Download Self-Interaction of the Herpes Simplex Virus Type 1

Virology 257, 341–351 (1999)
Article ID viro.1999.9698, available online at http://www.idealibrary.com on
Self-Interaction of the Herpes Simplex Virus Type 1 Regulatory Protein ICP27
Yan Zhi, Kathryn S. Sciabica, and Rozanne M. Sandri-Goldin1
Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4025
Received December 14, 1998; returned to author for revision January 28, 1999; accepted March 10, 1999
The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a nuclear phosphoprotein required for viral lytic
infection, which acts partly at the posttranscriptional level to affect RNA processing and export. In the present study, we show
that ICP27 can interact with itself in vivo. Immunofluorescent staining of cells expressing both an ICP27 mutant with a
deletion of the major nuclear localization signal and wild-type ICP27 showed that the mutant protein was efficiently imported
into the nucleus in the majority of the cotransfected cells, suggesting heterodimer formation between the wild-type and
mutant proteins. Coimmunoprecipitation experiments using epitope-tagged wild-type ICP27 and a series of ICP27 mutants
with deletions and insertions in important functional regions of the protein revealed that the C-terminal cysteine-histidine-rich
zinc-finger-like region of ICP27 was required for the self-association. Furthermore the self-association was also shown in
yeast using two-hybrid assays, and again, an intact C-terminal zinc-finger-like region was required for the interaction. This
study provides biochemical evidence that ICP27 may function as a multimer in infected cells. © 1999 Academic Press
Key Words: ICP27 self-interaction; HSV-1 regulatory protein.
teins may yield products that exhibit dominant negative
phenotypes because these proteins contain multiple functional sites that can be mutated independently (Herskowitz,
1987). This has been shown to occur for a number of viral
regulatory proteins, including HIV tat and rev (Malim et al.,
1989; Mermer et al., 1990; Pearson et al., 1990), and the
HSV-1 regulatory proteins VP16 (Friedman et al., 1988), ICP4
(Shepard et al., 1990), and ICP8 (Gao and Knipe, 1991). This
has also been shown for ICP27 (Smith et al., 1991). Specifically, mutations in the activation region of ICP27, between
amino acids 260 and 434 exhibited a trans-dominant negative phenotype in transfection experiments and during
infection (Smith et al., 1991). In some instances, it has been
shown that the dominant negative phenotype arises by the
formation of heterodimers containing one wild-type and
one mutant subunit (Shepard et al., 1990; Hope et al., 1992).
HIV Rev and HSV-1 ICP4 have been shown to form dimers
or multimers (Metzler and Wilcox, 1985; Michael and Roizman, 1989; Shepard et al., 1990; Shepard and DeLuca, 1991;
Zapp et al., 1991; Hope et al., 1992; Gallinari et al., 1994) that
include mutant and wild-type proteins (Shepard et al., 1990;
Hope et al., 1992). Therefore because activation mutants of
ICP27 can be trans-dominant, we tested whether ICP27
also forms dimers or multimers.
Here, we show that ICP27 can interact with itself in vivo.
Immunofluorescent staining of a mutant ICP27 protein that
lacks the major nuclear localization signal (NLS) showed
that import into the nucleus was facilitated when wild-type
ICP27 was co-expressed, suggesting heterodimer formation. Self-interaction as assayed by coimmunoprecipitation
of epitope-tagged wild-type and mutant forms of ICP27 and
by the yeast two-hybrid system implicated the C-terminal
INTRODUCTION
The herpes simplex virus type 1 (HSV-1) regulatory
protein ICP27 is a 512 amino acid, nuclear phosphoprotein that has an apparent molecular mass of 63 kDa
(Ackerman et al., 1984). ICP27 is essential for viral replication (Sacks et al., 1985; Rice and Knipe, 1988, 1990,
Rice et al., 1989; McMahan and Schaffer, 1990), and it
has been shown to perform some of its regulatory functions at the posttranscriptional level by influencing RNA
processing and export (Smith et al., 1992; McLauchlan et
al., 1992; Sandri-Goldin and Mendoza, 1992; Phelan et al.,
1993; Hardwicke and Sandri-Goldin, 1994; Hardy and
Sandri-Goldin, 1994; Sandri-Goldin et al., 1995; McGregor
et al., 1996; Sandri-Goldin and Hibbard, 1996; Soliman et
al., 1997; Phelan and Clements, 1997; Mears and Rice,
1998; Sandri-Goldin, 1998). That is, ICP27 appears to
contribute to the shut off of host protein synthesis by
impairing host cell splicing (Hardwicke and Sandri-Goldin, 1994; Hardy and Sandri-Goldin, 1994) and to the
efficient expression of HSV-1 early and late gene products by affecting 39 RNA processing (McLauchlan et al.,
1992; McGregor et al., 1996) and viral RNA export (Phelan
et al., 1996; Soliman et al., 1997; Sandri-Goldin, 1998).
The inhibition of function of a wild-type gene product can
be accomplished by the expression of a variant of the same
protein. This is because mutation of some regulatory pro-
1
To whom correspondence should be addressed at Department of
Microbiology and Molecular Genetics, College of Medicine, B240 Medical Sciences I, University of California, Irvine, CA 92697-4025. Fax:
(949) 824-8598. E-mail: [email protected].
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All rights of reproduction in any form reserved.
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ZHI, SCIABICA, AND SANDRI-GOLDIN
zinc-finger-like region as directly involved in the self-interaction. Furthermore mutants with lesions in this region
were not found to be trans-dominant (Smith et al., 1991),
which supports the notion that the zinc-finger-like region
may be required for multimerization.
