Fig. 4 suggest that ERp57 acts principally as
an isomerase in producing the virus-derived VP1 species.
In contrast to ERp57, PDI (Fig. 4D, compare lane 4 to 2)
and ERp72 (Fig. 4E, compare lane 4 to lane 2) generated the
Py-derived species potently regardless of whether they were
incubated with WT or NEM-treated virus. The prominent 72-
kDa band that appeared in Fig. 4E (lanes 2 and 4) was also
present in a sample without Py (data not shown), indicating
that the VP1 antibody used in this immunoblot (i.e., M1)
cross-reacts with ERp72. As free cysteines in the Py were not
necessary for the PDI- or ERp72-dependent reactions, these
data demonstrate that PDI and ERp72 function here as reduc-
tases. We note that ERp57 also functions as an isomerase on
Characterization of the C11A, C15A, and C11A-C15A Py
mutants. The finding that Py’s free cysteines and ERp57 may
engage in an isomerization reaction to disrupt the virus disul-
fide bonds prompted us to examine the nature of the previously
uncharacterized C11 and C15 residues. Two single mutants
were generated, C11 mutated to A [Py (C11A)] and C15 mu-
tated to A [Py (C15A)], and one double mutant was generated,
C11 and C15 mutated to A [Py (C11A-C15A)]. We found that
when similar levels of WT and mutant viruses were used (Fig.
5A, lanes 1 to 4), infection induced by all three mutants was
FIG. 5. Characterization of the C11A, C15A, and C11A-C15A Py mutants. (A) VP1 C11 and C15 are important for Py infection. NIH 3T3 cells
were incubated with similar levels of crude WT Py, Py (C11A), Py (C15A), and Py (C11A-C15A), and the infection efficiency was analyzed as for
Fig. 1C. (B) Mutant viruses are not misfolded globally. The viruses in panel A were subjected to limited proteolysis as for Fig. 4B. (C) Native
agarose gel analyses of mutant Py. The viruses in panel A were subjected to 0.4% native agarose gel electrophoresis, transferred to a nitrocellulose
membrane, and immunoblotted with an antibody against VP1. (D) VP1 C11 and C15 mediate interpentamer interaction for a subset of the
pentamers. The viruses in panel A were subjected to nonreducing and reducing SDS-PAGE, followed by immunoblotting with an antibody
VOL. 85, 2011A PDI FAMILY NETWORK MEDIATES POLYOMAVIRUS INFECTION2393
decreased by approximately 50% compared to that induced by
WT Py (Fig. 5A, right). Limited proteolysis demonstrated that
all three mutant viruses displayed protease sensitivity patterns
similar to that of WT Py (Fig. 5B, compare lanes 5 to 8, 9 to 12,
and 13 to 16 to lanes 1 to 4). Moreover, these mutant viruses
migrated similarly to WT Py, as analyzed by a native agarose
gel system (Fig. 5C, compare lanes 2 to 4 to lane 1). These
findings demonstrate that the decrease in infection caused by
the mutant viruses is not due to global disruption of their
structural integrity. Instead, the results indicate a specific role
of C11 and C15 in infection, consistent with a requirement for
viral free cysteines during ERp57-mediated isomerization.
Interestingly, when the WT and mutant Pys were subjected
to nonreducing SDS-PAGE, a species corresponding to a VP1
higher oligomer was detected only in the Py (C11A-C15A)
double mutant (Fig. 5D, compare lane 4 to lanes 1 to 3). A
similar VP1 level appeared when the viruses were subjected to
reducing SDS-PAGE (Fig. 5D, lanes 5 to 8). These findings
indicate that C11 and C15 stabilize interpentamer interactions
for at least a subset of the pentamers. This observation raises
the possibility that while some C11 and C15 residues within a
Py particle are in the free reduced state, other C11 and C15
residues within the same particle likely form interpentamer
disulfide bonds between some of the pentamers.
Nonenveloped virus penetration of the limiting membrane
represents a decisive step in virus infection. Host factors facil-
itate this process by imparting conformational changes to the
virus to initiate membrane penetration. Although multiple host
factors likely work together to mediate this process, until now,
only individual cellular components responsible for these re-
modeling events have been identified; more complex mecha-
nisms remain undefined. In the present study, we pinpoint a
network of ER-resident proteins that act on Py distinctly and
coordinately to facilitate infection. They do so by modifying
the viral disulfide bonds to promote its ER membrane pen-
etration. We also identify specific viral cysteines that play
pivotal roles in this process.
