Previous Article | Next Article 
J Virol, February 1998, p. 1297-1307, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Protein Linkage Map of the P2 Nonstructural
Proteins of Poliovirus
Andrea
Cuconati,
Wenkai
Xiang,
Frederick
Lahser,
Thomas
Pfister, and
Eckard
Wimmer*
Department of Molecular Genetics and
Microbiology, School of Medicine, State University of New York at
Stony Brook, Stony Brook, New York 11794
Received 2 July 1997/Accepted 5 November 1997
 |
ABSTRACT |
The yeast two-hybrid system was used to catalog all detectable
interactions among the P2 nonstructural cleavage products of poliovirus
type 1 (Mahoney). Evidence has been obtained for specific associations
among 2Apro, 2BC, 2C, and 2B. Specifically,
2Apro can interact with itself and 2BC and its cleavage
products (2B and 2C) interact in all possible combinations, with the
exception of 2C/2C. Detected interactions were confirmed in vitro by a
glutathione S-transferase pulldown assay, which allowed us
to detect 2C/2C association. trans-dominant-negative
mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol.
65:4341-4349, 1991) were examined and were found to retain interaction
with wild-type 2B, perhaps reflecting a need for 2B multimerization in
viral RNA replication. The multimerization of 2B was examined further
by screening a mutagenized library for 2B variants that have lost the
ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as
truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome
of the virus conferred a quasi-infectious phenotype, an observation
strongly suggesting that the 2B/2B interaction is required for
replication of the viral genome.
| |
INTRODUCTION |
The reproduction of a positive-sense
RNA genome presents special problems for viruses in that the cell does
not contain an RNA replication mechanism that can be subverted during
the viral life cycle. Unlike DNA viruses, the RNA viruses must
establish an RNA replication pathway under the limitations of an
error-prone RNA replication process, giving rise to the necessity for
small genomes (59). One answer to these constraints is the
evolution of minimized open reading frames specifying polyproteins
yielding multifunctional protein products. The well-studied
positive-sense RNA animal viruses, the Picornaviridae,
encode a single polyprotein that is proteolytically processed to yield
the final cleavage products plus several cleavage intermediates.
Interestingly, some of these intermediates have functions distinct from
those of their products (27, 43, 59). In this manner,
minimal coding capacity is maximized. However, the intertwining of
different functions for the same polypeptide has rendered study of the
contribution of a polypeptide chain to replication difficult.
Polyprotein cleavage and the replication cycle have been thoroughly
studied for poliovirus, a prototype of Picornaviridae (59). Polioviral RNA replication occurs in association with membranous vesicles which are a prominent feature of the productively infected cell (10, 13). Biochemical and microscopic studies have demonstrated the association of the vesicles with the cleavage products of the P2 precursor, all of which have been shown to be
required for the replication process (9, 11, 52, 59). Indeed, both structural and nonstructural viral proteins can be coisolated in the membranous environment of the replication complex, indicating the possible formation of multimolecular complexes (18). P2 itself maps to the central region of the
polyprotein (Fig. 1A) and is processed to
yield three different end products (2Apro, 2B, and 2C) and
one long-lived precursor (2BC) (27, 59). 2Apro
is a cysteine proteinase with a catalytic triad reminiscent of those of
serine proteases (28). The enzyme catalyzes tyrosine-glycine cleavage at the junction of the P1 and P2 precursors (54).
2Apro is also involved in the shutoff of host cell
translation (51), has an undefined role in the initiation of
cap-independent translation of the viral genome (26), and
may also function directly in the replication of the genome (38,
41, 62). The functions and characteristics of 2BC and its
cleavage products, 2B and 2C, are less well defined. 2C contains a
functional nucleoside triphosphate-binding and hydrolysis domain
(39, 46) which is reminiscent of a helicase motif
(24) and which is required for RNA replication (40, 52a). 2C also binds nucleic acids (45) and contains a
zinc finger motif whose integrity is essential for RNA synthesis
(44). Exogenous expression of 2B in mammalian cells has been
associated with a strong block in secretory transport, permeabilization
of the plasma membrane (1, 17, 56, 58), and dissassembly of
the Golgi apparatus (47). Similar studies of 2BC and 2C
reveal the accumulation of vesicles reminiscent of those seen in
infected cells in the case of 2BC (4, 15) and
less-recognizable membrane rearrangements (invaginations of the
endoplasmic reticulum) by 2C (15). Whether vesicle induction
is absolutely required for efficient replication remains an unanswered
question, as is the precise function of 2BC and its cleavage products.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 1.
Gene organization and polyprotein processing of
poliovirus. (A) Structure of the genomic RNA of poliovirus and its
coding region. The 5' terminus is covalently linked to the VPg peptide
(3B). The highly structured 5' nontranslated region (NTR) regulates
translation and positive-sense RNA synthesis, while the 3' NTR
regulates negative-sense RNA synthesis. The product of the single open
reading frame, the polyprotein, is divided into three regions, as
shown. Proteolytic processing of the P2 nonstructural precursor yields
2Apro, 2BC, 2B, and 2C. (B) Representation of the 2B
polypeptide and the map positions of 2B mutants. LB21, W6, W36, W10,
LB5, and LB21 were isolated from a randomly mutagenized library of
GAD-2B assayed for loss of multimerization with the wild-type protein
(see Results). Italicized residue numbers indicate codons that have
been altered by mutation. Linker insertion mutant 2B(201) was generated
by Bernstein et al. (8). Linker insertion mutants 2B(204)
and 2B(205) were generated by Johnson and Sarnow (29). A
computer prediction of the 2B secondary structure was carried out with
PeptideStructure (Genetics Computer Group software package,
Biotechnology Center, University of Wisconsin). Features depicted
constitute a general consensus between the predictions of Chou and
Fasman and those of Garnier, Osguthorpe, and Robson.
|
|
In this study, our aim was to determine if the P2 products can interact
with each other and whether some of these interactions are required in
genomic replication. We have constructed a Saccharomyces cerevisiae two-hybrid "protein linkage map" of the P2 region
as part of a larger project that would ultimately include all the cleavage products of the polioviral polyprotein. On the basis of what
was achieved with the protein linkage map of bacteriophage T7
(7), we expected that cataloging the protein-protein
interactions among the P2 products would shed new light on previous
observations and suggest new roles in replication. Our results indicate
that 2BC, 2B, and 2C multimerize in a network of interactions which is
consistent with previous observations of trans-dominance by viral mutants of 2B (29). A genetic analysis of the 2B/2B
interaction strongly suggests that 2B multimerization is required for
the occurrence of viral replication.
| |
MATERIALS AND METHODS |
Construction of two-hybrid expression vectors and screening for
interaction.
The coding sequences of poliovirus type 1 (Mahoney)
(PVM) 2Apro, 2B, 2C, and 2BC were amplified by PCR with
Pfu polymerase (Stratagene) by using a set of primers meant
to introduce an XmaI site at the upstream (5') end and two
stop codons plus a SalI site at the downstream (3') end of
each coding sequence. The basic design of all upstream primers was
5'-GCG-XmaI-G-15 nucleotides (nt) of positive-sense 5'
coding sequence-3'. The basic design of all downstream primers was
5'-GCG-SalI-TTATCA-15 nt of negative-sense 3' coding
sequence-3'. These were used to amplify the appropriate coding
sequences from viral cDNA vector pT7PVM (14, 40), which contains unique XhoI and MluI sites in the 2C
coding region and a StuI site in the 2B N terminus coding
region. The PCR products were precipitated, digested with
XmaI and SalI, gel purified by the GeneClean II
method (Bio101, Inc.), and ligated into pGAD.GH (Gal4 activation
domain, or GAD) (55), pBTM116 (LexA DNA binding protein),
and pGBT9 (Gal4 DNA binding domain, or GBD) (6) vectors that
had been digested with XmaI and SalI and treated
with calf intestinal phosphatase. The 2B PCR product was also ligated
into pBTM38, a derivative of pBTM116 in which the ADE2 gene
was inserted into the PvuII site. Plasmid DNA was prepared
from several subsequent bacterial transformants and screened for the
presence of the inserts, and positive clones were sequenced by standard
methods at the fusion junction to ensure correct reading frames.
Clones of pGAD.GH and pBTM116 constructs of 2A
pro, 2B, 2C,
and 2BC were transformed into
S. cerevisiae
L40-
ura3 (
48) in every
possible pairwise
combination. Control plasmids were pGAD.GH-retinoblastoma
and GAD empty
vector, pBTM116-lamin, and pBTM116-topoisomerase
I. This resulted in 28 pairwise transformations which were selected
on minimal synthetic plus
sucrose (SS) medium lacking Leu and
Trp. All two-hybrid media, buffers,
and protocols were used as
previously described (
6,
20).