RESULTS
An ICP27 NLS mutant protein can be efficiently
localized to the nucleus by heterodimer formation
with wild-type ICP27
We showed previously that mutations in the activation
region of ICP27 (residues 260–480) but not the splicing
repressor region were trans-dominant to the wild-type
activity, and the dominance was gene dosage dependent
(Smith et al., 1991). These results suggested that ICP27
may interact with itself to form dimers or multimers in
vivo. To investigate this possibility, cells were cotransfected with a plasmid that expresses a mutant ICP27
protein lacking the major nuclear localization signal
(NLS) and with a plasmid expressing wild-type ICP27.
The NLS deletion mutant D2DS5 (Fig. 1) is inefficiently
imported to the nucleus (Hibbard and Sandri-Goldin,
1995). We reasoned that if ICP27 can self-associate,
cotransfection with a construct expressing the wild-type
protein would increase the nuclear localization of D2DS5
by heterodimer formation. To be able to distinguish the
wild-type protein from the mutant protein in the same
cell, a Flag-epitope-tagged version of D2DS5, in which a
synthetic Flag epitope was fused to the N terminus was
constructed (Fig. 1). Wild-type ICP27 does not encode
this epitope and thus does not stain with the anti-Flag
monoclonal antibody M2Ab. Further, the deletion in
D2DS5 extends from amino acid 104 to 178 and encompasses the epitope to which the anti-ICP27 monoclonal
antibody H1113 reacts (Hibbard and Sandri-Goldin, 1995;
Mears et al., 1995). Thus both the wild-type and mutant
protein can be distinguished when expressed in the
same cells. For wild-type ICP27, nuclear staining was
seen as expected (Figs. 2A and 2B). While diffuse nuclear staining is seen in Fig. 2A, a somewhat punctate
distribution, along with more diffuse nuclear staining,
can be seen in Fig. 2B. This pattern reflects the finding
that a portion of the nuclear ICP27 has been shown to
colocalize with reassorted splicing factors (Phelan et al.,
1993; Sandri-Goldin et al., 1995). In transfections with
D2DS5 alone, overall diffuse nuclear and bright cytoplasmic staining was observed (Figs. 2C and 2D). The same
staining pattern was reported previously with this mutant
(Hibbard and Sandri-Goldin, 1995). Although D2DS5
lacks the major NLS, the protein is not excluded from the
nucleus. Similar results were reported for other NLS
mutants by Mears et al. (1996) who postulated that there
may be minor nuclear localization sequences that map to
the C-terminal portions of ICP27 that enable inefficient
import. In contrast to the bright cytoplasmic staining
FIG. 1. Diagrammatic description of ICP27 constructs. (A) Schematic
representation of the 512 amino acid coding region of ICP27 shows the
positions of deletion (horizontal lines) and insertion (vertical lines)
mutations. The regions encompassing the nuclear export signal (NES)
(Sandri-Goldin, 1998), the nuclear localization signal (NLS) (Mears et
al., 1995), the arginine-rich regions (R1/R2) (Hibbard and Sandri-Goldin,
1995), the activation and splicing repression regions (Hardwicke et al.,
1989), and the zinc-finger-like region (CCHC) (Vaughan et al., 1992) are
shown. Mutant DNES contains a deletion of amino acids 4–27 (SandriGoldin, 1998), R9DN6 has a deletion of amino acids 28–163, D2DS5 has
a deletion of amino acids 104–178 (Hibbard and Sandri-Goldin, 1995),
N6DS13 has a deletion of amino acids 164–262, N6R is a frame-shift
mutant and encodes the N-terminal portion of the protein from amino
acids 1–163, and H17 has a deletion of amino acids 288–444 (Hardwicke et al., 1989). Mutant N2 contains both a substitution of 2 amino
acids and an insertion of 2 amino acids between residues 459 and 460,
and S18 contains an insertion of 4 amino acids between residues 504
and 505 (Hardwicke et al., 1989). (B) A schematic representation of the
Flag-epitope-tagged D2DS5 mutant and wild-type ICP27 constructs
used in the coimmunoprecipitation experiments shows the location
and sequence of the Flag epitope fused to mutant and wild-type
proteins.
observed with D2DS5 alone, when wild-type ICP27 was
cotransfected with D2DS5 at a ratio of 5:1, .50% of the
cells were found to have a predominant nuclear fluorescence with light cytoplasmic staining (Fig. 2E) or no
detectable cytoplasmic staining (Fig. 2F). These staining
patterns were never observed in transfections with
D2DS5 alone, suggesting that heterodimer formation
with wild-type protein facilitated NLS mutant import.
Self-interaction of ICP27 in vivo can be shown by
coimmunoprecipitation of Flag-epitope-tagged wildtype ICP27 and various mutant polypeptides
To verify the self-interaction of ICP27 in vivo and to
enable mapping of the region of the protein involved in
ICP27 INTERACTS WITH ITSELF IN VIVO
343
FIG. 2. Immunofluorescent staining of wild-type ICP27 and an NLS deletion mutant. RSF cells were transfected with plasmids encoding wild-type
ICP27 and an NLS deletion mutant termed D2DS5, either singly or in combination. A Flag-epitope-tag was inserted in-frame into the N-terminus of
D2DS5 (Fig. 1B). (A and B) Cells were transfected with the wild-type ICP27 expressing plasmid pSG130B/S and were stained with anti-ICP27
monoclonal antibody H1113. (C and D) Cells were transfected with pFlag-D2DS5 and stained with anti-Flag monoclonal antibody M2Ab. (E and F) Cells
were transfected with pSG130B/S and pFlag-D2DS5 at a ratio of 5:1 and were stained with the anti-Flag M2Ab antibody.
the interaction, we designed a Flag-epitope-tagged
ICP27 construct in which a synthetic Flag epitope was
fused to the N terminus of the wild-type ICP27 polypeptide (Fig. 1B). The Flag-epitope-tagged construct was
cotransfected with plasmids expressing wild-type ICP27
and a series of ICP27 mutants containing in-frame deletions such that their mobility on SDS–polyacrylamide
gels was distinguishable from the Flag-epitope-tagged
protein (Fig. 1A). Two major protein species were precipitated with anti-Flag M2Ab from cells cotransfected
with plasmids expressing the Flag-epitope-tagged ICP27
and either wild-type ICP27 or each individual mutant,
including: DNES, D2DS5, N6DS13, and H17 (Fig. 3). One
band corresponded to the Flag-epitope-tagged ICP27
polypeptide with an apparent molecular weight of ;65
kDa (indicated by arrowheads), and the other band corresponded to either wild-type or mutant ICP27 (Fig. 3).
