Download The herpesvirus saimiri ORF73 gene product interacts with host

Journal of General Virology (2004), 85, 147–153
Short
Communication
DOI 10.1099/vir.0.19437-0
The herpesvirus saimiri ORF73 gene product
interacts with host-cell mitotic chromosomes and
self-associates via its C terminus
Michael A. Calderwood,1 Kersten T. Hall,2 David A. Matthews3
and Adrian Whitehouse1
Correspondence
Adrian Whitehouse
[email protected]
Received 18 June 2003
Accepted 3 October 2003
1,2
School of Biochemistry and Molecular Biology1, and Institute of Cardiovascular Research2,
University of Leeds, Leeds LS2 9JT, UK
3
Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK
The herpesvirus saimiri (HVS) ORF73 gene product shares limited homology with the ORF73
protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). ORF73 is expressed in an in vitro
model of HVS latency, where the genome persists as a non-integrated circular episome. This
suggests it may have a similar role to KSHV ORF73 in episomal maintenance, by tethering
viral genomes to host-cell chromosomes. Here, the association of ORF73 with host mitotic
chromosomes is described. Deletion analysis demonstrates that the distal 123 aa of the ORF73
protein are required for mitotic chromosomal localization and for self-association. Moreover,
deletion of the extreme C terminus disrupts both self-association and host mitotic chromosome
colocalization. This suggests that HVS ORF73 has a similar role to KSHV ORF73 in episomal
maintenance and that association of ORF73 to host mitotic chromosomes is dependent on its
ability to form multimers.
Herpesvirus saimiri (HVS) is the prototype gamma-2
herpesvirus and has significant homology with other gammaherpesviruses including Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV) and murine
gammaherpesvirus-68 (MHV-68) (Albrecht et al., 1992;
Russo et al., 1996; Virgin et al., 1997). During latent infection the HVS genome, like that of other herpesviruses,
persists as a non-integrated circular episome. Analysis has
shown that a cluster of genes, namely open reading frames
(ORFs) 71, 72 and 73, are transcribed as a polycistronic
mRNA from a common latency-associated promoter in an
in vitro model of HVS latency (Hall et al., 2000a). Similar
profiles have been observed in various KSHV latently
infected cell lines (Dittmer et al., 1998; Talbot et al., 1999).
In both HVS and KSHV, ORF71 and ORF72 encode an
anti-apoptotic FLICE inhibitory protein (Thome et al.,
1997) and a cyclin D homologue (Chang et al., 1996; Jung
et al., 1994), respectively. However, ORF73 encodes a protein
with no known cellular homologue.
KSHV ORF73 encodes the latency-associated nuclear antigen
(LANA) (Kedes et al., 1997; Kellam et al., 1997; Rainbow
et al., 1997). HVS ORF73 has only limited sequence
homology with KSHV ORF73; however, both proteins
share a number of common features. Both consist of a large
central acidic repeat domain, flanked by a small N-terminal
region and a larger C terminus (Hall et al., 2000b). A major
function of LANA is to maintain the extrachromosomal
viral genome during KSHV latent infection (Ballestas et al.,
0001-9437 G 2004 SGM
1999; Cotter & Robertson, 1999). LANA has been shown to
associate with host-cell mitotic chromosomes via a region
at its N terminus encompassing aa 5–22 (Piolot et al.,
2001) and to specifically bind KSHV terminal repeat DNA.
Therefore, it acts as a tether attaching the KSHV latent viral
genome to the host-cell chromosomes (Ballestas & Kaye,
2001). This suggests that KSHV ORF73 is functionally analogous to the EBV nuclear antigen 1 (EBNA1) (Kedes et al.,
1997; Leight & Sugden, 2000). It has been well established
that EBNA1 is the only viral protein required for EBV viral
maintenance (Lee et al., 1999; Leight & Sugden, 2000; Lupton
& Levine, 1985). EBNA1 binds to the family repeat element
within the EBV genome (Yates et al., 1984, 1985) and
interacts with metaphase chromosomes via human EBNA1
binding protein 2 (EBP2) (Shire et al., 1999; Kapoor &
Frappier, 2003; Kapoor et al., 2001; Wu et al., 2002).
To date it is unknown if HVS ORF73 colocalizes with host
mitotic chromosomes and if so which domains are required.