Each plate was subjected to
a
-galactosidase filter lift assay to
ensure that all or nearly
all colonies would show the same activity
(
6). Paper filters
(VWR Scientific) were laid upon the
surface of the plate containing
transformed colonies, lifted, and
frozen for 10 s in liquid N
2;
upon thawing they were
placed on another filter wetted with Z
buffer (
6) containing
5-bromo-4-chloro-3-indolyl-
-
D-galactoside
(X-Gal) and
incubated at 37°C until the color change was fully
developed. From
each plate, approximately 12 colonies were then
picked and combined
into a single streak which was then tested
for activity by filter lift,
and restreaked onto SS media lacking
Leu, Trp, and His to test for
HIS3 expression.
-Galactosidase
activities in positive
pairwise combinations were quantified by
colorimetric measurement of
Miller units (
6) with chlorophenol
red-
-
D-galactopyranoside and by measurement of
absorbance at
574 nm.
The Y153 strain (Table
1) was transformed in an analogous manner with
pGAD.GH and pGBT9 constructs, along with a GAD fusion
of an active-site
mutant of 2A
pro [2A
pro(C109S)]
(
28). Transformants were tested for activity as described
above and grown on SS medium lacking Leu, Trp, and His in the
presence
of 20 mM 3-amino-1,2,4-triazole (an inhibitor of basal
levels of
HIS3 caused by leaky Gal1-dependent reporter expression)
to
test for
HIS3 reporter expression (
6,
20).
LexA and GAD fusions of 2B mutants 2B(201), 2B(204), and 2B(205) were
constructed essentially as described above for wild-type
2B, except PCR
products were generated from the full-length cDNAs
pT7201
(
8), pT7205, and pT72B204, respectively (
29).
The mutation carried by the W36 isolate (see Results) was introduced
into pBTM116 and pGAD.GH by PCR with coding sequences
for 2B and 2BC
from pT7PVM(W36) and the appropriate primers as
described above,
generating LexA and GAD fusions 2B(W36) and 2BC(W36).
The integrity of
positive clones was confirmed by sequencing the
junctions and the
mutated codon.
Expression of GST fusion proteins and pulldown assay for
interaction.
Glutathione S-transferase (GST) fusion
proteins of the P2 cleavage products were cloned and expressed as
follows. The pT7PVM vector (14, 40) was used as a template
for PCR amplification (with Pfu polymerase) of coding
sequences for 2A, 2B, 2C, and 2BC with primers designed to introduce
flanking EcoRI and HindIII sites (principally
the same design as that described above). The resulting products were
digested with EcoRI and HindIII and ligated into the appropriately digested pGEX-KG vector (25) to yield pGEX-KG2A, pGEX-KG2B, pGEX-KG2C, and pGEX-KG2BC.
Positive clones were transformed into the BL-21(DE3) strain of
Escherichia coli, and isolated colonies were grown overnight
in 2× yeast extract-tryptone (YT) plus ampicillin medium. These
were
diluted 50-fold and grown in 50 ml of the same medium to
an optical
density at 600 nm of 1.0, at which point expression
was induced with
0.1 mM isopropyl-
-
D-galactopyranoside (IPTG)
for 4 h at 25°C. Cultures were centrifuged and resuspended in
2.5 ml of
ice-cold HBS (20 mM HEPES [pH 7.5], 140 mM NaCl) supplemented
with 5 mM 2-mercaptoethanol and 1 µg each of pepstatin A and leupeptin
(Sigma) per ml. Cells were lysed by sonication, Triton X-100 was
added
to a final concentration of 1.5%, and samples were rotated
at 4°C
for 30 min and centrifuged at 8,000 ×
g for 10 min. A
50-µl
bed volume (bv) of glutathione-Sepharose (GSH) 4B beads
(equilibrated
in an equal volume of HBS supplemented with 5 mM
dithiothreitol
and 0.1% Triton X-100; referred to as binding buffer)
was added
to the supernatant fraction (all bead pipetting was done with
cut tips), and the mixture was rotated at 4°C for 45 min. Beads
were
washed four times with ice-cold binding buffer, resuspended
in 50 µl
of the same buffer, and stored at 4°C. A 2-µl bv of beads
was
analyzed by sodium dodecyl sulfate-15% polyacrylamide gel
electrophoresis (SDS-PAGE) and Coomassie blue staining to check
the
expression and recovery of fusion proteins.
To assay for the binding of the GST fusion proteins with
35S-labeled P2 cleavage products, the P2 proteins were
translated in
vitro in rabbit reticulocyte lysate (Promega) in the
presence
of 35S-Translabel (ICN Pharmaceuticals) from synthetic
transcripts
under the control of the encephalomyocarditis virus
internal ribosomal
entry site (IRES). A total of 1.0 × 10
6 cpm of trichloroacetic acid-precipitable material
(approximately
equal to 20 µl of translation reaction mixture) was
precleared
with a 5-µl bv of empty GSH beads by incubation at 30°C
for 20
min, with gentle mixing every 5 min. Following centrifugation
for 30 s, the supernatant was diluted with 20 µl of binding
buffer
and a 10-µl bv of beads plus fusion protein was added. The
mixture
was incubated for 30 min at 30°C, the supernatant was
removed,
and an additional 20-µl bv of empty GSH beads was added as a
carrier.
Beads were washed six times with 1 ml of binding buffer
(rotating
10 min at 4°C each time), resuspended in 1× Laemmli
buffer, boiled
for 5 min, and analyzed by SDS-PAGE and autoradiography.
Construction of a mutagenized GAD-2B library and reverse
screening for interaction.
We used buffer conditions derived by
Leung et al. (35) to mutagenize the 2B coding sequence by
PCR using Taq polymerase (Boehringer Mannheim) at expected
misincorporation rates of 0.4, 1.0, and 2.0%. Three different sets of
reaction mixtures were cycled 25 times at 95°C for 1 min, 42°C for
1 min, and 72°C for 2 min. Primers used were of the same design as
that described above to amplify the 2B sequence. The PCR products were
processed as described above and ligated into pGAD.GH. The ligated
products were transformed into electrocompetent stocks of E. coli DH10B (GIBCO BRL) by electroporation, and bacterial
transformants were grown on selective media. Colonies were counted,
scraped into liquid media and grown for 6 h at 37°C. Plasmid DNA
was then prepared to obtain the library.
For each library produced at the three different rates, the procedure
was repeated and the preparations were combined until
each contained
>4,700 members. The libraries were then cotransformed
into
L40-
ura3 with pBTM38-2B, and transformants were tested for
activity by filter lift assay. For the library constructed at
the 0.4%
misincorporation rate, the procedure was repeated until
>5,000
transformants had been screened. Fifty negative colonies
were picked,
replated, and retested; 25 of these were then grown
in liquid SS medium
lacking Leu to facilitate loss of the pBTM38-2B
plasmid and plated on
low-concentration adenine (10% of normal
concentration) SS medium
lacking Leu. Colonies of each isolate
that lost the plasmid (indicated
by a red color) were picked and
grown in liquid media, and extracts
were prepared and tested for
2B expression by SDS-PAGE and
immunoblotting with anti-2B monoclonal
antibody. Plasmid DNA was
prepared from positive isolates, and
the 2B coding region of each
plasmid was sequenced by standard
methods. Those isolates showing
alterations of the amino acid
sequence were reintroduced into pGAD.GH
and retested for loss
of interaction by filter lift and growth on media
lacking HIS.
Construction of mutant viral cDNA vectors.
The megaprimer
method (16) was used to generate the point mutations seen in
isolates W10 and W36 in the corresponding 2B codons of full-length cDNA
vector pT7PVM, generating plasmids pT7PVM(W10s) and pT7PVM(W36).
Primers that allowed the mutagenized PCR product to be ligated into the
XhoI site within the 2C coding region and into the blunt end
at the start of the 2B coding region left by StuI digestion
were generated. The resulting constructs lost the StuI site
and the ensuing amino acid change of isoleucine to leucine at position
2 (I2L) (40); this restored the wild-type amino acid
sequence at position 2 of 2B. The LB21 point mutation was introduced by
one-step PCR using an upstream primer that encodes that base change
into codon 3 of the 2B coding sequence, generating pT7PVM(LB21). To
construct an appropriate wild-type control (called pT7PVMwt), we used
one-step PCR with the upstream and downstream primers from the above
constructions to generate the wild-type sequence at position 3 of the
2B coding sequence.
Mutant viral replicons containing the luciferase gene in place of the
P1 region were constructed by ligating the
KpnI fragment
of
the pPV1(M)/Luc construct (
36) into the large
KpnI fragments
(vector background) of the pT7PVMwt,
pT7PVM(LB21), pT7PVM(W10s),
and pT7PVM(W36) constructs. This
resulted in the substitution
of the IRES and P1 regions in the
full-length viral cDNAs with
the IRES and luciferase genes of
pPV1(M)/Luc, generating pPV1(M)/Luc(wt),
pPV1(M)/Luc(LB21),
pPV1(M)/Luc(W10s), and pPV1(M)/Luc(W36).
In vitro translation, transfection, and analysis of viral
phenotypes.
