Molecular Medicine Unit, University of Leeds, St Jamess University Hospital, Leeds LS9 7TF, UK1
Author for correspondence: Adrian Whitehouse. Fax +44 113 244 4475. e-mail a.whitehouse{at}leeds.ac.uk
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Abstract |
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Introduction |
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Gene expression during the HVS lytic replication cycle is regulated by the products of the two major transcriptional regulating genes encoded by open reading frames (ORFs) 50 and 57 (Nicholas et al., 1991 ; Whitehouse et al.,1997a
, 1998a
, b
). The ORF 50 or R gene encodes two proteins which are homologous to the EBV BRLF1 protein (Rta), which is sufficient for disruption of latency in EBV-permissive B cells and epithelial cells (Ragoczy et al.,1998
; Zalani et al.,1996
), and the KSHV R protein, which induces lytic replication in transformed B cells (Lukac et al.,1998
; Sun et al., 1998
). The HVS ORF 50 proteins activate transcription directly following interactions with promoters containing a specific sequence motif, the consensus ORF 50 recognition sequence, CCN9GG (Whitehouse et al.,1997b
). Furthermore, ORF 50 contains a conserved carboxy-terminal activation domain required for ORF 50 transactivation and for the interaction between the ORF 50 proteins and the general cellular transcription factor TATA-binding protein (Hall et al.,1999
).
The second transcriptional activator encoded by HVS is ORF 57, which is homologous to genes identified in all classes of herpesviruses, including the EBV Mta protein transactivator encoded by BMLF1, ORF 57 of KSHV, IE63 or ICP27 of herpes simplex virus (HSV-1), ORF 4 of varicella-zoster virus (VZV) and UL69 in human cytomegalovirus (Albecht et al.,1992 ; Bello et al.,1999
; Kenney et al.,1989
; Nicholas et al.,1988
; Perera et al.,1994
; Russo et al., 1996
; Winkler et al.,1994
). ORF 57 is a 52 kDa multifunctional trans-regulatory protein. Transactivation of late viral genes by ORF 57 occurs independently of target gene promoter sequences and appears to be mediated at a post-transcriptional level (Whitehouse et al.,1998a
). Recent analysis has demonstrated that the ORF 57 protein has the ability to shuttle between the nucleus and cytoplasm and is required for efficient cytoplasmic accumulation of virus mRNA, suggesting that ORF 57 plays a role in mediating the nuclear export of viral transcripts (Goodwin et al.,1999
).
In addition to its transactivation properties, ORF 57 is responsible for repression of viral gene expression, which appears to correlate with the presence of introns within the target gene (Whitehouse et al.,1998a , b
). ORF 57 also redistributes both U2 and SC-35 splicing factors during an HVS infection into intense distinct nuclear aggregations. ORF 57 causes a more intense SC-35 staining with larger more intense nuclear spotting resulting in a less diffuse SC-35 background staining in the nucleus as compared with control cells (Cooper et al.,1999
). These observations further suggest that ORF 57 plays a role in RNA processing and shares some common properties with the IE63 and Mta proteins (Phelan & Clements, 1998
; Ruvolo et al.,1998
; Semmes et al.,1998
). In addition, the more widely studied IE63 protein also contributes to the shut-off of host-cell protein synthesis and to a decrease in cellular mRNA levels during infection (Hardwicke & Sandri-Goldin, 1994
; Hardy & Sandri-Goldin, 1994
).
Analysis of IE63 has shown that it contains a number of functional domains including an RGG box required for RNA binding (Mears & Rice, 1996 ), an amino-terminal nuclear localization signal (NLS) (Hibbard & Sandri-Goldin, 1995
; Mears et al., 1995
), a leucine-rich nuclear export signal (NES) (Sandri-Goldin, 1998
), and carboxy-terminal transactivation and repression domains (Sandri-Goldin et al., 1995
; Sandri-Goldin & Hibbard, 1996
). However, although the HVS ORF 57 protein has shown to possess some common properties with IE63, there is a limited degree of sequence homology between these proteins. As such, there is little information regarding the functional domains contained within the ORF 57 protein. At present, a leucine-rich NES has been identified which is required for the nuclear cytoplasmic shuttling of ORF 57. Moreover, utilizing ORF 57 fusion proteins, we have demonstrated that the RNA-binding determinant is contained within the amino terminus of ORF 57 (Goodwin et al., 1999
).