Therefore, due to the lack of sequence homology between
HVS and KSHV ORF73, we aimed to identify and characterize the HVS ORF73 chromosomal binding mechanism.
To determine whether HVS ORF73 associates with host-cell
mitotic chromosomes, the complete ORF73 coding region
was PCR-amplified and inserted into the eukaryotic expression vector pEGFP-C2 (Invitrogen) to yield pEGFP-73,
which contains an N-terminal enhanced green fluorescent
protein (EGFP) tag (Fig. 1a). To determine the subcellular
localization of ORF73, a transient transfection was
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M. A. Calderwood and others
performed and the resulting pattern of expression observed. Cos-7 cells were transfected using Lipofectamine 2000
(Invitrogen) and 2 mg of construct DNA and incubated
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for 36 h. Cells were then fixed and permeabilized before
mounting in Vectashield containing DAPI (Vector).
The slides were then examined using a laser confocal
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Journal of General Virology 85
Herpesvirus saimiri ORF73 gene product
Fig. 1. Deletion analysis of the HVS ORF73 protein. (a) Schematic representation of deletion series of ORF73 protein in
pEGFP. PCR amplification was used to generate wild-type ORF73 and mutants containing only the N-terminal domain or a
fusion of the N- and C-terminal domains, deleting the central acidic domain. These were ligated into the eukaryotic expression
vector pEGFP-C2 to produce pEGFP-73, pEGFP-73N and pEGFP-73N-C. (b) To determine the subcellular localization of
these constructs Cos-7 cells were transfected with pEGFP-73 or pEGFP-73N-C. Cells were fixed 36 h later and visualized
by laser scanning confocal microscopy. (c) Schematic representation of further deletion mutants of the C terminus prepared
as described previously and ligated into pEGFP-NLS to produce pEGFP-NLS-73C and pEGFP-NLS-73CD1–D3. (d) To
determine the subcellular localization of the C-terminal deletion mutants, Cos-7 cells were transfected with each mutant, fixed
and visualized by laser scanning confocal microscopy.
microscope (Leica TCS SP) and a PlanApo 1006 UV oilimmersion lens.
As expected pEGFP displayed a fluorescent signal throughout the cell nucleus and cytoplasm. In contrast, pEGFP-73
resulted in a distinct nuclear speckling pattern as previously
reported (Hall et al., 2000b). More interesting was the
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observation that EGFP-73 fluorescence was coincident with
the DAPI-stained mitotic chromosomes (Fig. 1b). This
result suggests that HVS ORF73 encodes a chromosomebinding domain (CBD) and is capable of association with
host-cell chromosomes during mitosis.
In order to further characterize the ORF73 CBD, a range of
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ORF73 deletion mutants (Fig. 1a) was produced by a similar PCR method as described above and assessed for the
ability to colocalize with host mitotic chromosomes. Results
demonstrated that a deletion containing the ORF73 N
terminus (pEGFP-73N) localized to the nucleus of cells but
failed to associate with mitotic chromosomes. This confirms
previous data identifying two nuclear-localization signals
(NLSs) in the N terminus (Hall et al., 2000b) but suggests
that neither of them is capable of acting as a CBD (data
not shown). Previous studies have also demonstrated that
deletion of the central acidic domain resulted in a wild-type
distribution of fluorescence (Hall et al., 2000b). Analysis of
the clone pEGFP-73N-C in mitotic cells also demonstrated a
wild-type association with host-cell chromosomes (Fig. 1b).
These two mutants therefore suggest that the CBD is located
in the C terminus. Although we have previously suggested
that an additional NLS may reside within the C terminus
(Hall et al., 2000b), the C-terminal deletions used were all
produced in a second vector, pEGFP-NLS, which contains
the well-characterized NLS from SV40 (Kalderon et al.,
1984) (Fig. 1c). Transfection of clone pEGFP-NLS-73C
resulted in a strong nuclear distribution of fluorescence
which co-localized with the host-cell chromosomes
(Fig. 1d). This indicates that the CBD is located in the
ORF73 C terminus. This was further supported using
pEGFP-NLS-73CD1, which lacks the first 44 aa of the C
terminus: fluorescence was observed in the nucleus which
co-localized with the host-cell chromosomes during mitosis.
However, pEGFP-NLS-73CD2 and pEGFP-NLS-73CD3,
which contain small deletions from the C or N terminus
of pEGFP-NLS-73CD1 respectively, both localized to the cell
nucleus but failed to associate with mitotic chromosomes.