Full-length genomic cDNA constructs pT7PVMwt,
pT7PVM(W10s), pT7PVM(W36), and pT7PVM(LB21) were linearized with
EcoRI and transcribed with T7 polymerase. The resulting RNA
was purified, quantified, and translated in cytoplasmic HeLa cell
extracts in the presence of 35S-Translabel (ICN
Pharmaceuticals) as previously described (42). After
overnight incubation, the reaction mixtures were mixed with equal
volumes of Laemmli buffer and analyzed by SDS-15% PAGE and autoradiography.
The RNAs were also transfected (in quadruplicate) into subconfluent
(80%) monolayers of HeLa R19 by the Lipofectin method
(GIBCO BRL). A
mock control was exposed to transfection mixture
minus RNA. Transfected
cultures were incubated in Dulbecco modified
Eagle medium (GIBCO BRL)
supplemented with 10% fetal bovine serum
at 37°C in a 5%
CO
2 atmosphere. Cells were observed by phase-contrast
microscopy for signs of cytopathic effect (CPE). When CPE appeared
in
80 to 100% of the cellular monolayer, the culture was frozen
at
80°C and subjected to three cycles of freezing and thawing.
The
resulting supernatants were titrated on confluent (90 to 95%)
HeLa R19
monolayers, and the cultures were incubated in nutrient
agar overlay
for 48 h to determine plaque size and titer by crystal
violet
staining. One set of plaques was stained with INT-MTT [0.001%
p-iodonitrotetrazolium violet-0.005%
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide]
(
49,
50) at 37°C for 30 min to visualize plaques
in viable
cultures, and seven plaques each were isolated to purify
progeny of
PVM(W10s) and PVM(W36). These were used to reinfect
HeLa monolayers,
and total RNA was extracted from 200-µl aliquots
of the resulting
supernatants. The 2B sequence from viral RNA
was amplified by reverse
transcription with avian myeloblastosis
virus reverse transcriptase
(Promega) and by PCR with
Pfu polymerase
and was sequenced
by standard methods.
The effects of the 2B mutations on viral RNA replication were assayed
by transfecting transcript RNAs of all four luciferase-expressing
replicons into 35-mm-diameter HeLa R19 monolayers either in the
presence or absence of guanidine hydrochloride. The cultures were
lysed
at different time points with 300 µl of lysis buffer (10
mM
Tris-HCl [pH 7.5], 140 mM NaCl, 1.5 mM MgCl
2, 0.5%
Nonidet
P-40), and reporter activity was determined with the luciferase
assay system (Promega) on a Gem Optocomp 1 luminometer (MGM
Instruments).
| |
RESULTS |
Protein linkage within the P2 products.
In an attempt to
determine whether protein-protein interactions occur among the
polypeptide cleavage products of the P2 region, we have engineered
fusions of all P2 products with the GAD and the LexA DNA binding
protein specifically for use in LexA-dependent two-hybrid interaction
assays (strain L40-ura3; Table
1). Initial studies revealed that the
LexA-2Apro fusion by itself was a powerful activator of
reporter gene transcription in this system, rendering the construct
useless. To circumvent this obstacle, we constructed a fusion of the
GBD with 2Apro, for use in Gal4-dependent (Gal1
promoter-containing) two-hybrid assays (strain Y153, Table 1).
Subsequently, it became apparent that expression of the
GAD-2Apro fusion was most likely toxic in either system, as
evidenced by our total failure to recover any viable colonies when this
construct was included in transformation experiments. Such toxicity is
consistent with the results of previous observations (5,
32). Replacement of the wild-type 2Apro coding
sequence with that of a mutant encoding a substitution of the putative
active-site cysteine [2Apro(C109S)] (28)
resulted in a fusion construct that yielded viable transformants.
However, the resulting colonies were still very slow growing compared
to those resulting from other transformant pairings.
All possible pairwise combinations of 2BC and its cleavage products
were transformed into the L40-
ura3 strain, and
2A
pro fusion constructs were likewise cotransformed into
Y153 with
other P2 products. The results of the assays for reporter
expression
in the L40-
ura3 transformation experiments are
shown in Fig.
2.
As is apparent, 2BC and
its cleavage products produce a positive
signal in every possible
combination with the exception of 2C/2C.
These interactions resulted in
expression of both the
lacZ (Fig.
2A and B) and
HIS3 (Fig.
2C) reporter genes. Interestingly, we
noted that
the GAD-2C fusion failed to give a positive signal
in any combination,
whereas LexA-2C was positive for interaction
with GAD-2B and 2BC (Fig.
2B). Such "polarity" of two-hybrid interactions
is frequently
observed (
61) and was also noted when the relative
amounts
of
-galactosidase expression were quantified by measurement
of
Miller units (Fig.
2B). For example, the pairing of LexA-2B
and GAD-2BC
resulted in very weak reporter activity compared to
the reciprocal
combination.
View larger version (52K):
[in this window]
[in a new window]
View larger version (52K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 2.
Two-hybrid assays for interactions among 2BC and its
cleavage products in strain L40-ura3 transformed with LexA
and GAD fusions of 2B, 2C, and 2BC. GAD-retinoblastoma (Rb), GAD empty
vector (Vector), and LexA-lamin were used as negative controls. (A)
Filter lift assay for lacZ expression with X-Gal as the
substrate. (B) Colorimetric quantitation in Miller units of positive
pairings from panel A with chlorophenol
red--D-galactopyranoside as the substrate for
-galactosidase. Bars represent the means of duplicate samples; error
bars represent maximum measured values. (C) Assay for expression of the
HIS3 reporter gene by selection of transformants on minimal
media lacking His.
|
|
In strain Y153, 2A
pro is positive for multimerization but
negative for interaction with the other P2 products. However, the
relative
level of activity was very low in the case of
2A
pro multimerization (Fig.
3A), ranging from 2 to 8 Miller units for
different sample cultures (Fig.
3B). We were not able to detect
activation of the second reporter gene,
HIS3, possibly
because
the level of expression was too low to allow the transformants
to survive on media lacking His under stringent conditions (20
mM
3-amino-1,2,4-triazole; see Materials and Methods). The reason
for the
weakness of this interaction is not known. It may be related
to the use
of a mutant sequence of 2A
pro in the GAD fusion protein, to
an intrinsically weak interaction
between 2A
pro polypeptide
chains, or to an inherent consequence of the two-hybrid
assay.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3.
Two-hybrid assays for interaction of 2Apro
with cleavage products of P2 in strain Y153 transformed with GBD and
GAD fusions of 2Apro, 2B, 2C, and 2BC. (A) Filter lift
assay for lacZ expression. (B) Colorimetric quantitation of
the 2Apro multimerization seen in panel A. Bars and error
bars are as defined for Fig. 2B.
|
|
Interaction of putative interacting pairs in vitro.
To obtain
corroborating evidence for the interactions detected by two-hybrid
analysis, we employed a GST pulldown assay. Fusion proteins of GST with
2Apro, 2B, 2C, and 2BC were generated, expressed in
bacteria, and bound to GSH beads. Over the course of several
experiments, we noted that the level of recovery of the fusion proteins
was highest for 2B and 2C and lowest for 2A. Significant amounts of the
proteins were found in the insoluble fractions of the lysed bacterial
cells, and in the case of GST-2Apro almost all the
expressed protein was insoluble (as assayed by SDS-PAGE and Coomassie
blue staining) and only very small amounts (<5%) could be recovered
on the beads (data not shown).
Each of the P2 cleavage products was synthesized in vitro in rabbit
reticulocyte lysate with
35S-Translabel present. Such
reactions were programmed with synthetic
transcripts encoding the P2
proteins, under the translational
control of the encephalomyocarditis
virus IRES. Interactions were
assayed by mixing the translation
reaction mixtures with the GST
fusion protein-bead complexes.
35S-labeled material retained on the beads was visualized
by SDS-PAGE
and autoradiography. The results are shown in Fig.
4. Interactions
that were detected by the
two-hybrid system could be reproduced
with this biochemical assay,
although the strengths of positive
signals did not necessarily
correlate with the corresponding signals
from the two-hybrid system.
Surprisingly, the 2B/2C interaction,
which was relatively strong in
yeast, was barely detectable in
vitro and the GST-2C/2C pairing was
found to be positive in the
GST pulldown assay (Fig.
4A) but negative
in the two-hybrid system.
The latter observation suggests that the
failure to detect a positive
2C/2C signal in yeast was due to the
generation of a nonfunctional
GAD-2C, a conclusion supported by the
lack of interaction in any
pairings which involved GAD-2C (in contrast
to LexA-2C).
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 4.
GST pulldown assay of putative interacting pairs,
performed as described in Materials and Methods. Lanes marked
"translation products" were loaded with 0.5 µl of in vitro
translation reaction mixture with the indicated contents. (A) Assays
for the interaction of 35S-labeled 2B, 2C, and 2BC with
GST, GST-2B, GST-2C, and GST-2BC. (B) Assays for the interaction of
35S-labeled 2Apro with GST and
GST-2Apro.
|
|
As was the case for the two-hybrid analysis, the GST pulldown assay
detected only a weak interaction signal for the
GST-2A
pro/2A
pro pairing (Fig.