In this report, we have further investigated the properties of the ORF 57 protein and identified the functional domains responsible for the transactivation and repression properties of ORF 57. We demonstrate that the carboxy terminus is required for ORF 57 transactivation, repression and the more intense SC-35 nuclear spotting. This region contains two highly conserved motifs amongst its homologues, a zinc finger-like motif and a hydrophobic GLFF domain. Moreover, we show that the GLFF domain is required for transactivation by ORF 57 and is also involved in nuclear localization of the ORF 57 protein, whereas the zinc finger-like domain is required for transactivation, repression and the intense SC-35 nuclear spotting.
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Methods |
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Site-directed mutants containing alterations in elements of the zinc finger-like motif were produced by a method similar to that above. Histidine and cysteine residues were altered to phenylalanine and serine, respectively (underlined), using the forward primer CFOR2 and a series of reverse primers, ZF17REV (Fig. 1a). These 500 bp fragments incorporated MunI restriction sites, allowing the convenient cloning of each PCR product into p57 previously digested with MunI, deriving the zinc finger-like mutations, pZF17.
Viruses, cell culture and transfections.
HVS (strain A11) was propagated in Owl Monkey kidney (OMK) cells which were maintained in Dulbeccos modified Eagles medium (DMEM) (Life Technologies) supplemented with 10% foetal calf serum (FCS). Cos-7 and 293T cells were also maintained in DMEM supplemented with 10% FCS. Plasmids used in the transfections were prepared using Qiagen plasmid kits according to the manufacturers directions. Cells were seeded at 5x105 cells per 35 mm diameter Petri dish 24 h before transfection. Transfections were performed using Lipofectamine (Life Technologies) as described by the manufacturer, using 2 µg of the appropriate DNAs.
CAT assay.
Cell extracts were prepared 48 h after transfection and incubated with [14C]chloramphenicol in the presence of acetyl coenzyme-A as described previously (Gorman et al., 1982 ). The percentage acetylation of chloramphenicol was quantified by scintillation counting (Packard) of appropriate regions of the thin-layer chromatography plate.
RNA extraction.
Total RNA was isolated from control and transfected cells using Trizol reagent (Life Technologies). Chloroform (0·2 ml) was then added, and the solution vortex-mixed for 20 s and stored at 20 °C for 15 min. Samples were centrifuged for 15 min at 4 °C, and the aqueous phase containing nucleic acids was precipitated using 0·5 ml of isopropanol. After this, the pellet was washed with 70% ethanol, resuspended in 50 µl of water and stored at -70 °C.
RTPCR.
Total RNA (0·1 or 1 µg) was reversed transcribed with Superscript II (Life Technologies) for 1 h at 42 °C using an oligo(dT) primer. cDNA was then amplified by PCR using ORF 57 gene-specific primers, 57FOR and 3REV. As a RNA control GAPDH specific primers were also utilized. The amplification conditions were: 1 cycle of 5 min 95 °C, 30 cycles of 1 min 95 °C, 1 min 55 °C and 1 min 72 °C, followed by 10 min at 72 °C.
Polyclonal antibody generation.
Polyclonal antiserum was raised against a portion of recombinant ORF 57 protein. The ORF 57 fragment was expressed as a GST fusion protein in E. coli DH5 and purified from crude lysates by affinity chromatography with glutathioneSepharose 4B according to the manufacturers specification (Pharmacia), as previously described (Goodwin et al.,1999
). The purified recombinant protein was used to generate a polyclonal antibody in New Zealand White rabbits using standard protocols.
Immunofluorescence analysis.
Cells were fixed with 4% formaldehyde in PBS, washed in PBS and permeabilized in 0·5% Triton X-100 for 5 min. The cells were rinsed in PBS and blocked by preincubation with 1% (w/v) non-fat milk powder for 1 h at 37 °C. A 1:50 dilution of anti-ORF 57 antibody was layered over the cells and incubated for 1 h at 37 °C. Fluorescence-conjugated anti-rabbit immunoglobulin (Dako), 1:50 dilution, was added and the mixture incubated for 1 h at 37 °C. For SnRNP redistribution experiments, colocalization studies were performed using a 1:1000 dilution of anti-SC-35 MAb (Sigma) and 1:250 dilution of Texas red-conjugated anti-mouse immunoglobulin (Vector Laboratories). After each incubation step, cells were washed extensively with PBS. The immunofluorescence slides were observed using a Zeiss Axiovert 135TV inverted microscope with a Neofluar 40x oil-immersion lens.