The amount of the H17 protein that coimmunoprecipitated with Flag-ICP27 appeared to be somewhat less
than the amounts seen with wild-type ICP27 or the other
mutants. While the amount varied somewhat in different
experiments, $10% of the H17 protein was coprecipitated with anti-Flag M2Ab, indicating that the H17 protein
could interact with wild-type Flag-epitope-tagged ICP27.
In addition, deletion mutant H7, which maps to the activation region (deletion of amino acids 384–450) (Hardwicke et al., 1989) was also coimmunoprecipitated with
the anti-Flag M2Ab antibody (data not shown). Because
only Flag-epitope-tagged ICP27 can be immunoprecipitated with anti-Flag M2Ab (Fig. 3), the coimmunoprecipitation of wild-type and mutant ICP27 polypeptides verified that ICP27 can interact with itself in vivo. Furthermore these data demonstrate that the nuclear export
signal from amino acids 4 to 27 (Sandri-Goldin, 1998), the
nuclear localization signal, and the arginine-rich regions
that follow it from amino acids 104 to 178 (Hibbard and
Sandri-Goldin, 1995; Mears et al., 1995), the internal
region of the protein from amino acids 164 to 262, and
the activation region from amino acids 288 to 444 (Hardwicke et al., 1989) were all not involved in the interaction
because these regions were deleted in the mutants
DNES, D2DS5, N6DS13, and H17, respectively (Fig. 1A).
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ZHI, SCIABICA, AND SANDRI-GOLDIN
FIG. 3. Immunoprecipitation of 32P-labeled proteins with anti-ICP27 or anti-Flag monoclonal antibodies. RSF cells were transfected with plasmids
encoding a series of ICP27 mutants as indicated or cotransfected with these mutants along with Flag-epitope-tagged wild-type ICP27 plasmid.
Twenty-four hours after transfection, the cells were infected with 27-LacZ virus to boost expression of the transfected plasmids. 32P-labeling was
performed for 5 h, after which cells were harvested. For R9DN6, [35S]methionine labeling was performed for 5 h. In all cases except two, nuclear
extracts were prepared. When D2DS5 and R9DN6 were used, whole cell extracts were prepared because of the nuclear translocation defect of these
mutant proteins (Hibbard and Sandri-Goldin, 1995). One half of each extract was immunoprecipitated with monoclonal antibodies H1113 and H1119
to ICP27, and the other half was immunoprecipitated with monoclonal antibody M2Ab to the Flag epitope, as indicated. For R9DN6, a polyclonal
antibody to the C-terminal half of ICP27 was used (Hardwicke et al., 1989). The antigen-antibody complexes were resolved on 7.5% SDS–
polyacrylamide gels, which were exposed to film. The positions of the Flag-epitope-tagged ICP27 polypeptides are indicated by arrowheads.
However, the frame-shift mutant N6R, which contains
only the N-terminal portion of ICP27 from amino acids 1
to 163, was not able to be coimmunoprecipitated with
M2Ab in lysates from cells that were cotransfected with
N6R and Flag-epitope-tagged ICP27 (Fig. 3). This mutant
protein also encodes 30 amino acids that are not found
in ICP27 from the site of the frame-shift to the first stop
codon. This result suggests that the N-terminus of ICP27
is either not involved or is not sufficient for interaction
with wild-type ICP27. It should be noted that anti-Flag
M2Ab did immunoprecipitate a major degradation product of wild-type ICP27 with an apparent size of ;30 kDa,
but this band was clearly distinguishable from mutant
N6R (Fig. 3). This degradation product was also immunoprecipitated by the anti-ICP27 monoclonal antibodies,
H119 and H113, which react with two different N-terminal
epitopes (Mears et al., 1995), suggesting that the N
terminus of wild-type ICP27 including the Flag epitope
was intact in the truncated degradation product.
These results implicated the C terminus of the protein
as the region required for the interaction. It seemed
possible that we might better define the region, and
confirm that it was the only region required for self
interaction, by expressing increasing truncations of
ICP27 until only the multimerization region was present,
as has been done for ICP4 (Gallinari et al., 1994). These
investigators found that a truncated ICP4 protein containing amino acids 343–490 retained the capacity to
form a heterodimer with a longer ICP4 peptide. Two
additional in-frame deletion mutants were generated,
R9DN6, containing a deletion of residues 28–163 (Fig.
1A), and R9DS13, containing a deletion of residues 28–
262. Their capability of interacting with wild-type ICP27
was tested in the coimmunoprecipitation assay. Because
the major nuclear localization signal (NLS) of ICP27 is
located from amino acids 110 to 137 (Mears et al., 1995),
both mutant proteins were defective in nuclear translocation. Furthermore several major phosphorylation sites
of ICP27 appear to cluster at the very N terminus (Zhi and
Sandri-Goldin, 1999), and the epitopes recognized by
monoclonal antibodies H1113 and H1119, have also been
mapped to the N-terminal half of the protein (Mears et al.,
ICP27 INTERACTS WITH ITSELF IN VIVO
1995). Therefore whole cell extracts were prepared from
cells that were transfected with the mutant constructs
and the cells were labeled with [35S]methionine instead
of [32P]orthophosphate as was done in the previous
experiments. Mutant proteins were immunoprecipitated
with a polyclonal antibody raised against the C-terminal
half of ICP27 (Hardwicke et al., 1989). A higher background of nonspecific bands was found with the polyclonal antibody; however, the mutant protein R9DN6 was
clearly resolved as a 40-kDa species (Fig. 3). As expected, the mutant protein alone was not precipitated
with the anti-Flag antibody, however R9DN6 was coprecipitated with Flag-epitope-tagged ICP27, indicating
that a truncated protein containing the first 28 amino
acids fused to amino acids 164–512 was sufficient for
association with wild-type ICP27 to occur, indicating that
the region between amino acids 28 and 164 was not
involved in multimerization.