Using the siRNA knockdown strategy, we demonstrate
that individually downregulating ERp57, ERp72, or PDI
decreased infection, while simultaneously downregulating
any two of these proteins attenuated infection more severely.
The fact that infection was not blocked completely is likely due
to incomplete downregulation of the PDI family proteins. Ad-
ditionally, compensation by other PDI family members may
contribute to this effect. Under none of the knockdown con-
ditions did we observe induction of significant ER stress, sug-
gesting that the decrease in virus infection is not due to non-
specific effects. Instead, these results point to the possibility
that the PDI family members engage Py directly. Although
knockdown of PDI in a human cell line was shown previously
to block Py infection (6), whether PDI interacts with Py di-
rectly is not known.
For SV40, downregulation of ERp57 and PDI attenuated
infection (20), consistent with our observation for Py. How-
ever, different results using two independent siRNAs to down-
regulate ERp72 were observed: one enhanced infection, while
the other did not have an effect (20). For Py, downregulating
ERp72 using two different siRNAs decreased infection. The
reason for this discrepancy is not clear, but it may be due to the
different disulfide bond arrangements in the two viruses. In
addition, the PDI-like protein ERp29 does not appear to act
on SV40 (20), in contrast to its established role in Py infection
(11). As ERp29 extrudes Py’s VP1 C-terminal arm to initiate
viral disassembly (11), the precise disassembly mechanisms in
Py and SV40 may be different.
To test the hypothesis that ERp57, PDI, and ERp72 affect
Py directly, we expressed and purified the three proteins to
homogeneity. When incubated with Py, these proteins individ-
ually induced the formation of virus-derived products consist-
ing of VP1 monomer, trimer, pentamer, and higher oligomers.
VP1 monomer is likely generated when the five disulfide bonds
within a pentamer (formed between C19 of one monomer and
C114 of another monomer) are disrupted. Interestingly, a dou-
ble band pattern corresponding to the size of the monomer was
observed. This pattern could be explained by the formation
of a new intramonomer disulfide bond within a subset of the
monomers, causing a different mobility pattern. This doublet
pattern is more obvious for the ERp57- and ERp72-mediated
reactions than the PDI-triggered reaction. Precisely how this
new intramonomer disulfide bond is formed is unknown. One
possibility is that a free cysteine in VP1 of the native Py is used
to form a new disulfide bond. Alternatively, a new free cysteine
generated in VP1 after reduction may possibly catalyze
intramonomer disulfide bond formation. Why PDI-mediated re-
duction of Py did not generate the doublet pattern as efficiently
as the ERp72-dependent reaction is not clear.
How is a trimer, but not a dimer or a tetramer, generated?
In principle, the trimers could be formed from three monomers
within a pentamer that are disulfide bonded to each other. Al-
ternatively, given our findings that suggest the presence of
interpentamer disulfide bonds, the trimers could be formed
from two monomers from a pentamer (linked to each other by
a disulfide bond), with one of the monomers forming an inter-
pentamer disulfide bond with a monomer from an adjacent
During disassembly by the PDI family proteins, transient
viral intermediates may form that expose certain disulfide
bonds while rendering others less exposed. This creates a sit-
uation in which certain disulfide bonds are preferentially dis-
rupted over others. Indeed, the intrinsic asymmetric property
of Py—with 12 pentamers surrounded by 5 pentamers and 60
pentamers surrounded by 6 pentamers—favors the presence of
nonequivalent disulfide bonds during viral disassembly. Al-
though it is not entirely clear why trimers, but not tetramers or
dimers, were generated in our experiments, this finding sug-
gests that disulfide bonds within a trimer are likely less exposed
than a tetramer or dimer during the course of disassembly,
thereby rendering the disulfide bonds linking the trimers less
easy to disrupt.