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Results |
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The carboxy terminus is required for the transactivation and repression properties of the ORF 57 protein
We have previously shown that ORF 57 transactivates a range of HVS promoters, including those driving expression of the gB and capsid genes, but does not significantly alter the level of mRNA, suggesting that ORF 57 acts by a post-transcriptional mechanism. To identify the sequences which are required for ORF 57 transactivation activity, 1 µg of each deletion plasmid was cotransfected with 1 µg pgBCAT2. This plasmid contains the CAT coding region under the control of the HVS gB promoter (Whitehouse et al.,1998a ). Plasmid p57 was also used in the assay as a positive control. Both the HVS permissive OMK and the highly transfectable 293T cell lines were used. Cells were harvested after 48 h and assayed for CAT activity by standard methods (Gorman et al.,1982
) (Fig. 2a
). Severely reduced CAT activity was observed when each deletion construct was utilized in the assay. However, p57 was shown to transactivate the gB promoter to levels previously shown (Whitehouse et al.,1998a
). This suggested that the sequences contained within the ORF 57 carboxy terminus are required for transactivation by the ORF 57 protein.
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The ORF 57 carboxy-terminal deletions are transcribed at similar levels to the wild-type ORF 57 and produce nuclear proteins
In order to determine whether the lack of transactivation and repression by the ORF 57 deletion series was due to the removal of the ORF 57 carboxy terminus, or whether the removal of these sequences affected ORF 57 transcription or protein stability, immunofluorescence and RTPCR were performed. Initially, to determine whether each deletion construct produced a stable protein, Western blot and immunofluorescence analysis were attempted. Previously, we have studied the expression and cellular localization of the ORF 57 protein using the SB monoclonal antibody (Randall et al.,1983 ). However, this protein did not react with any of our deletion constructs using immunofluorescence, suggesting that the SB reactive epitope is contained within the carboxy portion of the ORF 57 protein (unpublished observations). Therefore, to analyse the ORF 57 carboxy terminus deletion series a polyclonal antiserum was raised against the amino portion of recombinant ORF 57 protein (Goodwin et al.,1999
). Unfortunately, the ORF 57 polyclonal antiserum did not react on a Western blot of HVS-infected or ORF 57-transfected cells (data not shown). Therefore, to determine if each deletion produced a protein product, immunofluorescence analysis of HVS-infected (m.o.i. of 1) and transiently transfected cells was performed using the polyclonal 57 antibody. Immunofluorescence analysis of HVS-infected cells resulted in a strong fluorescence of the nuclei of infected cells (data not shown). Similar results were observed with p57- and p57
13-transfected cells (Fig. 3a
); no reaction was observed with cells that had not been transfected (data not shown). This suggested that each deletion construct produced a protein which localized to the cell nucleus.
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The ORF 57 carboxy terminus is also required for an increase in the intensity of SC-35 nuclear spotting
We have previously demonstrated that the ORF 57 protein is also responsible for the redistribution of spliceosome components leading to intense spotting during HVS infection. ORF 57 causes a more intense SC-35 staining with larger more intense spotting, resulting in a less diffuse SC-35 background in the nucleus compared with control cells (Cooper et al.,1999 ). This redistribution correlates with the impairment of splicing by ORF 57. However, it is believed that splicing inhibition involves mechanisms more complex than simply redistributing spliceosome components. To further investigate this link we utilized members of the ORF 57 carboxy deletion series to determine if they maintained the ability to redistribute spliceosome components. Immunofluorescence was performed on untransfected, p57-transfected or p57
13-transfected cells. Cells were incubated for 24 h before being fixed and dual labelled with the polyclonal ORF 57 and SC-35 antibodies. Cells expressing the full-length ORF 57 protein resulted in a more intense nuclear spotting of the SC-35 spliceosome factor as previously described (Cooper et al.,1999
), whereas untransfected and p57
13-transfected cells showed no SC-35 redistribution, suggesting that the carboxy terminus is also required for the intense nuclear spotting of the SC-35 spliceosome component (Fig. 3a
).
Mutational analysis of the ORF 57 carboxy terminus
The above results indicate that the carboxy terminus is required for both the transactivation and repression properties of the ORF 57 protein. Fig. 4(a) shows amino acid sequence alignments of the carboxy termini of ORF 57 and a number of its homologues. As highlighted, the carboxy terminus of the ORF 57 homologues contains two positionally conserved motifs: a zinc finger-like domain conserved in all herpesviruses (Vaughan et al.,1992
), and a hydrophobic GLFF domain highly conserved in gammaherpesviruses, except in both cases for the MHV-68 ORF 57 gene. To determine the importance of these conserved motifs for ORF 57 transactivation and repression activity, a range of site-directed mutations within the GLFF (Fig. 4b
) and elements of the zinc finger-like (Fig. 4c
) motifs were constructed. These were generated by a PCR-based method which incorporated the alteration of one or more of the conserved residues. All constructs were confirmed by DNA sequencing (data not shown).
