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Journal of Virology, June 2001, p. 5381-5384, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5381-5384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Biophysical Analysis of Natural Variants of the
Multimerization Region of Epstein-Barr Virus Lytic-Switch
Protein BZLF1
Matthew R.
Hicks,1
Sara
Balesaria,1
Cahora
Medina-Palazon,1
Maya J.
Pandya,1,2
Derek N.
Woolfson,1,2 and
Alison J.
Sinclair1,*
School of Biological
Sciences1 and Centre for Biomolecular
Design and Drug Development,2 School of
Biological Sciences, University of Sussex, Brighton, East Sussex
BN1 9QG, United Kingdom
Received 6 December 2000/Accepted 26 February 2001
 |
ABSTRACT |
BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV)
replication. On the basis of limited sequence homology and mutagenesis
experiments, BZLF1 has been described as a member of the bZip family of
transcription factors, but this prospect has not been rigorously tested
to date. Here, we present biophysical analysis of the multimerization
domain of BZLF1, from three natural variants of EBV, and demonstrate
for the first time that the region between amino acids 196 and 227 is
sufficient to direct folding as a coiled-coil dimer in vitro.
| |
TEXT |
BZLF1 (also known as Zebra, Zta, Z,
and EB1) is a key component of the machinery that induces the lytic
replicative cycle of Epstein-Barr virus (EBV) (3, 4, 6, 22,
27). Increased expression of BZLF1 is one of the first events
that can be detected following the induction of the lytic cycle in
EBV-harboring B lymphocytes, and the enforced expression of BZLF1 is
sufficient to induce the lytic cycle in cells containing EBV genomes.
BZLF1 functions as a transcription factor and activates its own
expression and that of a subset of EBV and cellular genes through
sequence-specific BZLF1 response elements within their respective
promoters. Furthermore, BZLF1 also acts as a replication factor by
interacting specifically with the viral lytic origin of replication
(23), again through specific BZLF1 response elements
(reviewed in references 21 and 24-26).
BZLF1 has been previously described as a member of the bZip family
(2, 5, 7, 13-15, 20, 25, 28). In support of this
conjecture, it has been shown that BZLF1 contains adjacent DNA-binding
(approximately residues 175 to 195) and multimerization (approximately
residues 196 to 245) regions, and the protein interacts with specific
DNA sequence elements (2, 5, 8, 13, 15, 20, 28, 30) as a
multimer (2, 8, 15). By analogy with other members of the
bZip family, multimerization has been assumed to occur through the
folding of a coiled-coil interface within this region (i.e., residues
196 to 245). The analysis of deletion mutants of BZLF1 and mutants
containing one or more amino acid substitutions at residues within the
proposed coiled-coil interface provided some support for this model
(2, 5, 7, 13-15, 20, 25, 28), although there has been
disagreement about the influence of some residues (Y200 and L225, for
example) on the ability to multimerize (7, 12). Therefore,
while the results of numerous studies have been consistent with the
existence of a coiled-coil interface on BZLF1, the studies have fallen
short of rigorously testing the model.
To date, three naturally occurring sequence variants within the
multimerization domain of BZLF1 have been described. Previous analyses
have focused on the BZLF1 sequence deduced from the B95-8 isolate of
EBV (1); using this as a reference sequence, the two
variants are A205S (19) and A206S (10). In
this study, we sought to determine whether further variants of the
multimerization region occur in natural isolates of EBV before
exploring biophysically whether BZLF1 is indeed able to form multimers
through a coiled-coil interface.
The regions surrounding exons 2 and 3 of BZLF1 were amplified by PCR
using DNA from Burkitt's lymphoma cell lines (Rael, Mutu, Jijoye, and
Akata) (9, 17, 27) and from nine lymphoblastoid cell lines
established from patients with EBV-associated diseases. The DNA
sequences were then determined. The BZLF1 coding sequence was
remarkably conserved, with 48 of the 50 residues invariant (Fig.
1). However, examples of each of the
known natural variants were also identified; six samples contained
A205S and two contained A206S, but none of the isolates contained the
double variation A205S-A206S.
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FIG. 1.
Sequence variation within the BZLF1 multimerization
domain. The deduced protein sequences encoded by BZLF1 exons 2 and 3 from 11 isolates of EBV are shown aligned with the B95-8 sequence.
Variations are shaded.
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The propensity of all three natural variants of BZLF1 to form coiled
coils was assessed using two structural modeling computer programs:
COILS (16) and MULTICOIL (31). Surprisingly,
neither program predicted the formation of a coiled-coil motif in BZLF1 approaching that of established members of the bZip family, such as
GCN4 and C/EBP (Fig. 2). As a more direct
test of whether the three natural variants of BZLF1 were able to fold
as coiled coils, synthetic peptides encompassing the minimal
multimerization domain BZLF1 (residues 196 to 227) were synthesized and
their secondary structures were assessed using circular dichroism (CD)
spectroscopy (Fig. 3A). The three BZLF1
peptides displayed spectra characteristic of 
-helical structures,
with minima around 208 and 222 nm (the more negative the signal at 222 nm, the higher the -helical content). For comparison, a
heat-denatured peptide with an unfolded, random structure displaying a
negligible signal in this region is also shown. The characteristic
double minima indicative of -helical structures were clear for all
three peptides, although a reduced signal intensity relative to that of
the B95-8 peptide was observed for A205S and, to a lesser extent,
A206S. This finding suggests that these natural variants are somewhat
less helical than B95-8. Further analysis of the stability of the
-helical structures revealed that all three BZLF1 peptides exhibited
sigmoidal unfolding curves typical of cooperatively folded structures,
such as coiled coils (Fig. 3B). However, the midpoint temperatures of
unfolding (Tm) of the peptides (25, 17, and
19°C for B95-8, A205S, and A206S, respectively, at a 100 µM
concentration) were much lower than that observed for the archetypal
leucine zipper peptide from GCN4, which ranges from ~50°C at 5 µM
to ~70°C at 500 µM (18, 29).