Overall, these results show that the C terminus is required
for chromosome association of HVS ORF73 and this
interaction requires the distal 123 aa. However, removal of
18 aa from the start or 12 aa from the end of this domain
abolishes chromosomal association and we have described
these domains as chromosome association sites (CAS) 1 and
2, respectively. This suggests that the chromosomal association of ORF73 may require multiple distinct elements,
hence explaining the relatively large domain required for
chromosomal association.
In order to further investigate the role of the essential
regions at each end of pEGFP-NLS-73CD1, sequence analysis was performed and identified a motif at aa 291–293
consisting of a proline followed by two lysines in CAS1. This
motif is similar to a SPKK motif identified in histone H1
and H2 proteins which is involved in histone binding to
the minor groove of the DNA (Churchill & Suzuki, 1989;
Suzuki, 1989). This PKK motif is also found in a number of
herpesvirus ORF73 homologues and in EBP2. To analyse
the possible significance of this motif in chromosomal
association, a series of constructs was produced in which the
amino acids in the PKK motif were replaced with alanines
(Fig. 2a). Substitution of all three amino acids resulted in
a complete loss of chromosome association (Fig. 2b). This
suggests that this motif is involved in the association of
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ORF73 with host-cell mitotic chromosomes. However,
single amino acid substitutions across this region failed to
inhibit chromosomal association, indicating a degree of
redundancy in this domain. In addition, deletion analysis
suggests that the extreme C terminus is also required for
chromosomal association; however, sequence analysis of
this region, CAS2, failed to identify any known motifs.
Therefore, single point mutations of selected amino acids
across the last 12 residues were generated and assessed for
chromosomal association. However, these point mutations
failed to disrupt the ability of ORF73 to associate with
mitotic chromosomes.
Another common feature displayed by gammaherpesvirus
ORF73 proteins is the ability to self-associate. To determine if HVS ORF73 formed multimers and which domains
were involved, we performed a co-immunoprecipitation
experiment using the EGFP-ORF73 deletion series and an
ORF73 Myc fusion protein as bait. To produce the ORF73
Myc fusion protein, the ORF73 gene was PCR-amplified
and inserted into the eukaryotic expression vector
pcDNA3.1myc-His-B (Invitrogen) to yield pMYC-73.
Transient transfection of 1 mg of pMYC-73 into Cos-7
cells resulted in the expression of full-length ORF73 fused
to the Myc peptide, as detected by Western blotting using
anti-c-Myc antibodies (data not shown). In order to perform the multimerization studies, Cos-7 cells were initially
co-transfected with pMYC-73 and pEGFP-NLS-73C; 36 h
post-transfection the cells were washed in ice-cold PBS,
before being harvested in 1 ml of lysis buffer (50 mM Tris/
HCl, pH 7?4, 150 mM NaCl, 1 mM EDTA, 1 % Triton
X-100, 1 mM AEBSF). The insoluble cellular debris was
removed and the supernatant was incubated with 50 ml of
anti-c-Myc agarose conjugate bead slurry (Sigma) for 2 h at
room temperature. The beads were then washed, separated
on a 10 % SDS-PAGE gel and blotted onto nitrocellulose
membranes. ORF73 was then detected using a 1 : 2000
dilution of anti-GFP antibody (Clontech), followed by
a 1 : 2000 dilution of anti-mouse immunoglobulin conjugated with horseradish peroxidase (Dako) and developed
using enhanced chemiluminescence (Amersham). Following
immunoprecipitation with the anti-Myc affinity resin, EGFPNLS-73C was detected when co-expressed with pMYC73
(Fig. 3). This demonstrated that the ORF73 protein can
self-associate and that the C terminus is sufficient for
multimerization. To further analyse the domains responsible for self-association, further EGFP-ORF73 constructs
covering the C terminus were utilized. Results showed that
pEGFP-NLS-73CD1 was sufficient for self-association and
that deletion of CAS1 did not disrupt self-association;
however, no interaction was observed upon deletion of
CAS2 (Fig. 3). Moreover, to determine whether the ORF73
co-immunoprecipitation is due to a common interaction
with DNA, rather than a specific protein–protein interaction, we repeated the homodimerization assay before
and after treatment of the ORF73 complexes with 10 mg of
DNase I (Fig. 3c). Confirmation that the digestion conditions
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Herpesvirus saimiri ORF73 gene product
Fig. 2. Point mutation analysis of CAS1 and 2. (a) Schematic representation of the series of point mutations within CAS1 and
2 indicating the amino acid substitutions produced. The effect of each mutation on chromosomal association is shown in the
table on the right. (b) Cos-7 cells were transfected with pEGFP-NLS-73CDPKK. Cells were fixed 36 h later and visualized by
laser scanning confocal microscopy.