4B).
Does wild-type 2B multimerize with
trans-dominant-negative 2B mutants?
Johnson and Sarnow
(29) have reported on the slow-replication phenotype of PVM
mutants that contained linker insertions after amino acid 28 of 2B.
These mutants, termed 2B(201) (8), 2B(204), and 2B(205)
(29) (Fig. 1B), could not be rescued by complementation. On
the contrary, they displayed dosage-dependent negative dominance
over the wild-type virus in mixed infections. There are two possible
interpretations for the trans-dominant phenotypes of these
mutants. (i) An interaction of 2B with a limiting (host or viral)
factor is normally required for replication. The mutant 2B
then interacts with this factor in competition with the wild-type
protein. (ii) The multimerization of wild-type 2B or 2BC is required
for replication, and mutant-wild-type heteromeric complexes may
be impaired in their normal function. Using the two-hybrid assay
described above, we tested the three insertional mutants
(29) for interaction with wild-type 2B and found that all
three have levels of activity similar to that for the wild-type interaction (Fig. 5). This observation
suggests (but does not prove) that the mutant protein exerts
trans-dominance by rendering a 2B multimer inactive.
View larger version (52K):
[in this window]
[in a new window]
View larger version (41K):
[in this window]
[in a new window]
View larger version (39K):
[in this window]
[in a new window]
|
FIG. 5.
Two-hybrid tests for the interaction of 2B with
trans-dominant-negative mutants in strain
L40-ura3 transformed with LexA and GAD fusions of 2B,
2B(201), 2B(204), and 2B(205) (Fig. 1B). (A) Filter lift assays for
lacZ expression. (B) Colorimetric quantitation of
interactions seen in panel A. Bars and error bars are as defined for
Fig. 2B. (C) Assay for expression of the HIS3 reporter
gene.
|
|
Mutational analysis of 2B multimerization.
In order to test
our hypothesis that 2B/2B interactions are essential in poliovirus
replication, we constructed a randomly mutagenized library of GAD-2B
fusion polypeptides and screened against the wild-type protein for loss
of interaction. The ultimate purpose of this "reverse screen"
was to identify noninteracting mutants of 2B that could be tested in
the context of the 2BC precursor. Moreover, these mutations could be
introduced into the genomic cDNA of the virus to ascertain their
effects on viral viability.
The coding sequence of PVM 2B contained in infectious poliovirus cDNA
was amplified by PCR under conditions previously estimated
to allow a
misincorporation rate of 0.4% (roughly 1 base change
per 291 nucleotides or per 2B coding sequence) (
35). Interestingly,
the use of higher mutagenesis rates (up to 2%) resulted in libraries
that had completely lost interaction with the wild-type protein,
probably indicating a high degree of sensitivity of the 2B protein
structure to changes in its primary sequence. A library (termed
GAD-2BM) in which 17% of the transformed yeast colonies were negative
for the 2B/2B interaction was finally selected. Then, 25 isolates
were
tested for GAD-2BM expression by Western blotting. Of these,
9 were
found to be expressing either full-length 2BM or apparently
truncated
products. Plasmid DNA was prepared from all nine and,
following
amplification of the DNA in bacteria, the 2BM coding
region was
sequenced to yield the results outlined in Fig.
1B.
The reverse screening identified one frameshift (LB5), two missense
(point) mutations (LB21 and W36), and two nonsense (amber
and opal)
mutations (W6 and LB3). One isolate (W10) carried both
a frameshift and
a missense change. In addition, three isolates
carried either wild-type
sequences or a silent mutation; we assumed
that these must have lost
the ability to activate transcription
through fortuitous changes in the
GAD sequence that were unrelated
to our library construction, and they
were not studied further.
To determine whether this was also the case
for the other GAD-2BM
isolates, the 2BM coding sequences were
reintroduced into the
GAD vector and reassayed for interaction.
Concurrently, we also
separated the missense and frameshift mutations
carried by W10,
thereby producing separate constructs, W10s and W10f,
respectively.
The results are presented in Fig.
6. After reconstruction, the
point
mutation carried by LB21 had no effect on 2B multimerization,
and the
shortest truncation of 2B (LB3) retained some activity,
while the rest
of the reconstructed 2BM isolates were negative
for interaction.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 6.
Two-hybrid assays for the interaction of wild-type 2B
with mutagenized library isolates that have been reconstructed into
pGAD.GH by using strain L40-ura3. The expression of
lacZ and HIS3 reporter genes was assayed by
filter lift and selection on media lacking HIS. The double mutations in
W10 were reconstructed into separate constructs (W10f and W10s). An
asterisk denotes the position of missense mutations outlined in Fig.
1B.
|
|
The results of the reverse screen indicate that the C-terminal half of
the protein is integral to the multimerization, either
because it
harbors the contact sites or because it is required
for the overall
structural integrity of 2B. The isolation of two
nonconservative point
mutations (carried by W10s and W36) in a
central region that is
predicted to form a hydrophobic
-sheet
suggests that the integrity
of this specific region is also required
for multimerization (Fig.
1B).
Linkage analysis of a noninteracting 2B mutant.
The mutation
carried by W36 (I53N) as a GAD fusion was introduced into LexA-2B, to
determine whether the mutant 2B could interact with itself or with the
other 2BC products in either polarity. The results of these interaction
tests are shown in Fig. 7A. The 2B(W36)
mutant does not multimerize and, in the context of this assay, fails to
interact with any 2BC product. These results suggest that determinants
for multimerization within 2B are also required for interaction with 2C
and 2BC.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 7.
Two-hybrid assays for the interaction with mutant 2B and
2BC. (A) Filter lift assays for the interaction of 2B constructs
carrying the W36 mutation (I53N) with 2B, 2C, and 2BC, and for the
multimerization of 2B(W36). The multimerization of wild-type 2B was
assayed in parallel as a positive control. (B) Two-hybrid assays for
the interaction of 2BC mutants carrying the W36 mutation (I53N) with
2B, 2C, 2BC, and for the multimerization of 2BC(W36). (C) Schematic
summary of interactions involving 2B(W36) and 2BC(W36). Double arrows
indicate positive reciprocal two-hybrid interactions, while single
arrows denote unidirectional (polar) interactions.
|
|
We also introduced the W36 mutation into GAD and LexA fusion constructs
of 2BC to determine the contribution of the 2B moiety
to interactions
involving the 2BC precursor. The resulting set
of positive pairings
(Fig.
7B) indicates, surprisingly, that the
mutant 2BC molecule retains
an interaction profile similar to
that of the wild-type protein. An
exception is the negative signal
produced by the
LexA-2B-GAD-2BC(W36) pairing, which is in contrast
to the signal
produced by the corresponding wild-type interaction
(Fig.
2A). The most
likely reason for this result may be the strength
of the reporter
signal. The wild-type pairing is one of the weakest
detected in the
linkage map (as opposed to the reciprocal pairing)
(Fig.
2B).
Therefore, it is possible that the presence of the
mutation in the 2BC
moiety reduces the overall strength of the
interaction between LexA-2B
and GAD-2BC(W36) below the limits
of detectability, resulting in the
polarity addressed above. The
quantitative measures of
-galactosidase expression in the interactions
with 2BC(W36) support
this explanation, since they were significantly
lower than the
corresponding values obtained with wild-type 2BC
(data not shown).
The overall scoring of pairings involving 2B(W36) and 2BC(W36)
(summarized in Fig.
7C) suggests that the positive interactions
that
include only 2BC(W36) are only partially dependent on the
presence of
the wild-type 2B moiety. This conclusion expands the
possible role of
2C in these interactions.
Genetic analysis of noninteracting mutants of 2B.
To determine
whether the substitution mutations carried by the LB21, W10s, and W36
isolates affect genomic replication, we constructed mutant and
wild-type polioviral replicons in which the P1 region was replaced with
the firefly luciferase gene. The accumulation of maximal levels of the
luciferase reporter protein in cell cultures transfected with
transcript RNAs is indicative of genome replication (2, 3,
36), since most of the viral protein in the infected cell
is synthesized from newly made RNA. Transfection of the wild-type and
LB21 replicons resulted in roughly equivalent levels of luciferase
activity, whereas the replicons harboring the W10s and W36 mutations
replicated at extremely low or nonexistent levels (Fig.
8). The inclusion of 2 mM guanidine hydrochloride in the culture media resulted in essentially equally low
levels of reporter activity among all replicons, an observation demonstrating that the luciferase activities of W10s and W36 replicons in the absence of the drug are indicative of a block in replication. Therefore, we conclude that the mutations in 2B which block
multimerization also severely reduce viral RNA replication.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 8.
Luciferase activity in cell cultures transfected with
luciferase-expressing viral replicons encoding either the wild-type
protein, LB21, W10s, or 2B(W36). Following transfection, cell cultures
were maintained either in the absence or presence of 2 mM guanidine
hydrochloride. Light units are arbitrary.
|
|
We then engineered the 2B mutations into the viral genome to determine
their effects on viability. Genomic cDNA clones encoding
these three
mutations and the wild-type protein sequence were
transcribed with T7
polymerase, and the resulting RNAs were translated
in vitro in a HeLa
cytoplasmic extract in the presence of
35S-Translabel
(
42). SDS-PAGE analysis revealed efficient translation
and
proteolytic processing patterns (Fig.