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FIG. 2.
Sequence-based coiled-coil propensity of the three
natural BZLF1 variants. The maximum probability (Pmax) of coiled-coil
formation was assessed for each of the three BZLF1 natural sequence
variants and the indicated proteins using the programs COILS
(16) and MULTICOIL (31).
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FIG. 3.
The BZLF1 peptides are -helical in solution. (A)
Three BZLF1
peptides pepB95-8...LLQHYREVAAAKSSENDRLRLLLKQMCPSLDV
pepA205S...LLQHYREVASAKSSENDRLRLLLKQMCPSLDV
pepA206S...LLQHYREVAASKSSENDRLRLLLKQMCPSLDV were
synthesized on an Applied Biosystems 432A automated, continuous-flow
peptide synthesizer using solid-phase 9-fluorenylmethoxy carbonyl
chemistry and purified by high-pressure liquid chromatography (as
described previously [11]). The secondary structures
were analyzed using CD spectroscopy using a Jasco J-715
spectropolarimeter fitted with a six-cell changer Peltier temperature
controller. The buffer system contained 25 mM potassium phosphate, 100 mM sodium chloride, and 1 mM dithiothreitol. The data are shown for
peptide concentrations of 100 µM measured at 5°C. pepB95-8 is shown
as closed circles, pepA205S as open circles, and pepA206S as open
diamonds. The spectrum for pepB95-8 at 95°C is shown as crosses. (B)
The signal of each BZLF1 peptide at 222 nm was determined using CD
spectroscopy across the indicated range of temperatures. pepB95-8 is
shown as closed circles, pepA205S as open circles, and pepA206S as open
diamonds.
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Although the data indicated that the three BZLF1 peptides are able to
form -helical structures, they did not address whether the
structures are multimeric, indicative of coiled coils. The Tm of each peptide was therefore determined by
CD spectroscopy at four different peptide concentrations (10, 30, 100, and 200 µM) (Fig. 4A). The increase in
Tm observed for each peptide is characteristic
of a cooperatively folded multimeric structure, since it reflects a
shift in the equilibrium between unfolded monomers and folded
multimers. We can therefore conclude that residues 196 to 227 of all
three natural variants of BZLF1 are sufficient to promote the formation
of -helical, cooperatively folded, oligomeric structures. The
preferred oligomeric states of the peptides were then assessed using
sedimentation equilibrium ultracentrifugation. The averaged molecular
weights of the three BZLF1 peptides were determined. A plot of the
calculated molecular weights relative to those of the respective
monomers is shown in Fig. 4B. The plots for all three peptides leveled
off at a ratio of 2, suggesting that the dominant species in solution
are dimers.
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FIG. 4.
The three BZLF1 peptides self-associate as dimers. (A)
The thermal stability of each peptide was measured by CD spectroscopy
at the four concentrations indicated. pepB95-8 is shown as closed
circles, pepA205S as open circles, and pepA206S as open diamonds. (B)
The weight-average molecular weight of the three BZLF1 peptides was
determined by sedimentation equilibrium centrifugation in a Beckman
XL-I analytical ultracentrifuge fitted with a titanium An60-Ti rotor,
using software supplied by Beckman Coulter UK. Three concentrations of
each peptide were used (100 to 500 µM) in a buffer system containing
25 mM potassium phosphate, 100 mM sodium chloride, and 1 mM
dithiothreitol. To relate this molecular weight to the extent of
oligomerization, the calculated average molecular weight was divided by
the monomer molecular weight for each peptide. The error bars show 95%
confidence intervals.
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Thus, although established coiled-coil prediction methods
suggested that the BZLF1 multimerization regions either are atypical coiled coils or have a low probability of forming such structures, our
subsequent biophysical analyses revealed (i) that residues 196 to 227 of BZLF1 are able to fold into helical multimers in solution and (ii)
that the molecular weights of the BZLF1 peptides in solution approach
those expected for dimers. In addition to providing the first
biophysical data in support of a coiled-coil dimerization interface for
BZLF1, our analyses revealed that the interface of all three natural
variants of BZLF1 is inherently less stable than that of archetypal
members of the bZip family. This suggests that other regions of BZLF1
may contribute to the formation of a robust dimer in vivo. Indeed, it
has been suggested that residues carboxy terminal to position 227 may
contribute to the stability of the BZLF1 multimer (20).
The relevance, if any, of the small differences in
Tm among the three natural variants of BZLF1
remains to be established.
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ACKNOWLEDGMENTS |
This research was funded by grants from the Medical Research
Council, the Wellcome Trust, and the Society of General Microbiology to
A. J. Sinclair and by grants from the Wellcome Trust and BBSRC to
D. N. Woolfson.
We thank K. Takada and M. Rowe for cell lines and A. Rickinson and
Quin-Yun Yao for the infectious mononucleosis and nasopharyngeal carcinoma series lymphoblastoid cell lines. Peptides were synthesized by Chris Kowalczyk (University of Sussex).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, University of Sussex, Brighton, E. Sussex BN1 9QG,
United Kingdom. Phone: (44) 1273 678 194. Fax: (44) 1273 678 433. E-mail: a.j.sinclair{at}Sussex.ac.uk.
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Journal of Virology, June 2001, p. 5381-5384, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5381-5384.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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