Mutant protein R9DS13 was resolved as a 35-kDa
species, but the level of expression, measured both by
immunoprecipitation with polyclonal antibody and by
Western blot analysis (data not shown), was extremely
low compared to the wild-type protein and to the other
mutant proteins that were used in this study. The very
low level of R9DS13 protein precluded our ability to
detect coprecipitation with full-length ICP27.
Taken together, these results suggest that the C-terminal splicing repressor region from amino acids 450 to
512 may be involved in the self-interaction in vivo because this is the only region of the protein that was
retained in all of the deletion mutants that interacted with
the wild-type protein.
The zinc-finger-like region of ICP27 is required for its
self-interaction in vivo
Two additional lines of evidence suggested that the
C-terminal splicing repressor region might be involved in
the self-interaction. First, mutants in this region were not
trans-dominant to the wild-type protein (Smith et al.,
1991), suggesting that mutants in this region may be
unable to form multimers that are compromised in activity. Second, the zinc-finger-like motif within the splicing
repressor region that maps to residues 483–508
(Vaughan et al., 1992) was shown to be required for the
interaction of ICP27 with splicing complex proteins (Sandri-Goldin and Hibbard, 1996), suggesting that this region of ICP27 can undergo protein–protein interactions.
Unfortunately, we were not able to directly test the coimmunoprecipitation of the C-terminal mutant with a deletion of residues 434–504 due to the instability of this
protein in vivo (data not shown). Instead, a mutant with a
small insertion in the C-terminal region was used. Mutant S18, which contains an insertion of 4 amino acids
within the zinc-finger-like motif, between residues 504
and 505 (Hardwicke et al., 1989; Sandri-Goldin and Hib-
345
FIG. 4. The zinc-finger-like motif in the C-terminal region of ICP27 is
involved in the self-interaction in vivo. (A) RSF cells were transfected
with pS18 or cotransfected with the same plasmid along with the
Flag-epitope-tagged wild-type ICP27 plasmid as indicated. (B) Cells
were transfected with a Flag-epitope-tagged version of S18 and untagged wild-type ICP27 (pSG130B/S) either or alone or in combination
as indicated. (C) Cells were transfected with pN2 alone or were cotransfected with pN2 and pFlag-ICP27. Immunoprecipitations were performed as described in the legend to Fig. 3. The immunoprecipitated
proteins were fractionated on 6% SDS–polyacrylamide gels and were
detected by autoradiography. Flag-epitope-tagged ICP27 and S18 are
indicated by arrowheads.
bard, 1996), yields a stable protein, although the level of
protein detected was considerably lower than that seen
with the other mutants tested in Fig. 3. Transfections
were performed with S18 alone or along with the Flagepitope-tagged ICP27 plasmid. The immunoprecipitated
proteins were resolved on a 6% SDS–polyacrylamide gel
to separate the S18 mutant protein and Flag-tagged
wild-type ICP27. The mutant protein was immunoprecipitated with anti-ICP27 monoclonal antibodies but not with
anti-Flag M2Ab as expected (Fig. 4A). More importantly,
S18 protein could not be detected in the coimmunoprecipitation with anti-Flag antibody in extracts from cells
that were cotransfected with plasmids expressing S18
and Flag-epitope-tagged ICP27 proteins (Fig. 4A). Because the level of the S18 protein was much lower than
that of wild type, we sought to confirm this result by
constructing a Flag-epitope-tagged S18 in which the Flag
epitope was fused to the N terminus of the mutant.
Wild-type ICP27 protein did not react with anti-Flag
M2Ab (Fig. 4B), and wild-type ICP27, which was much
more abundant than S18 protein, was not coimmunopre-
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ZHI, SCIABICA, AND SANDRI-GOLDIN
FIG. 5. A ts protein that is trans-dominant to wild-type ICP27 interacts with itself and with wild type ICP27. RSF cells were transfected with ptsLG4
(Smith et al., 1991), pFlag-ICP27 or pFlag-tsLG4, either alone or in combination. Twenty-four hours after transfection, cells were infected with 27-LacZ
at 39°C. Labeling with [32P]orthophosphate was performed for 5 h as described under Materials and Methods. Immunoprecipitations were performed
as described in the legend to Fig. 3. The immunoprecipitated proteins were fractionated on 6% SDS–polyacrylamide gels and were detected by
autoradiography. The position of Flag-ICP27 and Flag-tsLG4 are indicated by arrowheads.
cipitated with anti-Flag antibody in extracts from cells
that were cotransfected with plasmids expressing ICP27
and Flag-epitope-tagged S18 (Fig. 4B). In contrast, mutant N2, which contains a small insertion outside the
zinc-finger-like motif between residues 459 and 460
(Hardwicke et al., 1989), was able to be coimmunoprecipitated with anti-Flag M2Ab in cotransfections with the
Flag-epitope-tagged wild-type ICP27 expression plasmid
(Fig. 4C). Therefore these data indicate that the zincfinger-like motif of ICP27 appears to be required for
self-interaction in vivo.