How are VP1 pentamers and higher oligomers liberated
from the intact virus? One possibility is that, as the C19-C114
disulfide bonds within a pentamer clamp the invading VP1
C-terminal arm in place (24), disruption of this bond loosens
the C-terminal arm, thereby generating VP1 pentamers and
higher oligomers. Alternatively, interpentamer disulfide bonds
may be disrupted to produce these species. The X-ray structure
of Py, which did not indicate the presence of interpentamer
2394 WALCZAK AND TSAIJ. VIROL.
disulfide bonds (24), did not include information on the VP1
N-terminal C11 and C15 residues. Our present analyses sug-
gest that these residues stabilize interpentamer interactions for
a subset of the pentamers, possibly by forming interpentamer
disulfide bonds (see below).
Despite the finding that ERp57, PDI, and ERp72 can
individually act on Py to form the virus-derived species, only
ERp57 and PDI cooperate with ERp29 to extrude the VP1
C-terminal arm. We showed previously that this reaction gen-
erates a hydrophobic viral particle that binds and disrupts ER
membrane integrity (11, 17), events that initiate virus penetra-
tion across the ER membrane. Although PDI can use its chap-
erone activity to unfold cholera toxin (CT) in the ER to initiate
the toxin’s translocation into the cytosol (3, 4, 27), the catalytic,
but not chaperone, activity of PDI (and ERp57) is required to
assist ERp29 in unfolding the VP1 C-terminal arm. As there
are two pentamer types in Py (12 five coordinated and 60 six
coordinated) (9), it is possible that the ERp57-PDI-ERp29
network acts on one of these two types, while ERp72 engages
Our next key finding indicated that free viral cysteines play
an important role in Py infection, as an alkylated virus cannot
promote infection. Consistent with this observation, we found
that while PDI and ERp72 function as reductases in engaging
Py in vitro, ERp57 acts as an isomerase, a reaction that requires
free viral cysteines. We have yet to pinpoint the precise mech-
anism by which ERp57, PDI, and ERp72 act on Py in cells,
because less than 5% of the total internalized Py reaches the
ER from the cell surface (16). However, the biochemical anal-
yses, coupled with the infection result of the alkylated virus,
point to a role of isomerization facilitated by viral cysteines as
a potential critical reaction during infection. This idea is sup-
ported by our identification of a role for VP1’s C11 and C15
residues in infection, as mutating C11 to A, C15 to A, or both
residues to A decreased infection by 50%. These mutations did
not significantly affect the global viral conformations, suggest-
ing a specific role of these cysteines in infection. One interpre-
tation of these data is that some of the C11 and C15 residues
exist in the free and reduced state, enabling them to participate
Strikingly, using a nonreducing SDS-PAGE system, VP1
higher oligomers were generated when both C11 and C15 were
mutated to A, but not when only a single cysteine was altered.
This finding demonstrates that C11 and C15 stabilize interpen-
tamer interactions for a subset of the pentamers, possibly by
forming interpentamer disulfide bonds between some of the
pentamers. If these residues stabilized all interpentameric in-
teractions, a VP1 pentamer, but not a higher oligomer, would
appear. Moreover, because mutating a single cysteine did not
generate the higher oligomers, the potential interpentamer
disulfide bonds likely exist between two C11 residues and two
C15 residues. Py’s C11 is the homolog of SV40 C9, which is
implicated in C9-C9 interpentamer disulfide bonding (20). Be-
cause Py contains two types of pentamers, certain pentamers
will be situated in a different local environment than others.
This structural asymmetry likely allows some C11 and C15
residues to be disulfide bonded while others are in the reduced
state. This situation is similar to that of C104 in SV40’s VP1,
where it is thought to exist in both the reduced and disulfide-
bonded states (20).
While we cannot formally rule out the possibility that C11
and C15 are used to stabilize interpentamer contacts without
forming interpentamer disulfide bonds, we consider this pos-
sibility unlikely based on our data provided using NEM-Py.
Specifically, alkylation of these cysteine residues with NEM
would presumably disrupt their ability to make non-disul-
fide-bond-mediated stabilizing contacts, thereby generating
higher-order VP1 species. However, on nonreducing SDS-
PAGE, we did not observe higher-order VP1 species con-
taining only NEM-Py.