eliminated DNA was obtained by gel electrophoresis (data
not shown). Results demonstrate that the DNase treatment had little effect on the self-association of the ORF73
protein.
Taken together these results suggest that chromosome
association of HVS ORF73 may be dependent upon the
ability to form multimers. Deletion of CAS2 is sufficient
to disrupt self-association and host-cell mitotic chromosome association. However, chromosomal association also
requires additional elements, as deletion of CAS1 does not
disrupt self-association but is sufficient to inhibit the colocalization with host-cell chromosomes.
This C-terminal 123 aa region maps to the same region of
LANA required for dimerization (Schwam et al., 2000). The
last 205 aa of the LANA C terminus are required for selfassociation and this region is conserved in other LANA-like
proteins. Schwam et al. (2000) also suggest that this Cterminal region is required for the accumulation of LANA as
discrete nuclear speckles, suggesting an involvement in the
nuclear distribution of LANA. This was further explained by
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the identification of a 15 aa domain located close to the
LANA C terminus, deletion of which disrupted the interaction of LANA with nuclear heterochromatin and the
characteristic nuclear speckling pattern (Viejo-Borbolla
et al., 2003). However, on further analysis this region did
not associate with heterochromatin following high-salt
washes, suggesting that this was not sufficient for the
interaction with heterochromatin. The current view remains
that LANA interacts with nuclear heterochromatin via an
N-terminal region, aa 5–22 (Piolot et al., 2001); however
deletion of aa 1129–1143 from full-length LANA may result
in conformation changes which disrupt the interaction of
the N-terminal CBD (Viejo-Borbolla et al., 2003). Similarly,
deletion of the extreme 12 aa of HVS ORF73 abolishes
chromosomal binding and self-association; however, at
present this analysis cannot determine whether these
residues play a specific role in the conformation of the
protein which may affect these functions.
In summary, we have demonstrated that HVS ORF73
co-localizes with mitotic chromosomes and this localization is dependent upon the distal 123 aa. We have identified
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M. A. Calderwood and others
Fig. 3. Self-association of the ORF73 gene product. (a) Cos7 cells remained untransfected (lane 1), or transfected with
pEGFP-NLS-73C (lanes 2 and 4), pEGFP-NLS (lane 3), pEGFP-NLS-73CD1 (lane 5), pEGFP-NLS-73CD3 (lane 6) or
pEGFP-NLS-73CD2 (lane 7); in addition cells were co-transfected with either pcDNA3.1myc-His-B (lane 4) or pMYC-73
(lanes 2, 3, 5–7). Thirty-six hours post-transfection the cells were harvested, lysed and incubated with anti-c-Myc agarose for
2 h at room temperature. The proteins bound to the beads were then separated by 10 % SDS-PAGE, transferred to
nitrocellulose membrane and probed using an anti-GFP antiserum. (b) Expression of each GFP construct was confirmed by
probing the supernatant remaining following incubation with anti-c-Myc agarose using anti-GFP antiserum. (c) Cos7 cells
remained untransfected (lane 1), or transfected with pEGFP-NLS-73C and pMYC-73 (lane 2). Thirty-six hours post
transfection the cells were harvested, lysed and incubated with anti-c-Myc agarose for 2 h in the absence (i) or presence (ii)
of 10 mg of DNaseI. The proteins bound to the beads were then separated by 10 % SDS-PAGE, transferred to nitrocellulose
membrane and probed using an anti-GFP antiserum.
a PKK motif, termed CAS1, with similarity to a motif
found in other chromosome-associated proteins and shown
that substitution of this motif abolishes chromosomal
localization. A second essential domain for chromosome
association, termed CAS2, has also been shown to be
essential for the formation of ORF73 multimers, leading us
to speculate that the association of ORF73 with hostcell chromosomes is dependent upon its ability to form
homo-multimers.
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