Next we sought to better define the region required for
the association of ICP27 with itself by using additional
mutants. A viral temperature-sensitive mutant, termed
tsLG4 was tested. The mutation in tsLG4 maps to the
C-terminal region, where a histidine residue replaced the
arginine residue at position 480 (Smith et al., 1991). This
substitution occurs just 3 amino acids upstream of the
region that we have called the zinc-finger-like motif,
although there are additional histidine and cysteine residues in the region between amino acids 460 and 483. It
seemed possible that at nonpermissive temperature, the
ts protein might not interact with wild-type protein because of conformational alterations that might perturb
the zinc-finger-like region. A plasmid (ptsLG4) that contains the tsLG4 gene from the viral mutant tsLG4 (Smith
et al., 1991) was used. In addition, a Flag epitope was
added to this construct, to yield the plasmid, pFlagtsLG4. Cells were transfected with ptsLG4, pFlag-tsLG4,
and pFlag-ICP27, alone or in combination and then infected with 27-LacZ at permissive (34°C) or nonpermissive (39°C) temperature. The tsLG4 protein was found to
coimmunoprecipitate with Flag-epitope-tagged ICP27 at
34°C, as anticipated (data not shown), but was also
found to coimmunoprecipitate with Flag-epitope-tagged
wild-type and tsLG4 protein at 39°C (Fig. 5). This result
suggests that the histidine substitution in tsLG4 occurs
outside of the region of interaction. Although the tsLG4
protein is not defective at permissive temperature, it is
defective in several functions including late gene expression and host shut off at nonpermissive temperature,
where the protein is conformationally altered (Smith et
al., 1991, 1992; Hardy and Sandri-Goldin, 1994; Soliman et
al., 1997). These results suggest that inability to multimerize is not the cause of the defect in the tsLG4 protein
because it can interact with itself and with wild-type
ICP27 at nonpermissive temperature (Fig. 5). However,
these results are in accord with the finding that mutant
tsLG4 was found to be trans-dominant to the wild-type
phenotype (Smith et al., 1991), supporting the notion that
ICP27 functions in vivo as a multimer. The results seen
with tsLG4 also suggest that the ICP27 self-interacting
region does not extend as far as amino acid 480, the site
of the substitution.
Self-interaction of ICP27 can be demonstrated in
yeast
To further verify the self-interaction of ICP27 in vivo, we
used the yeast two-hybrid system (Fields and Song,
1989). A plasmid containing the ICP27 coding sequence
from amino acids 69 to 512 fused to the Gal4 DNAbinding domain (pDBD-Rsr27) was used as the wild-type
ICP27 bait plasmid. The N terminus of ICP27 from amino
acids 14 to 64 is highly acidic, which results in activation
of the reporter gene in the absence of the Gal4 transactivation domain (data not shown). Therefore this region
was removed to allow the detection of specific protein–
protein interactions. Plasmids encoding truncated versions of ICP27 fused to the Gal4 DNA-binding domain
were also constructed and cotransformed into yeast with
a plasmid containing the entire wild-type ICP27 coding
sequence fused to the Gal4 transactivation domain
(pAD-ICP27). The truncated ICP27 fusion constructs included: pDBD-Rsr27D192-261, containing amino acids
69–191 and amino acids 262–512 of ICP27; pDBDRsr27DC, containing amino acids 69–433 of ICP27;
pDBD-Rsr27N2, containing amino acids 69–514 of the
ICP27 insertion mutant N2; pDBD-Rsr27S18, containing
amino acids 69–516 of the ICP27 insertion mutant S18;
pDBD-179-512, containing amino acids 179–512 of ICP27;
and pDBD-C27, containing amino acids 262–512 of ICP27
(Fig. 6A). Interaction was detected by assaying transformed colonies for b-galactosidase expression. The ex-
ICP27 INTERACTS WITH ITSELF IN VIVO
347
a 4 amino acid insertion between residues 504 and 505
in the zinc-finger-like motif of ICP27 also failed to interact
with AD-ICP27. In contrast, the DBD-Rsr27N2 containing
a 2 amino acid substitution and a 2 amino acid insertion
between residues 459 and 460 outside the zinc-fingerlike motif was able to interact with AD-ICP27 (Fig. 6B).
Taken together, these results strongly support the notion
that the region encompassing the zinc-finger-like motif in
the C-terminal region of ICP27 is required for selfinteraction.
DISCUSSION
FIG. 6. Self-interaction of ICP27 demonstrated in the yeast twohybrid system. (A) A diagrammatic representation of the fusion constructs used to test ICP27 self-interaction in the yeast strain SFY526.
The Gal4 DNA-binding domain (DBD, amino acids 1–147) was fused to
various bait proteins. DBD-Rsr27 encodes most of ICP27 from amino
acids 69 to 512; DBD-Rsr27D192-261 encodes amino acids 69–191 and
amino acids 262–512; DBD-Rsr27DC encodes amino acids 69–433;
DBD-179-512 encodes amino acids 179–512; DBD-C27 encodes amino
acids 262–512; DBD-Rsr27N2 encodes amino acids 69–514 of insertion
mutant N2; and DBD-Rsr27S18 encodes amino acids 69–516 of insertion mutant S18. DBD-p53, encoding amino acids 72–390 of murine p53,
was used as a control plasmid. The Gal4 activation domain (AD, amino
acids 768–881) was fused to full-length ICP27 (AD-ICP27). AD-SV40 T
encoding the SV40 large T-antigen fused to the Gal4 activation domain
was used as a control plasmid. Transcription of the lacZ reporter gene
containing upstream Gal4 DNA binding sites was used to indicate
interaction between two proteins (Fields and Song, 1989). (B) Yeast
strain SFY526 was cotransformed with different fusion constructs as
indicated and b-galactosidase activity was determined by the colony lift
method. Cotransformation of DBD-p53 and AD-SV40 T served as a
positive control as these two proteins have been shown to interact with
each other (Li and Fields, 1993). Transformation with DBD-Rsr27 alone
did not result in b-galactosidase production and served as negative
control. 11 indicates colonies turned blue within 4 h; 1 indicates
colonies turned blue within 8 h; 2 indicates colonies did not turn blue
within 8 h.