The double Py mutant (C11A-C15A) did not display a more
pronounced decrease in infection than the single cysteine mu-
tants. This could be because C11 and C15 are normally used to
isomerize Py to generate the VP1 higher-oligomer intermedi-
ate that is critical for successful infection. Preformation of this
intermediate in the double mutant thus renders these cysteine
residues dispensable. However, the fact that the double mutant
was nonetheless defective in infection indicates that this mu-
tant harbors subtle structural alterations that do affect infec-
tion. The precise nature of this structural defect is not known,
but it may be due to premature disassembly in the endolyso-
somes prior to arrival at the ER.
Although we have identified three additional PDI family
members in the ER that engage and facilitate Py infection,
whether additional ER components act on Py is not known. In
this context, it is interesting that in the in vitro trypsin digestion
assay, ERp57 or PDI added in combination with a reduced
ERp29-enriched ER lumenal extract (which lacks ERp57 and
PDI) generated VP1b. The fact that reducing the ERp29-
enriched lumenal extract was required in this reaction suggests
that ERp29 in the extract must be in the reduced form to
unfold Py (ERp29 contains one cysteine). Indeed, there are
precedents for the redox state of a PDI protein controlling its
chaperone-unfolding activity (27). Alternatively, additional re-
ductases/isomerases in the extract may be involved in trigger-
ing a penetration-competent viral particle. Further experi-
ments are required to distinguish these possibilities.
In conclusion, our findings unveil a complex interplay be-
tween viral cysteine residues and host reductases, isomerases,
and chaperones of the PDI family that act coordinately and
distinctly on Py to facilitate infection. These reactions promote
transport of the virus from the ER into the cytosol, a pivotal
infection step. PDI family members not only engage endoge-
nous cellular substrates (7, 15, 32), they are often coopted by
toxins and viruses during infection (3, 8, 11, 12, 20, 22, 27). The
vast number of members within this family (1), coupled with
their functional versatility, thus renders them attractive targets
for pathogens during entry.
We thank Emily Rainey-Barger for critical review of the manuscript.
B.T. is a Burroughs Wellcome Fund Investigator in Pathogenesis of
Infectious Disease and is supported by the NIH (RO1-AI064296).
C.P.W. is funded partially by the Cellular and Molecular Biology
program at the University of Michigan.
1. Appenzeller-Herzog, C., and L. Ellgaard. 2008. The human PDI family:
versatility packed into a single fold. Biochim. Biophys. Acta 1783:535–548.
2. Chen, X. S., T. Stehle, and S. C. Harrison. 1998. Interaction of polyomavirus
internal protein VP2 with the major capsid protein VP1 and implications for
participation of VP2 in viral entry. EMBO J. 17:3233–3240.
VOL. 85, 2011A PDI FAMILY NETWORK MEDIATES POLYOMAVIRUS INFECTION2395
3. Forster, M. L., et al. 2006. Protein disulfide isomerase-like proteins play
opposing roles during retrotranslocation. J. Cell Biol. 173:853–859.
4. Forster, M. L., J. J. Mahn, and B. Tsai. 2009. Generating an unfoldase from
thioredoxin-like domains. J. Biol. Chem. 284:13045–13056.
5. Gilbert, J., and T. Benjamin. 2004. Uptake pathway of polyomavirus via
ganglioside GD1a. J. Virol. 78:12259–12267.
6. Gilbert, J. M., W. Ou, J. Silver, and T. Benjamin. 2006. Downregulation of
protein disulfide isomerase inhibits infection by the mouse polyomavirus.
J. Virol. 80:10868–10870.
7. Kang, K., B. Park, C. Oh, K. Cho, and K. Ahn. 2009. A role for protein
disulfide isomerase in the early folding and assembly of MHC class I mole-
cules. Antioxid. Redox Signal. 11:2553–2561.
8. Lee, S. O., et al. 2010. Protein disulphide isomerase is required for signal
peptide peptidase-mediated protein degradation. EMBO J. 29:363–375.
9. Liddington, R. C., et al. 1991. Structure of simian virus 40 at 3.8-A resolu-
tion. Nature 354:278–284.
10. Lilley, B. N., J. M. Gilbert, H. L. Ploegh, and T. L. Benjamin. 2006. Murine
polyomavirus requires the endoplasmic reticulum protein Derlin-2 to initiate
infection. J. Virol. 80:8739–8744.
11. Magnuson, B., et al. 2005. ERp29 triggers a conformational change in poly-
omavirus to stimulate membrane binding. Mol. Cell 28:289–300.