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The ORF 57 GLFF mutations are transcribed at similar levels to the wild-type ORF 57 and produce nuclear proteins
In order to determine whether the mutation of these residues affected the expression level or protein stability, RTPCR and immunofluorescence were again performed as described above. Firstly, RTPCR was performed using two serial dilutions of input RNA isolated from p57- or pGLFF14-transfected cells. The results (Fig. 6a) demonstrate that RTPCR products of the correct size and in similar quantities to the wild-type protein were amplified, suggesting that alteration of these residues did not affect ORF 57 transcription or RNA stability.
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The ORF 57 GLFF residues are not required for the intense SC-35 nuclear spotting
To determine if the GLFF residues are required for the ability of ORF 57 to redistribute spliceosome components, immunofluorescence was performed on untransfected, p57-transfected or pGLFF14-transfected cells. Cells were incubated for 24 h before being fixed and dual labelled with the polyclonal ORF 57 and SC-35 antibodies. Cells expressing the full-length ORF 57 protein but also the GLFF mutations resulted in a more intense nuclear spotting of the SC-35 spliceosome factor (Fig. 6b), suggesting that these residues are not required for redistribution of the SC-35 spliceosome component into the intense nuclear spots.
The zinc finger-like domain is required for both transactivation and repression
As previously mentioned, all ORF 57 homologues, except that of MHV-68, contain conserved residues which constitute a zinc finger-like domain (Vaughan et al.,1992 ). In order to investigate the role of this domain in the properties of the ORF 57 protein the histidine residue was changed to phenylalanine and the cysteine residues altered to serines, thereby introducing minimal overall structural or conformation perturbations in the polypeptide. To determine if the residues are required for the transactivation or repression properties of ORF 57, CAT assays were performed as described above. Initially, to identify if these residues are required for ORF 57 transactivation activity, 1 µg of each pZF17 was cotransfected with 1 µg of pgBCAT2. Plasmid p57 was also used in the assay as a positive control (Fig. 7a
). Results show that single mutations at the conserved histidine at 79526 bp and cysteines at 79538 and 79555 bp of the published sequence proved highly detrimental to ORF 57 transactivation, reducing CAT activity by approximately 90, 70 and 87%, respectively. Furthermore, multiple substitutions of these residues completely abrogated biological activity, showing that these conserved residues, which constitute elements of the zinc finger-like domain, are required for ORF 57 transactivation activity.
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The zinc finger-like mutations are expressed at similar levels to the wild-type ORF 57 and produce nuclear proteins
In order to determine whether the mutation of these residues affected expression levels or protein stability, RTPCR and immunofluorescence were again performed as described. Firstly, RTPCR was performed using two serial dilutions of input RNA isolated from p57- or pZF17-transfected cells. The results (Fig. 8a) demonstrate that RTPCR products of the correct size and in similar quantities to the wild-type protein were amplified, suggesting that the mutation of these residues does not affect RNA stability or transcription.
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The ORF 57 zinc finger-like domain is required for the intense SC-35 nuclear spotting
To determine if the zinc finger-like domain is required for the ability of the ORF 57 protein to redistribute spliceosome components, immunofluorescence was performed on untransfected, p57-transfected or p57ZF17-transfected cells. Cells were incubated for 24 h before being fixed and dual labelled with the polyclonal ORF 57 and SC-35 antibodies. As shown in Fig. 8(b), ZF mutations 1 and 3 showed no intense nuclear spotting suggesting, that this domain is required for spliceosome component redistribution. Similar results were observed for the remaining zinc finger-like mutations, ZF47 (data not shown). It was noted that ZF2, which mutated the cysteine residue at 79538 bp of the published sequence, resulted in what we have termed a partial spotting as shown in Fig. 8(b)
(panel vii).
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Discussion |
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Moreover, mutations within the GLFF hydrophobic domain result in a distinct subcellular localization. The majority of the hydrophobic GLFF proteins were observed in the nucleus; however, a proportion of the protein is present in the cytoplasm. This subcellular localization has never been observed with the wild-type ORF 57 protein or any other mutants tested to date. At present the implication of this result is yet to be elucidated. It is possible that these carboxy-terminal hydrophobic residues are involved in proteinprotein interactions with a nuclear cellular protein which retains the ORF 57 protein in the nucleus, or that this hydrophobic domain is itself a novel nuclear localization or retention signal. Further analysis of this domain is now required to fully determine its role in the multifunctional properties of the ORF 57 protein.