pression of fusion proteins in yeast was detected by
Western blot analysis using antibodies directed against
the Gal4 DNA-binding domain or the Gal4 transactivation
domain (data not shown). DBD-Rsr27, DBD-179-512, and
DBD-Rsr27D192-261 fusion proteins interacted with ADICP27, full-length ICP27 fused to the Gal4 activation domain (Fig. 6B). However, DBD-Rsr27DC fusion protein did
not interact with AD-ICP27, suggesting that the Cterminal region of ICP27 was involved in the selfassociation. In accord with the results found in the coimmunoprecipitation assays, DBD-Rsr27S18 containing
In this study, we have found that the HSV-1 immediate
early regulatory protein ICP27 can interact with itself in
vivo to form multimers. It was demonstrated previously
that two other HSV-1 immediate early regulatory proteins, ICP4 and ICP0 form dimers in vivo (Metzler and
Wilcox, 1985; Michael and Roizman, 1989; Shepard et al.,
1990; Shepard and DeLuca, 1991; Ciufo et al., 1994;
Gallinari et al., 1994). Heterodimer formation between
wild-type ICP27 and a mutant with a deletion of the NLS
was strongly suggested in immunofluorescent staining
experiments, where the mutant protein was efficiently
localized to the nucleus when wild-type ICP27 was also
present (Fig. 2). Further, coimmunoprecipitation between
Flag-epitope-tagged wild-type ICP27 and nontagged mutant and wild-type proteins verified the interaction and
pointed to a requirement for an intact C-terminal zincfinger-like region (Figs. 3 and 4). These findings were
further supported by the yeast two-hybrid experiments,
which showed that an insertion within but not outside the
zinc-finger-like region resulted in a loss of the interaction
between the ICP27 moieties fused to Gal4 DNA-binding
and activation domains (Fig. 6). Finer mapping was obtained with mutant tsLG4. The substitution mutation occurs just outside of the region we have defined as the
zinc-finger-like motif at position 480. Self-interaction was
found at both permissive and nonpermissive temperatures, indicating that the region required for interaction
does not extend as far as residue 480.
Unlike the dimerization domain of ICP4 that can be
removed from the context of the ICP4 protein and fused
to a heterologous protein and still result in dimerization
(Gallinari et al., 1994), the context of the C-terminal region surrounding the zinc-finger-like domain of ICP27
may be important for proper folding or protein stability.
The C-terminal half of ICP27 fused to the Gal4 DNA
binding domain was unable to interact with wild-type
protein in yeast even though the zinc-finger-like domain
was intact (Fig. 6B). We were unable to confirm this result
in mammalian cells due to the extremely low expression
or stability of a peptide encoding the C-terminal half of
ICP27. We also noted that a C-terminal deletion mutant
protein, although stable and easily detectable in yeast,
was highly unstable and undetectable in mammalian
348
ZHI, SCIABICA, AND SANDRI-GOLDIN
cells. This is in contrast to a frame-shift mutant that
expresses a truncated peptide encoding only the first
163 amino acids of ICP27 and to an N-terminal degradation product that is frequently found (Fig. 3). Both of these
truncated peptides were found to be abundant and stable. Thus expression of the C-terminal portion of ICP27
alone or in the context of a fusion protein or deletion of
the C-terminus may result in misfolding that alters protein interactions and stability. Misfolded proteins may be
more susceptible to proteases.
Several previous studies support the notion that protein context and structure is important for ICP27 to interact with proteins and RNA. For example, it has been
shown that different regions of ICP27 interact with ICP4
as measured in vitro using GST-ICP27 fusion proteins
(Panagiotidis et al., 1997). In those studies, portions of
ICP27 were fused to GST so that different protein contexts were presented. In addition, the region between
amino acids 140 and 175 contains two arginine-rich regions (Hibbard and Sandri-Goldin, 1995), one of which
resembles RGG boxes found in RNA-binding proteins
(Kiledjian and Dreyfuss, 1991; Ghisolfi et al., 1992; Burd
and Dreyfuss, 1994; Liu and Dreyfuss, 1995). This region
has been shown to be necessary for RNA binding by
ICP27 both in vitro (Mears and Rice, 1996) and in vivo
(Sandri-Goldin, 1998). Mears and Rice (1996) found that
RNA binding of ICP27 in vitro was much stronger if the C
terminus was not present in GST fusion proteins. These
authors postulated that masking of the RGG box may
occur to some extent in the context of the full-length
protein. In fact, we also measured the self-association of
ICP27 in vitro by far Western analysis (data not shown).
We found two anomalies in those experiments. First, the
interaction between mutant forms of ICP27 translated in
vitro and a full-length, bacterially expressed ICP27-thiofusion protein was always stronger than the interaction
between wild-type ICP27 protein translated in vitro and
the ICP27 fusion protein (data not shown). Second, the
far Western analysis suggested that the N-terminal region containing the NLS and two arginine-rich regions
was required for the interaction. That this is not the case
in vivo was shown in the immunofluorescent staining
experiment with the NLS deletion mutant (Fig. 2), in the
coimmunoprecipitation experiments (Fig. 3), and in the
yeast two-hybrid assays (Fig. 6). This may suggest that
the denaturing and renaturing process in the far Western
procedure could result in improper folding of the protein
where interacting regions are more readily masked, and
perhaps other regions become more prominent or exposed. Thus regions of ICP27 found to interact with
proteins or RNA by in vitro far Western studies may not
correspond to the regions involved in those interactions
in vivo.
For these reasons, we determined the region of ICP27
required for self-interaction in vivo. The advantages of
using in vivo assays are that the protein is allowed to be
fully modified to achieve its native conformation and
self-association is examined under physiologically relevant conditions. However, the intrinsic disadvantage is
that protein expression can not be tightly controlled in
transfected cells or yeast cells. This results in difficulty in
directly comparing the strength of the association between different protein pairs and in determining the stoichiometry of the interaction. Thus we cannot determine
whether ICP27 forms dimers or multimers.