9).
A slight anomaly
in processing was observed in the case of W10 and W36,
namely,
a slight accumulation of precursor 3BCD (
14), and
some reduction
in the quantity of P3 processing products (3C, 3D, and
3AB) compared
to those for the wild type and LB21 was also observed.
However,
all structural and nonstructural proteins can be easily
identified
among the products of all four reactions.
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 9.
SDS-15% PAGE analysis of in vitro-translated
full-length transcript RNAs harboring the LB21, W10s, and W36
mutations, as compared to the wild type. The "Marker" lane consists
of the (35S-Translabel) pulse-labeled products from
PVM-infected HeLa R-19 cells.
|
|
Transfection of all RNAs into HeLa R19 cells resulted in drastic
differences in the onset of CPEs (Table
2). For cultures
transfected with the
wild-type and LB21 RNAs, CPE was observed
at between 23 and 25 h
and between 21 and 24 h, respectively.
Interestingly, LB21 genomic
RNA reproducibly produced CPE slightly
earlier than the wild-type
standard, a phenomenon that we cannot
explain at present. The W10s and
W36 mutants displayed CPE at
90 to 97 h and at 60 to 66 h,
respectively. Of quadruplicate cultures,
three transfected with W10s
produced CPE, while all four transfected
with W36 produced CPE.
The cell culture supernatants were examined by a plaque assay, and we
observed that the plaque morphologies of LB21, W10,
and W36 were
indistinguishable from those of the wild-type virus
(Table
2). We also
observed equivalent titers in one-step growth
experiments (data not
shown). The single W10 culture which did
not give rise to CPE did not
yield any plaques. Seven isolates
each of W10s and W36 were plaque
purified and amplified once in
cell culture. Viral RNAs were then
isolated from each culture,
and their 2B coding sequences were
analyzed. In all cases, the
mutated codons of W10 and W36 mutant RNAs
had reverted to the
wild-type genotype, an observation indicating that
the W10s and
W36 mutants exhibited a quasi-infectious (qi) phenotype
(
22).
We have examined the possibility that the observed viruses were
wild-type contaminants by repeating the transfections under
nutrient
agar overlays, reasoning that contamination would give
rise to isolated
plaques at the same time as (but at a much lower
titer than) the
wild-type control; we never observed plaques generated
by W10s and W36
in such assays, which involved incubation until
the monolayers began to
lose viability (110 h in both mock and
RNA-containing samples). This
observation indicates that infectious
particles were generated too late
in the incubation to yield visible
plaques (results not shown). On the
other hand, reversion yielding
a very small number of wild-type
genotypes would yield infectious
particles that can readily spread in
liquid media, allowing a
detectable onset of CPE.
| |
DISCUSSION |
Protein-protein interactions among the cleavage products of the P2
precursor.
The two-hybrid system (21) has recently been
used to construct a protein linkage map of bacteriophage T7 in an
attempt to catalog all detectable protein-protein interactions among
the gene products of the virus (7). Using an analogous
strategy, we have carried out experiments to detect protein-protein
interactions among the cleavage products of the poliovirus polyprotein
specifying the nonstructural proteins. These include some cleavage
precursors that may have functions distinct from those of their
cleavage products. Previously, a protein linkage map of the products of the P3 precursor was established by using a semi-inducible version of
the two-hybrid system (60). In this work, protein linkage mapping among the P2 products was carried out by using two widely adopted versions of the two-hybrid system in which the fusion proteins
are expressed constitutively. Our results strongly suggest that
functional interactions occur among 2BC and its cleavage products.
Moreover, 2Apro may bind to itself.
Interactions detected by two-hybrid pairings were reconstituted in
vitro by a GST pulldown assay (Fig.
4). The strength of
the interaction
signal did not always correspond to that from
the two-hybrid
observations, as in the case of the 2C/2B interaction
(see Results). We
also found one interaction (2C/2C) which we
were not able to detect in
yeast. At present, we cannot explain
the variances between the results
obtained with the two systems,
but these observations underline the
need for alternative experimentational
approaches in any study designed
to detect protein-protein interactions.
It is important to note that
two-hybrid measurements of interaction
strength are not always in
correspondence with biochemically determined
values, presumably because
many different variables affect the
level of a two-hybrid signal (i.e.,
polarity of interaction, stability
of fusion proteins, and promoter
strength, etc.) (
19). Moreover,
experiments carried out with
different cell preparations (but
a constant set of plasmids) may not
necessarily produce the same
levels of interaction signal.
Interestingly, the weakness of the
2C/2B interaction in vitro and the
weak signal obtained for the
2B/2BC interaction suggest that the 2BC
multimerization is principally
due to the 2C/2C interaction, a
conclusion supported by two-hybrid
studies of 2BC mutants (see below).
The observed interactions from pairings of two-hybrid fusion constructs
and from GST pulldown assays can be divided into two
classes (Fig.
10), which may be relevant to the
functions of the
P2 products during viral RNA replication. Class I
describes the
formation of intermolecular complexes that can be further
divided
into homomultimeric interactions (2B/2B, 2C/2C, 2BC/2BC, and
2A
pro/2A
pro) and heteromultimeric interactions
(2B/2BC and 2C/2BC). Class
II consists of a mode of interaction (2C/2B)
that may, in fact,
reflect the intrinsic affinity between two separate
domains within
the uncleaved precursor. Although all two-hybrid
interactions
are intermolecular, the separate 2C and 2B hybrids may be
interacting
in a manner which normally exists within the 2BC
polypeptide,
reflecting intramolecular binding between distinct
domains. Such
intramolecular associations have been detected in the
protein
linkage map of bacteriophage T7, on which basis it was
suggested
that the two-hybrid system may aid in the elucidation of
protein
folding (
7). Of course, the 2C/2B interaction may
also be intermolecular
in the infected cell, since our analysis does
not permit us to
differentiate between these possibilities.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 10.
Protein linkage map of the cleavage products of the P2
protein precursor, suggested by data obtained by two-hybrid and GST
pulldown assays. See the text for an explanation of the two classes of
interaction. Double arrows indicate positive reciprocal interactions,
whereas single arrows denote unidirectional (polar) interactions.
|
|
As mentioned above, we were unable to detect an interaction between 2C
and 2C in the two-hybrid system. Indeed, we noted that
GAD-2C did not
interact with any LexA fusion. In contrast, the
LexA-2C fusion was
positive for interaction with GAD-2B and GAD-2BC.
Explanations for this
polar phenomenon include the possibility
that the noninteracting fusion
protein (GAD-2C) is misfolded or
inherently unstable, that the
expression is poor, and that transport
to the nucleus does not occur.
The expression of GAD-2C, as assayed
by Western blotting of yeast
extracts, was efficient (data not
shown), eliminating the possibility
that the protein was not present.
Although we have failed to directly
observe 2C/2C interaction,
the 2C moiety of 2BC nevertheless appears to
contribute to an
interaction between 2BC and its cleavage products.
This we conclude
from the positive signals, albeit weak, that we
obtained between
2BC(W36) and 2B, 2C, and 2BC, whereas 2B(W36) had lost
interaction
with any of the possible partners (Fig.
7C). These
observations
suggest that the 2BC/2BC interaction is aided by, or is
even dependent
on, the 2C moieties and does not occur exclusively
through 2B/2B
or 2B/2C interaction. Indeed, the 2BC/2BC interaction in
vitro
appears to be mediated mainly by the 2C moieties (Fig.
4A). In
addition, the overall reduction in the signal may explain the
polarity
of the 2B/2BC(W36) interaction, which was already weak
in the pairing
with wild-type 2BC and is absent in the pairing
with the mutant.
Polypeptide 2C has been proposed to be a helicase required in RNA
replication (
23,
24,
59), although experimental evidence
for
such a function is as yet missing. It is possible that such
a function
could be performed by 2BC and not by its cleavage product
2C.
Oligomerization has been described as being critical to the
function of
most, but not all, DNA and RNA helicases (
30). However,
the
question of whether 2C and 2BC are helicases must await further
biochemical study of these proteins.
Observations by Tolskaya et al. (
53) that
guanidine-resistant viral mutants of 2C could complement
guanidine-sensitive and
-dependent mutants under restrictive conditions
have been interpreted
to suggest a functional oligomerization of 2C,
perhaps at the
level of 2BC. However, the rescue of a function in
trans does
not necessarily require oligomerization, merely
the presence of
the functional version of the protein, to carry out the
activity.
The signal for the 2A
pro/2A
pro interaction,
even for mutants debilitated in proteinase activity, was so weak that
HIS3 expression
was undetectable, a phenomenon that we
cannot explain at present.