12. McKee, M. L., and D. J. FitzGerald. 1999. Reduction of furin-nicked
Pseudomonas exotoxin A: an unfolding story. Biochemistry 38:16507–16513.
13. Oda, Y., et al. 2006. Derlin-2 and Derlin-3 are regulated by the mammalian
unfolded protein response and are required for ER-associated degradation.
J. Cell Biol. 172:383–393.
14. Poranen, M. M., R. Daugelavicius, and D. H. Bamford. 2002. Common
principles in viral entry. Annu. Rev. Microbiol. 56:521–538.
15. Puig, A., and H. F. Gilbert. 1994. Protein disulfide isomerase exhibits chap-
erone and anti-chaperone activity in the oxidative refolding of lysozyme.
J. Biol. Chem. 269:7764–7771.
16. Qian, M., D. Cai, K. J. Verhey, and B. Tsai. 2009. A lipid receptor sorts
polyomavirus from the endolysosome to the endoplasmic reticulum to cause
infection. PloS Pathog. 5:e1000465.
17. Rainey-Barger, E. K., B. Magnuson, and B. Tsai. 2007. A chaperone-acti-
vated nonenveloped virus perforates the physiologically relevant endoplas-
mic reticulum membrane. J. Virol. 81:12996–13004.
18. Rainey-Barger, E. K., S. Mkrtchian, and B. Tsai. 2007. Dimerization of
ERp29, a PDI-like protein, is essential for its diverse functions. Mol. Biol.
19. Rainey-Barger, E. K., S. Mkrtchian, and B. Tsai. 2009. The C-terminal
domain of ERp29 mediates polyomavirus binding, unfolding, and infection.
J. Virol. 83:1483–1491.
20. Schelhaas, M., et al. 2007. Simian virus 40 depends on ER protein folding
and quality control factors for entry into host cells. Cell 131:516–529.
21. Smith, A. E., H. Lilie, and A. Helenius. 2003. Ganglioside-dependent cell
attachment and endocytosis of murine polyomavirus-like particles. FEBS
22. Spooner, R. A., et al. 2004. Protein disulphide-isomerase reduces ricin to its
A and B chains in the endoplasmic reticulum. Biochem. J. 383:285–293.
23. Stehle, T., Y. Yan, T. L. Benjamin, and S. C. Harrison. 1994. Structure of
murine polyomavirus complexed with an oligosaccharide receptor fragment.
24. Stehle, T., and S. C. Harrison. 1996. Crystal structures of murine polyoma-
virus in complex with straight-chain and branched-chain sialyloligosaccha-
ride receptor fragments. Structure 4:183–194.
25. Stehle, T., S. J. Gamblin, Y. Yan, and S. C. Harrison. 1996. The structure of
simian virus 40 refined at 3.1 A resolution. Structure 4:165–182.
26. Stehle, T., and S. C. Harrison. 1997. High-resolution structure of a poly-
omavirus VP1-oligosacccharide complex: implications for assembly and re-
ceptor binding. EMBO J. 16:5139–5148.
27. Tsai, B., C. Rodighiero, W. I. Lencer, and T. A. Rapoport. 2001. Protein
disulfide isomerase acts as a redox-dependent chaperone to unfold cholera
toxin. Cell 104:937–948.
28. Tsai, B., et al. 2003. Gangliosides are receptors for murine polyoma virus and
SV40. EMBO J. 22:4346–4355.
29. Tsai, B. 2007. Penetration of nonenveloped viruses into the cytoplasm.
Annu. Rev. Cell Dev. Biol. 23:23–43.
30. Tsai, B., and M. Qian. 2010. Cellular entry of polyoma viruses. Curr. Top.
Microbiol. Immunol. 343:177–194.
31. Uemura, A., M. Oku, K. Mori, and H. Yoshida. 2009. Unconventional splic-
ing of XBP1 mRNA occurs in the cytoplasm during the mammalian unfolded
protein response. J. Cell Sci. 122:2877–2886.
32. Wang, C. C., and C. L. Tsou. 1993. Protein disulfide isomerase is both an
enzyme and a chaperone. FASEB J. 7:1515–1517.
2396WALCZAK AND TSAIJ. VIROL.