In addition to the role in transactivation, the zinc finger-like domain is required for the repression of intron-containing viral genes by ORF 57. Similar observations that the carboxy terminus has a role in repression of IE63 have been reported. Mutant viruses defective in the repression of an early target plasmid have been mapped to within the carboxy-terminal 78 amino acids of IE63 (Hardwicke et al.,1989 ; Smith et al.,1991
). In addition, Brown et al. (1995)
observed that the mutations within the carboxy terminus conserved residues, which abrogated transactivation, were also responsible for the repression properties of the IE63 protein. However, in contrast, other studies utilizing IE63 proteins containing mutations within the carboxy terminus maintain the ability to repress gene expression whereas amino-terminal deletions were deficient in the repression function (Rice et al.,1993
; Rice & Lam, 1994
). At present, we have not performed mutational analysis on the amino-terminal portion of the ORF 57 protein and it cannot be excluded that this may also include additional repression domains. The amino-terminal similarity between ORF 57 and its homologues is very limited, however; they all contain a cluster of acidic residues, which suggests that this acidic region has a common function. We have also demonstrated that the ORF 57 zinc finger-like domain is required for the intense nuclear spotting of the SC-35 spliceosome component. This increase in spotting may correlate with the impairment of splicing by ORF 57; however, it is believed that splicing inhibition involves mechanisms more complex than simply redistributing spliceosome components. Similar results have been observed in IE63, demonstrating that the carboxy terminus is required for interaction with SnRNPs and the redistribution of SnRNPs and SC-35, but does not inhibit host cell splicing (Sandri-Goldin et al.,1995
; Sandri-Goldin & Hibbard, 1996
). Interestingly, analysis of the zinc finger-like mutant 2, which alters the cysteine at 79738 of the published sequence to a serine, results in what we have termed a partial spotting effect. Moreover, this mutation results in a limited repression of intron-containing genes which correlates with the partial spotting effect. These results suggests that this is the least important residue contained with the zinc finger domain mutated in this report and draws to a parallel with the conservation in this domain. This residue is the only one not totally conserved: as highlighted in Fig. 4
, the MHV-68 carboxy terminus contains a lysine residue in this position. It would be of interest to determine if these domains have similar functions in other gammaherpesvirus ORF 57 homologues.
At present we cannot determine the exact role of these domains in the transactivation and repression properties of ORF 57 protein. These functional domains within the carboxy terminus of the ORF 57 gene may have several functions. (i) They may be required for the self-interaction of the ORF 57 protein, enabling the ORF 57 protein to function as a multimer. Analysis of HSV-1 IE63 has shown that this region is involved in the self-interaction of the protein, suggesting that IE63 acts as a multimer in infected cells (Zhi et al.,1999 ; Wadd et al.,1999
). At present studies are being undertaken to determine if ORF 57 self-interacts. (ii) These domains are required for the proper folding or protein stability of the ORF 57 protein. However, the limited analysis we could perform using our ORF 57 reagents suggest that these mutations have a limited effect on RNA transcription or protein stability. (iii) These domains are required for the binding of RNA by ORF 57. Although, we believe the ORF 57 RNA binding determinant is contained within the amino-terminal portion (Goodwin et al.,1999
), it cannot be excluded that ORF 57 binds directly to RNA via its zinc finger-like domain. (iv) These domains are required for specific proteinprotein interactions with cellular proteins which are required for the multifunctional properties. For example, the EBV Mta protein interacts with exportin 1, which mediates the function and intracellular localization of the Mta protein (Boyle et al.,1999
). Moreover, HSV IE63 has been shown to interact with the heterogeneous ribonucleoprotein K, a multifunctional protein with the ability to shuttle between the nucleus and cytoplasm, and casein kinase 2 (Wadd et al., 1999
). At present, we are determining if ORF 57 interacts with any cellular proteins and it will be of interest to establish if the functional domains identified in this report are important for these interactions.
In summary, we demonstrate that the carboxy terminus is required for ORF 57 transactivation, repression and the intense spotting of the SC-35 spliceosome component. This region contains two highly conserved motifs amongst its homologues, a hydrophobic GLFF domain and a zinc finger-like motif. We further show that the hydrophobic domain is required for transactivation and is also involved in nuclear localization of the ORF 57 protein, whereas the zinc finger-like domain is required for transactivation, repression and the intense SC-35 nuclear spotting.
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Acknowledgments |
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References |
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Received 12 January 2000;
accepted 26 May 2000.