In general, we noted that the version of the
two-hybrid system
utilizing the Y153 strain (with
GAL1
promoter-dependent reporter
genes) was significantly less sensitive
than the L40-
ura3 system.
This suggests that the limiting
factor in the level of the signal
may be the promoter strength of the
reporter system. Liebig et
al. (
37) reported that purified
2A
pro of human rhinovirus 2 may exist as a dimer. Work with
purified
recombinant 2A
pro of PVM indicates that under some
conditions, the protein can
exist as a monomer, dimer, and tetramer,
but these forms have
not been detected in infected cells
(
31). Our observation that
the GST
2A
pro/2A
pro interaction is weak but detectable
corroborates the two-hybrid
results and suggests that the interaction
may be intrinsically
weak.
Genetic analysis of 2B multimerization.
Two observations have
led us to conclude that the 2B/2B interaction is required for
poliovirus replication. First, dominant-negative mutants of 2B studied
by Johnson and Sarnow (29) interacted with the wild-type
protein in the two-hybrid system at levels comparable to those for
wild-type 2B multimerization. If the normal function of 2B requires
multimerization, the hybrid wild-type-mutant complex would presumably
be inactive, resulting in a lower level of replication of wild-type as
well as mutant viral genomes. One example of such an effect is that of
dominant-negative mutants of the p53 tumor suppressor protein, in which
function appears to be lost through the assembly of heterotetramers
consisting of wild-type and mutant monomers (12, 33).
Second, two mutations in 2B, identified by randomly mutagenizing 2B and
selecting for variants that are negative for 2B multimerization,
nearly
abolished replication of luciferase-expressing viral replicons.
When
introduced into the full-length genome of PVM, these mutations
conferred a qi phenotype. After transfection of HeLa cells with
mutant
transcript RNAs, CPE was observed only after prolonged
incubation. From
these cultures, only virus in which the mutation
had reverted to the
wild-type sequence was recovered. We interpret
this to mean that the 2B
mutants, as expressed by translation
of the corresponding transcript
RNAs in transfected HeLa cells,
may still interact weakly, allowing a
very low level of genomic
replication and reversion. This property is
the signature of the
qi phenotype (
22). Thus, the absence of
a 2B/2B interaction
in the two-hybrid system covaries with severe
impairment in replication.
In addition, the linkage analysis of
2BC(W36) suggests that this
phenotype may result from loss of function
at the level of 2B,
since 2BC(W36) can still multimerize and interact
with 2B and
2C. The mild processing anomaly seen by in vitro
translation of
the mutant genomes is unlikely to cause the qi
phenotype, since
all the nonstructural proteins are produced. For
comparison, mutants
of coxsackie B3 virus mapping to the 2B/2C cleavage
site that
exhibited a severe defect in 2B and 2C production were still
viable,
if slow-growing (
57).
The two amino acid changes in the polypeptide chain of 2B that abolish
multimerization are located inside a region which is
predicted to form
a hydrophobic
-sheet (Fig.
1B; residues 46
to 56). We cannot
conclude that the specific residues in question
(I-53 and I-54)
constitute contact points for interaction. Since
the hydrophobicity of
the region has changed, the overall structure
of the region may be
distorted. However, computer predictions
indicate that the mutant
sequences retain the propensity for a
-sheet, suggesting that the
effect of the mutations on the tertiary
structure is minimal.
Therefore, the general hydrophobicity of
this region may be required
for multimerization. The fact that
the C-terminal truncation mutants
were also negative for 2B/2B
binding in the two-hybrid system may
implicate large segments
of the C terminus in 2B multimerization.
Previous work from other groups has shown that exogenously expressed
enteroviral 2B can permeabilize bacterial and mammalian
cells (
1,
17,
34,
56) and can block secretory transport
at the endoplasmic
reticulum/
cis-Golgi step in the latter (
17).
Permeabilization suggests that 2B may form a pore in cellular
membranes; such a structure would almost certainly require
oligomerization
of the protein, possibly dependent upon the C-terminal
hydrophobic
domain. The observation that 2B multimerizes in the
two-hybrid
assay and the broad structural requirements for this
multimerization
support this model of 2B function. Van Kuppeveld et al.
(
58)
have implicated this property of 2B in the release of
progeny
virions from the cell during the final stages of infection.
What
purpose permeabilization would serve in the life cycle of the
virus and whether the 2BC precursor would be the true functional
entity
in this activity (
1) are questions that remain to be
answered.
| |
ACKNOWLEDGMENTS |
We thank Rohit Duggal for critical reading of the manuscript,
Rolf Sternglanz for the gift of the L40-ura3 strain, Stanley Fields and his colleagues for providing valuable advice on two-hybrid vectors, protocols, control plasmids, and the Y153 strain, and Kurt
Bienz for providing anti-2B monoclonal antibody. We are particularly indebted to Xuemei Cao and Meijia Yang for the pBTM38 plasmid and
suggestions.
A.C. was a member of the graduate training program of the Department of
Molecular Genetics and Microbiology. T.P. was supported, in part, by
the Swiss National Foundation, the Freie Akademische Gesellschaft
Basel, and the Theodor Engelmann-Stiftung, Basel, Switzerland. F.L. was
supported by a grant from Schering-Plough. This work was supported in
part by NIH-5R37AI15122 and AI32100.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics and Microbiology, School of Medicine, State
University of New York at Stony Brook, Stony Brook, NY 11794-5222. Phone: (516) 632-8787. Fax: (516) 632-8891. E-mail:
wimmer{at}asterix.bio.sunysb.edu.
Present address: Molecular Pathogenesis Program, Skirball
Institute, New York, NY 10016.
| |
REFERENCES |
| 1.
|
Aldabe, R.,
A. Barco, and L. Carrasco.
1996.
Membrane permeabilization by poliovirus proteins 2B and 2BC.
J. Biol. Chem.
271:23134-23137[Abstract/Free Full Text].
|
| 2.
|
Alexander, L.,
H.-H. Lu, and E. Wimmer.
1994.
Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene.
Proc. Natl. Acad. Sci. USA
91:1406-1410[Abstract/Free Full Text].
|
| 3.
|
Andino, R.,
G. E. Rieckhof,
P. L. Achacoso, and D. Baltimore.
1993.
Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.
EMBO J.
12:3587-3598[Medline].
|
| 4.
|
Barco, A., and L. Carrasco.
1995.
A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.
EMBO J.
14:3349-3364[Medline].
|
| 5.
|
Barco, A., and L. Carrasco.
1995.
Poliovirus 2Apro expression inhibits growth of yeast cells.
FEBS Lett.
371:4-8[Medline].
|
| 6.
|
Bartel, P. L.,
C. Chien,
R. Sternglanz, and S. Fields.
1993.
Using the two-hybrid system to detect protein-protein interactions, p. 153-179. In
D. A. Hartley (ed.), Cellular interactions in development: a practical approach.
Oxford University Press, Oxford, United Kingdom.
|
| 7.
|
Bartel, P. L.,
J. A. Roecklein,
D. SenGupta, and S. Fields.
1996.
A protein linkage map of Escherichia coli bacteriophage T7.
Nat. Genet.
12:72-77[Medline].
|
| 8.
|
Bernstein, H. D.,
P. Sarnow, and D. Baltimore.
1986.
Genetic complementation among poliovirus mutants derived from an infectious cDNA clone.
J. Virol.
60:1040-1049[Abstract/Free Full Text].
|
| 9.
|
Bienz, K.,
D. Egger, and L. Pasamontes.
1987.
Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.
Virology
160:220-226[Medline].
|
| 10.
|
Bienz, K.,
D. Egger, and T. Pfister.
1994.
Characteristics of the poliovirus replication complex.
Arch. Virol. Suppl.
9:147-157[Medline].
|
| 11.
|
Bienz, K.,
D. Egger,
M. Troxler, and L. Pasamontes.
1990.
Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.
J. Virol.
64:1156-1163[Abstract/Free Full Text].
|
| 12.
|
Brachmann, R. K.,
M. Vidal, and J. D. Boeke.
1996.
Dominant-negative p53 mutations selected in yeast hit cancer hot spots.
Proc. Natl. Acad. Sci. USA
93:4091-4095[Abstract/Free Full Text].
|
| 13.
|
Caliguiri, L. A., and I. Tamm.
1969.
Membranous structures associated with translation and transcription of poliovirus RNA.
Science
166:885-886[Abstract/Free Full Text].
|
| 14.
|
Cao, X.,
R. J. Kuhn, and E. Wimmer.
1993.
Replication of poliovirus RNA containing two VPg coding sequences leads to a specific deletion event.
J. Virol.
67:5572-5578[Abstract/Free Full Text].
|
| 15.
|
Cho, M. W.,
N. Teterina,
D. Egger,
K. Bienz, and E. Ehrenfeld.
1994.
Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells.
Virology
202:129-145[Medline].
|
| 16.
|
Datta, A. K.
1995.
Efficient amplification using `megaprimer' by asymmetric polymerase chain reaction.
Nucleic Acids Res.
23:4530-4531[Free Full Text].
|
| 17.
|
Doedens, J. R., and K. Kirkegaard.