We have shown previously that tsLG4 is trans-dominant at the nonpermissive temperature (Smith et al.,
1991). We postulated that the interference with wild-type
function resulted from the formation of heterodimers that
are impaired in function. Here, we have provided biochemical support for that hypothesis, in that the tsLG4
protein was able to interact with itself and with wild-type
ICP27 at both permissive and nonpermissive temperature. This finding is in accord with the notion that the
dominant negative phenotype of activation mutants results from heterodimer formation.
ICP27 has been shown to be a multifunctional protein.
The region of ICP27 required for interaction with splicing
factors was mapped to the zinc-finger-like motif between
amino acids 483–512 (Sandri-Goldin and Hibbard, 1996).
We postulated that this region may be involved in protein–protein interactions. Here, we presented strong biochemical evidence demonstrating that ICP27 self associates in vivo and that the C-terminal region beginning at
residues past 480 and extending to position 508 must be
intact for multimerization to occur. Although the boundaries have not been fully defined, these results implicate
the zinc-finger-like region of ICP27 in self-interaction and
suggest that ICP27 functions in vivo as a multimer.
MATERIALS AND METHODS
Cell lines and viruses
Rabbit skin fibroblast (RSF) cells, used for the transfections were grown as described previously (Hardwicke
et al., 1989). HSV-1 wild-type strain KOS 1.1, the ICP27 null
mutant 27-LacZ and which has an insertion of the LacZ
gene in the ICP27 locus were described previously
(Smith et al., 1992).
Recombinant plasmids
Plasmid pSG130B/S, which encodes the wild-type
ICP27 gene; pH17, which contains an ICP27 mutant with
a deletion of residues 288–444; pN2, which encodes an
ICP27 mutant with a 2 amino acid substitution and an
insertion of 2 amino acids between residues 459 and
460; and pS18, which encodes an ICP27 mutant with an
insertion of 4 amino acids between residues 504 and 505
were described previously (Hardwicke et al., 1989). Plasmid pD2DS5, containing a deletion of amino acids 104–
178, was described previously (Hibbard and Sandri-Gol-
ICP27 INTERACTS WITH ITSELF IN VIVO
din, 1994). Plasmid pN9M, which contains a 12-bp EcoRI
linker at a NaeI site that occurs at amino acid residue 27
in the ICP27 coding sequence (Hardwicke et al., 1989),
was partially digested with DrdI, and an 8-bp BglII linker
was inserted into the DrdI site that occurs five nucleotides upstream of the initiating methionine codon at the
translational start site of ICP27. The resulting plasmid
termed pN9B/D was digested with BglII and EcoRI and
an oligonucleotide encoding the first three amino acids
of ICP27 was inserted into these sites. The resulting
plasmid, pDNES contains a deletion of residues 4–27.
The deletion was verified by DNA sequencing. Plasmids
pN6, which encodes an ICP27 mutant with 1 amino acid
substitution and an insertion of 4 amino acids between
residues 163 and 164, and pS13, which encodes an
ICP27 mutant with an insertion of 4 amino acids between
residues 262 and 263, were described previously (Hardwicke et al., 1989). Plasmids containing these mutations
were digested at the BamHI site and the EcoRI site,
which occurs in the linker present in these mutants. The
BamHI-EcoRI fragment of pN6, containing the 59 noncoding region and the sequence encoding the N-terminal
portion of ICP27, from residues 1 to 163, was joined with
the EcoRI-SstI fragment of pS13, which contains the
sequence encoding the C-terminal portion of ICP27 from
residues 263 to 512 and the 39 noncoding region in the
pUC18 vector into which all of these mutants were originally cloned (Hardwicke et al., 1989). This resulted in a
frame-shift deletion mutant termed pN6R that encodes
the N-terminal portion of ICP27 from residues 1 to 163.
Plasmid pN6DS13 was created by insertion of a EcoRI
linker into EcoRI site in pN6R to restore the protein
reading frame, which resulted in a deletion of residues
164–262 of ICP27. In addition, the BamHI-EcoRI fragment
of pN9M, containing the 59 noncoding region and the
sequence encoding the N-terminus of ICP27, from residues 1 to 27, was joined with the same EcoRI-SstI fragment of pS13 described above, after appropriate modification of the DNA ends with Klenow. This created a
mutant termed pR9DS13 containing a deletion of residues 28–262 of ICP27. The BamHI-EcoRI fragment of
pN9M was also joined with the EcoRI-SstI fragment of
pN6, which contains the sequence encoding the C-terminal portion of ICP27 from residues 164 to 512, and
subsequently an EcoRI linker was inserted into EcoRI
site to restore the protein reading frame. The resulting
mutant, termed pN9DN6 contained a deletion of residues
28–163 of ICP27. All of these mutants were sequenced
around the sites of the deletions. Cloning and sequencing of plasmid ptsLG4 was described previously (Smith
et al., 1991).
For the yeast two-hybrid experiments, plasmid pGBT9
(Clontech) containing the Gal4 DNA-binding domain and
TRP1 marker was modified by inserting a 38-bp linker
containing SalI, DrdI, DraIII, and SstI sites to create
plasmid pGBT9(2). Plasmid pDBD-C27 was constructed
349
by inserting a 1.23 kb SalI-SstI fragment of ICP27 in-frame
into plasmid pGBT9(2). Plasmid pGBT9(2) was further
modified by inserting a 15-bp linker with an RsrII site into
the EcoRI and SalI sites to create plasmid pGBT9(3).