1995.
Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.
EMBO J.
14:894-907[Medline].
|
| 18.
|
Egger, D.,
L. Pasamontes,
R. Bolten,
V. Boyko, and K. Bienz.
1996.
Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis.
J. Virol.
70:8675-8683[Abstract].
|
| 19.
|
Estojak, J.,
R. Brent, and E. A. Golemis.
1995.
Correlation of two-hybrid affinity data with in vitro measurements.
Mol. Cell. Biol.
15:5820-5829[Abstract].
|
| 20.
|
Fields, S.
1993.
The two-hybrid system to detect protein-protein interactions., p. 116-124.
Methods: a companion to Methods in Enzymology, vol. 5.
Academic Press, New York, N.Y.
|
| 21.
|
Fields, S., and O. Song.
1989.
A novel genetic system to detect protein-protein interactions.
Nature
340:245-246[Medline].
|
| 22.
|
Gmyl, A. P.,
E. V. Pilipenko,
S. V. Maslova,
G. A. Belov, and V. I. Agol.
1993.
Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.
J. Virol.
67:6309-6316[Abstract/Free Full Text].
|
| 23.
|
Gorbalenya, A. E.,
E. V. Koonin,
A. P. Donchenko, and V. M. Blinov.
1989.
Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes.
Nucleic Acids Res.
17:4713-4730[Abstract/Free Full Text].
|
| 24.
|
Gorbalenya, A. E.,
E. V. Koonin, and Y. I. Wolf.
1990.
A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.
FEBS Lett.
262:145-148[Medline].
|
| 25.
|
Guan, K. L., and J. E. Dixon.
1991.
Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase.
Anal. Biochem.
192:262-267[Medline].
|
| 26.
|
Hambidge, S. J., and P. Sarnow.
1992.
Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A.
Proc. Natl. Acad. Sci. USA
89:10272-10276[Abstract/Free Full Text].
|
| 27.
|
Harris, K. S.,
C. U. T. Hellen, and E. Wimmer.
1990.
Proteolytic processing in the replication of picornaviruses.
Semin. Virol.
1:323-333.
|
| 28.
|
Hellen, C. U.,
M. Facke,
H. G. Krausslich,
C. K. Lee, and E. Wimmer.
1991.
Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.
J. Virol.
65:4226-4231[Abstract/Free Full Text].
|
| 29.
|
Johnson, K. L., and P. Sarnow.
1991.
Three poliovirus 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus.
J. Virol.
65:4341-4349[Abstract/Free Full Text].
|
| 30.
|
Kadare, G., and A. Haenni.
1997.
Virus-encoded RNA helicases.
J. Virol.
71:2583-2590[Medline].
|
| 31.
| Kakegawa, H., and E. Wimmer. 1997. Unpublished
results.
|
| 32.
|
Klump, H.,
H. Auer,
H.-D. Liebig,
E. Kuechler, and T. Skern.
1996.
Proteolytically active 2A proteinase of human rhinovirus 2 is toxic for Saccharomyces cerevisiae but does not cleave the homologues of eIF-4g in vivo or in vitro.
Virology
220:109-118[Medline].
|
| 33.
|
Ko, L. J., and C. Prives.
1996.
p53: function and paradigm.
Genes Dev.
10:1054-1072[Free Full Text].
|
| 34.
|
Lama, J., and L. Carrasco.
1992.
Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A.
J. Biol. Chem.
267:15932-15937[Abstract/Free Full Text].
|
| 35.
|
Leung, D. W.,
E. Chen, and D. V. Goeddel.
1989.
A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction.
Technique
1:11-15.
|
| 36.
|
Li, X.,
H.-H. Lu,
S. Mueller, and E. Wimmer.
1997.
.
Functional analysis of poliovirus 2Apro C-terminal domain.
Unpublished data.
|
| 37.
|
Liebig, H. D.,
E. Ziegler,
R. Yan,
K. Hartmuth,
H. Klump,
H. Kowalski,
D. Blaas,
W. Sommergruber,
L. Frasel,
B. Lamphear,
R. Rhoads,
E. Kuechler, and T. Skern.
1993.
Purification of two picornaviral 2A proteinases: interaction with eIF-4g and influence on in vitro translation.
Biochemistry
32:7581-7587[Medline].
|
| 38.
|
Lu, H. H.,
X. Li,
A. Cuconati, and E. Wimmer.
1995.
Analysis of picornavirus 2Apro proteins: separation of proteinase from translation and replication functions.
J. Virol.
69:7445-7452[Abstract].
|
| 39.
|
Mirzayan, C., and E. Wimmer.
1994.
Biochemical studies on poliovirus polypeptide 2C: evidence for ATPase activity.
Virology
199:176-187[Medline].
|
| 40.
|
Mirzayan, C., and E. Wimmer.
1992.
Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.
Virology
189:547-555[Medline].
|
| 41.
|
Molla, A.,
A. V. Paul,
M. Schmid,
S. K. Jang, and E. Wimmer.
1993.
Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication.
Virology
196:739-747[Medline].
|
| 42.
|
Molla, A.,
A. V. Paul, and E. Wimmer.
1991.
Cell-free, de novo synthesis of poliovirus.
Science
254:1647-1651[Abstract/Free Full Text].
|
| 43.
|
Pallansch, M. A.,
O. M. Kew,
B. L. Semler,
D. R. Omilianowski,
C. W. Anderson,
E. Wimmer, and R. R. Rueckert.
1984.
Protein processing map of poliovirus.
J. Virol.
49:873-880[Abstract/Free Full Text].
|
| 44.
|
Pfister, T., and E. Wimmer.
1997.
.
Poliovirus 2C contains a zinc finger motif involved in RNA replication.
Unpublished data.
|
| 45.
|
Rodriguez, P. L., and L. Carrasco.
1995.
Poliovirus protein 2C contains two regions involved in RNA binding activity.
J. Biol. Chem.
270:10105-10112[Abstract/Free Full Text].
|
| 46.
|
Rodriguez, P. L., and L. Carrasco.
1993.
Poliovirus protein 2C has ATPase and GTPase activities.
J. Biol. Chem.
268:8105-8110[Abstract/Free Full Text].
|
| 47.
|
Sandoval, I. V., and L. Carrasco.
1997.
Poliovirus infection and expression of the poliovirus protein 2B provoke the dissassembly of the Golgi complex, the organelle target for the antipoliovirus drug Ro-090179.
J. Virol.
71:4679-4693[Abstract].
|
| 48.
|
SenGupta, D. J.,
B. Zhang,
B. Kraemer,
P. Pochart,
S. Fields, and M. Wickens.
1996.
A three-hybrid system to detect RNA-protein interactions in vivo.
Proc. Natl. Acad. Sci. USA
93:8496-8501[Abstract/Free Full Text].
|
| 49.
| Shepley, M. 1997. Personal communication.
|
| 50.
|
Shepley, M. P.,
B. Sherry, and H. L. Weiner.
1988.
Monoclonal antibody identification of a 100-kDa membrane protein in HeLa cells and human spinal cord involved in poliovirus attachment.
Proc. Natl. Acad. Sci. USA
85:7743-7747[Abstract/Free Full Text].
|
| 51.
|
Sonenberg, N.
1987.
Regulation of translation by poliovirus.
Adv. Virus Res.
33:175-204[Medline].
|
| 52.
|
Takegami, T.,
B. L. Semler,
C. W. Anderson, and E. Wimmer.
1983.
Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides.
Virology
128:33-47[Medline].
|
| 52a.
|
Teterina, N. L.,
K. M. Kean,
A. E. Gorbalenya,
V. I. Agol, and M. Girard.
1992.
Analysis of the functional significance of amino acid residues in the putative NTP-binding pattern of the poliovirus 2C protein.
J. Gen. Virol.
73:1977-1986[Abstract/Free Full Text].
|
| 53.
|
Tolskaya, E. A.,
L. I. Romanova,
M. S. Kolesnikova,
A. P. Gmyl,
A. E. Gorbalenya, and V. I. Agol.
1994.
Genetic studies on the poliovirus 2C protein, an NTPase. A plausible mechanism of guanidine effect on the 2C function and evidence for the importance of 2C oligomerization.
J. Mol. Biol.
236:1310-1323[Medline].
|
| 54.
|
Toyoda, H.,
N. J. Niclin,
M. G. Murray,
C. W. Anderson,
J. J. Dunn,
F. W. Studier, and E. Wimmer.
1986.
A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein.
Cell
45:761-770[Medline].
|
| 55.
|
Van Aelst, L.,
M. Barr,
S. Marcus,
A. Polverino, and M. Wigler.
1993.
Complex formation between RAS and RAF and other protein kinases.
Proc. Natl. Acad. Sci. USA
90:6213-6217[Abstract/Free Full Text].
|
| 56.
|
van Kuppeveld, F. J.,
W. J. Melchers,
K. Kirkegaard, and J. R. Doedens.
1997.
Structure-function analysis of coxsackie B3 virus protein 2B.
Virology
227:111-118[Medline].
|
| 57.
|
van Kuppeveld, F. J.,
P. J. van den Hurk,
J. Zoll,
J. M. Galama, and W. J. Melchers.
1996.
Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication.
J. Virol.
70:7632-7640[Abstract].
|
| 58.
|
van Kuppeveld, F. J. M.,
J. G. J. Hoenderop,
R. L. L. Smeets,
P. H. G. M. Willems,
H. B. P. M. Dijkman,
J. M. D. Galama, and W. J. G. Melchers.
1997.
Coxsackie protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release.
EMBO J.
16:3519-3532[Medline].
|
| 59.
|
Wimmer, E.,
C. U. Hellen, and X. Cao.
1993.
Genetics of poliovirus.
Annu. Rev. Genet.
27:353-436[Medline].
|
| 60.
| Xiang, W., A. Cuconati, D. Hope, K. Kirkegaard, and E. Wimmer. A protein linkage map of poliovirus P3 proteins. Submitted
for publication.
|
| 61.
|
Xiang, W.,
A. Cuconati,
A. V. Paul,
X. Cao, and E. Wimmer.
1995.
Molecular dissection of the multifunctional poliovirus RNA-binding protein 3AB.
RNA
1:892-904[Abstract].
|
| 62.
|
Yu, S. F.,
P. Benton,
M. Bovee,
J. Sessions, and R. E. Lloyd.
1995.
Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity.
J. Virol.
69:247-252[Abstract].
|
J Virol, February 1998, p. 1297-1307, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Adams, P., Kandiah, E., Effantin, G., Steven, A. C., Ehrenfeld, E.
(2009). Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity. J. Biol. Chem.
284: 22012-22021
[Abstract]
[Full Text]
-
de Jong, A. S., de Mattia, F., Van Dommelen, M. M., Lanke, K., Melchers, W. J. G., Willems, P. H. G. M., van Kuppeveld, F. J. M.
(2008). Functional Analysis of Picornavirus 2B Proteins: Effects on Calcium Homeostasis and Intracellular Protein Trafficking. J. Virol.
82: 3782-3790
[Abstract]
[Full Text]
-
Martinez-Salas, E., Pacheco, A., Serrano, P., Fernandez, N.
(2008). New insights into internal ribosome entry site elements relevant for viral gene expression. J. Gen. Virol.
89: 611-626
[Abstract]
[Full Text]
-
Taylor, M. P., Kirkegaard, K.
(2007). Modification of Cellular Autophagy Protein LC3 by Poliovirus. J. Virol.
81: 12543-12553
[Abstract]
[Full Text]
-
Yin, J., Liu, Y., Wimmer, E., Paul, A. V.
(2007). Complete protein linkage map between the P2 and P3 non-structural proteins of poliovirus. J. Gen. Virol.
88: 2259-2267
[Abstract]
[Full Text]
-
Moffat, K., Knox, C., Howell, G., Clark, S. J., Yang, H., Belsham, G. J., Ryan, M., Wileman, T.
(2007). Inhibition of the Secretory Pathway by Foot-and-Mouth Disease Virus 2BC Protein Is Reproduced by Coexpression of 2B with 2C, and the Site of Inhibition Is Determined by the Subcellular Location of 2C. J. Virol.
81: 1129-1139
[Abstract]
[Full Text]
-
Teterina, N. L., Levenson, E., Rinaudo, M. S., Egger, D., Bienz, K., Gorbalenya, A. E., Ehrenfeld, E.
(2006). Evidence for functional protein interactions required for poliovirus RNA replication.. J. Virol.
80: 5327-5337
[Abstract]
[Full Text]
-
Kaiser, W. J., Chaudhry, Y., Sosnovtsev, S. V., Goodfellow, I. G.
(2006). Analysis of protein-protein interactions in the feline calicivirus replication complex. J. Gen. Virol.
87: 363-368
[Abstract]
[Full Text]
-
Zell, R., Seitz, S., Henke, A., Munder, T., Wutzler, P.
(2005). Linkage map of protein-protein interactions of Porcine teschovirus. J. Gen. Virol.
86: 2763-2768
[Abstract]
[Full Text]
-
Uetz, P., Rajagopala, S. V., Dong, Y.-A., Haas, J.
(2004). From ORFeomes to Protein Interaction Maps in Viruses. Genome Res
14: 2029-2033
[Abstract]
[Full Text]
-
de Jong, A. S., Melchers, W. J. G., Glaudemans, D. H. R. F., Willems, P. H. G. M., van Kuppeveld, F. J. M.
(2004). Mutational Analysis of Different Regions in the Coxsackievirus 2B Protein: REQUIREMENTS FOR HOMO-MULTIMERIZATION, MEMBRANE PERMEABILIZATION, SUBCELLULAR LOCALIZATION, AND VIRUS REPLICATION. J. Biol. Chem.
279: 19924-19935
[Abstract]
[Full Text]
-
Campanella, M., de Jong, A. S., Lanke, K. W. H., Melchers, W. J. G., Willems, P. H. G. M., Pinton, P., Rizzuto, R., van Kuppeveld, F. J. M.
(2004). The Coxsackievirus 2B Protein Suppresses Apoptotic Host Cell Responses by Manipulating Intracellular Ca2+ Homeostasis. J. Biol. Chem.
279: 18440-18450
[Abstract]
[Full Text]
-
Harris, J. R., Racaniello, V. R.
(2003). Changes in Rhinovirus Protein 2C Allow Efficient Replication in Mouse Cells. J. Virol.
77: 4773-4780
[Abstract]
[Full Text]
-
de Jong, A. S., Wessels, E., Dijkman, H. B. P. M., Galama, J. M. D., Melchers, W. J. G., Willems, P. H. G. M., van Kuppeveld, F. J. M.
(2003). Determinants for Membrane Association and Permeabilization of the Coxsackievirus 2B Protein and the Identification of the Golgi Complex as the Target Organelle. J. Biol. Chem.
278: 1012-1021
[Abstract]
[Full Text]
-
Agirre, A., Barco, A., Carrasco, L., Nieva, J. L.
(2002). Viroporin-mediated Membrane Permeabilization. PORE FORMATION BY NONSTRUCTURAL POLIOVIRUS 2B PROTEIN. J. Biol. Chem.
277: 40434-40441
[Abstract]
[Full Text]
-
de Jong, A. S., Schrama, I. W. J., Willems, P. H. G. M., Galama, J. M. D., Melchers, W. J. G., van Kuppeveld, F. J. M.
(2002). Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis. J. Gen. Virol.
83: 783-793
[Abstract]
[Full Text]
-
Mueller, S., Cao, X., Welker, R., Wimmer, E.
(2002). Interaction of the Poliovirus Receptor CD155 with the Dynein Light Chain Tctex-1 and Its Implication for Poliovirus Pathogenesis. J. Biol. Chem.
277: 7897-7904
[Abstract]
[Full Text]
-
Guo, D., Rajamäki, M.-L., Saarma, M., Valkonen, J. P. T.
(2001). Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system. J. Gen. Virol.
82: 935-939
[Abstract]
[Full Text]
-
López, L., Urzainqui, A., Domínguez, E., García, J. A.
(2001). Identification of an N-terminal domain of the plum pox potyvirus CI RNA helicase involved in self-interaction in a yeast two-hybrid system. J. Gen. Virol.
82: 677-686
[Abstract]
[Full Text]
-
Shimizu, H., Agoh, M., Agoh, Y., Yoshida, H., Yoshii, K., Yoneyama, T., Hagiwara, A., Miyamura, T.
(2000). Mutations in the 2C Region of Poliovirus Responsible for Altered Sensitivity to Benzimidazole Derivatives. J. Virol.
74: 4146-4154
[Abstract]
[Full Text]
-
Klein, M., Hadaschik, D., Zimmermann, H., Eggers, H. J., Nelsen-Salz, B.
(2000). The picornavirus replication inhibitors HBB and guanidine in the echovirus-9 system: the significance of viral protein 2C. J. Gen. Virol.
81: 895-901
[Abstract]
[Full Text]
-
Pfister, T., Jones, K. W., Wimmer, E.
(2000). A Cysteine-Rich Motif in Poliovirus Protein 2CATPase Is Involved in RNA Replication and Binds Zinc In Vitro. J. Virol.
74: 334-343
[Abstract]
[Full Text]
-
Tacken, M. G. J., Rottier, P. J. M., Gielkens, A. L. J., Peeters, B. P. H.
(2000). Interactions in vivo between the proteins of infectious bursal disease virus: capsid protein VP3 interacts with the RNA-dependent RNA polymerase, VP1. J. Gen. Virol.
81: 209-218
[Abstract]
[Full Text]
-
Pfister, T., Wimmer, E.
(1999). Characterization of the Nucleoside Triphosphatase Activity of Poliovirus Protein 2C Reveals a Mechanism by Which Guanidine Inhibits Poliovirus Replication. J. Biol. Chem.
274: 6992-7001
[Abstract]
[Full Text]
Top
Abstract
Introduction