Plasmids pDBD-Rsr27, pDBD-Rsr27N2, and pDBDRsr27S18 were constructed by inserting a 1.8 kb RsrIISstI fragment of wild-type ICP27 from pSG130B/S, of the
N2 mutant of ICP27 from pN2, or the S18 mutant of ICP27
from pS18, respectively (Hardwicke et al., 1989), in-frame
into the plasmid pGBT9(3). Plasmid pDBD-Rsr27 was
digested with NcoI and SalI, and a 10-mer oligonucleotide linker was inserted to create the in-frame deletion
plasmid pDBD-Rsr27D192-261. Plasmid pDBD-Rsr27 was
digested with MscI and SstI, treated with Klenow, and
ligated to create plasmid pDBD-Rsr27DC. Plasmid
pGBT9 was modified by inserting a 33-bp linker into the
EcoRI and PstI sites to generate a new multiple cloning
site with SmaI, EcoRI, BamHI, SalI, and PstI sites to
create the plasmid pGBT9(5). Plasmid pDBD-179-512
was constructed by inserting a 1.49-kb SmaI-PstI fragment of ICP27 from pGBT9(3)Rsr27 in-frame into
pGBT9(5). Plasmid pAD-ICP27 was constructed by inserting a 2.4-kb BglII-SstI fragment from plasmid
pSG130D/B, described above, and a 9-bp adapter with
SstI and SalI sticky-ends into the BamHI and SalI sites of
the plasmid pGAD424 (Clontech), which contains the
Gal4 activation domain and LEU2 marker. All of the
constructs were sequenced around the sites of insertions. Plasmids pDBD-p53 and pAD-SV40 T (Clontech)
served as the positive control for detection of protein–
protein interactions (Li and Fields, 1993).
Yeast two-hybrid assay
Plasmids containing ICP27 constructs fused to the
Gal4 DNA-binding domain and activation domain, as
described above, were cotransformed into Saccharomyces cerevisiae strain SFY526 (Clontech), which carries
the Gal1-Lac Z reporter gene. Transformants were plated
on yeast media lacking tryptophan and leucine. Primary
transformants were screened for b-galactosidase expression by a colony lift filter assay (Clontech) using
X-gal as a chromogenic substrate. The color of the colonies was determined within 4 h of the addition of X-gal.
Flag-epitope-tagged ICP27 protein
A 34-mer oligonucleotide encoding amino acids
MDYKDDDDK (Hopp et al., 1988) was inserted in frame
into the DrdI site upstream of the translational start site
of ICP27. The resulting plasmid, pFlag-ICP27 expresses
a Flag epitope in frame at the amino terminus of the
ICP27 protein (Fig. 1). The pFlag-ICP27 was sequenced
around the site of the insertion. The same oligonucleotide was also inserted into the DrdI site of the plasmid
pD2DS5 (Hibbard and Sandri-Goldin, 1995), which contains a deletion of residues 104–178 encoding the major
350
ZHI, SCIABICA, AND SANDRI-GOLDIN
nuclear localization signal (Mears et al., 1995) and two
arginine-rich regions that directly follow it (Hibbard and
Sandri-Goldin, 1995), and into the plasmid pS18, with an
insertion between amino acids 504 and 505. The resulting plasmids were termed pFlag-D2DS5 and pFlag-S18,
respectively.
Virus infection, transfection, and radiolabeling
RSF cells were grown on 100-mm-diameter tissue culture dishes. Cells were transfected with lipofectamine
reagent (Life Technologies) used at 50 ml/dish, and the
plasmid DNA concentration was 10 mg/dish as described previously (Sandri-Goldin and Hibbard, 1996).
Twenty-four hours after transfection, cells were infected
with 27-LacZ at a multiplicity of infection of 10 PFU/cell.
RSF cells were routinely labeled for 5 h beginning 60 min
after infection. Labeling with either [32P]orthophosphate
(NEN) or [35S]methionine (NEN) was carried out at a
concentration of 100 mCi/ml in either phosphate-free
modified Eagle medium (ICN) or methionine-free modified Eagle medium (Life Technologies) containing 2%
fetal calf serum.
Immunofluorescence staining
RSF cells were grown on glass coverslips for 16 h and
then transfected with plasmids pSG130B/S (Sekulovich
et al., 1988), which expresses wild-type ICP27 and pFlagD2DS5 (described above), either singly or in combination
as described in the legend to Fig. 2. Transfections were
performed using lipofectamine according to the manufacturer’s directions (Life Technologies). Cells were fixed
48 h after transfection in 3.7% formaldehyde in PBS for 10
min at room temperature. Before staining, cells were
permeabilized in PBS containing 0.5% Nonidet P-40. Indirect immunofluorescent staining was performed as described (Sandri-Goldin et al., 1995). Primary antibodies
included the ICP27 monoclonal antibodies H1113 (Goodwin Institute, Plantation, FL), used at a dilution of 1:500,
and the anti-Flag epitope antibody M2Ab (Kodak) at a
dilution of 1:400. Coverslips were mounted in 90% glycerol and cells were examined with a Nikon UFX-II epifluorescent microscope with a 1003 objective lens with
a numerical aperture of 1.25.
Immunoprecipitation
RSF cells were rinsed and scraped into cold PBS and
nuclear extracts were prepared as described previously
(Sandri-Goldin and Hibbard, 1996). Nuclear and cytoplasmic extracts were mixed when cells were transfected
with mutant ICP27 containing deletions in the nuclear
localization signal (NLS). Immunoprecipitations were
performed with either anti-ICP27 monoclonal antibodies
H1113 and H1119 (Goodwin Institute), an anti-ICP27 polyclonal antibody (Hardwicke et al., 1989) or anti-Flag
epitope monoclonal antibody M2Ab (Kodak) under high-
salt conditions (0.5 M NaCl) as described previously
(Sandri-Goldin and Hibbard, 1996; Sandri-Goldin, 1998).
The immunoprecipitated proteins were fractionated on
SDS–polyacrylamide gels and detected by autoradiography.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI-21515
from the National Institute of Allergy and Infectious Diseases to R.M.S.-G.
K.S.S. was supported by NIAID Virology training grant AI-07815.
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