Institute of Virology, Robert-Koch-Str. 17, 35037 Marburg, Germany1
Author for correspondence: Elke Bogner. Fax +49 6421 2865482. e-mail bogner{at}mailer.uni-marburg.de
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Abstract |
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Introduction |
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In a previous report, partial characterization of the gene product of HCMV ORF UL56 demonstrated that the encoded product, pUL56, is a 130 kDa, nucleocapsid-associated protein (Bogner et al., 1993 ). Recently, we showed that pUL56 binds specifically to viral DNA-packaging motifs and cleaves DNA bearing these motifs (Bogner, 1999
; Bogner et al., 1998
), suggesting an involvement of pUL56 in HCMV DNA packaging. These observations are in line with studies on ICP18.5, the homologous protein of herpes simplex virus type 1 (HSV-1) (Pellett et al., 1986
) and pseudorabies virus (PrV) (Pederson & Enquist, 1989
). By the use of virus mutants, it was demonstrated that the deletion of ICP18.5 leads to nuclear accumulation of naked nucleocapsids and uncleaved concatemeric DNA (Addison et al., 1990
; Tengelsen et al., 1993
; Mettenleiter et al., 1993
). In view of these observations, nuclear translocation of HCMV pUL56 is expected to be essential for virus maturation.
Targetting of nuclear proteins has been shown to depend on intrinsic nuclear localization signals (NLS). The best-characterized NLS motifs are the classical monopartite NLS of simian virus 40 (SV40) large T antigen (PKKKRKV; Kalderon et al., 1984 ), which consists of a single stretch of basic amino acids, and the bipartite nucleoplasmin NLS of Xenopus laevis, which has two basic motifs separated by a mutation-tolerant 10 amino acid spacer (KRPAATKKAGQAKKKK; Robbins et al., 1991
). Although there is no clear consensus, it is known that a simple cluster of basic amino acids is generally not sufficient to serve as a nuclear transport signal (Chelsky et al., 1989
; Dingwall & Laskey, 1991
; Makkerh et al., 1996
).
Active transport of such proteins across the nuclear pore complex (NPC) is achieved by the importin/karyopherin system. Several cellular proteins have been identified that are involved in this process (reviewed by Görlich, 1997 ). It is known that nuclear transport exhibits energy and signal dependence and is carrier mediated (Dingwall & Laskey, 1991
; Newmeyer et al., 1986
; Zasloff, 1983
). Several sequential steps may be distinguished in the nuclear translocation of proteins. As an initial step, the import substrate binds via its NLS to importin
, which forms a stable heterodimeric complex with the importin
subunit in the cytoplasm (Adam & Gerace, 1991
; Görlich et al., 1995
). Importin
subsequently mediates targetting of the importin
substrate complex to filaments of the NPC (Moroianu et al., 1995
). For translocation through the NPC, GTP (Weis et al., 1996b
) and two additional factors are required, the GTPase Ran/TC4 (Melchior et al., 1993
) and p10/NTF2 (Moore & Blobel, 1994
). Once inside the nucleus, the complex dissociates into an importin
substrate complex and importin
, followed by a further dissociation of importin
from the import substrate (Görlich et al., 1996
). Both importin subunits are relocated separately to the cytoplasm, whereas the import substrate is retained in the nucleus (Weis et al., 1996a
). In higher eukaryotes, several isoforms of importin
have been isolated, which have distinct substrate specificities and can be grouped into three subfamilies (Köhler et al., 1999
).
NLS sequences have been identified in several herpesvirus proteins such as the HSV-1 regulatory protein ICP27 (Mears et al., 1995 ), EpsteinBarr virus nuclear antigen 1 (EBNA-1; Ambinder et al., 1991
) and the assembly protein precursor (pUL80.5; Plafker & Gibson, 1998
) and the tegument protein pp65 (Schmolke et al., 1995
) of HCMV. However, with the exception of EBNA-1 (Fischer et al., 1997
), little is known about the pathway of herpesvirus protein translocation into the nucleus. In this report, experiments are described regarding the identification of a pUL56 NLS sequence and its interaction with one of the best-characterized human importin
homologues, hSRP1
(Weis et al., 1995
).
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Methods |
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Plasmid construction.
Restriction enzymes were purchased from New England Biolabs and used according to the instructions of the manufacturer. The construction of expression plasmid pRC/CMV-UL56 encoding wild-type HCMV pUL56 has been described previously (Bogner et al., 1993 ). To produce cDNA inserts for cloning, PCR was performed with the GeneAmp Kit (Perkin Elmer). All constructs in which PCR was used to generate coding sequences were analysed by DNA sequencing performed on a 377 DNA Sequencer (Applied Biosystems).
Construction of an epitope-tagged pUL56 construct.
To generate the construct HispUL56, which expressed 5'-Xpress epitope-tagged pUL56, plasmid pcDNA3.1/HisC (Invitrogen) was digested with KpnI and XbaI prior to insertion of a gene fragment encoding the ORF of HCMV pUL56. The respective fragment was obtained by using plasmid pRC/CMV-UL56 as the template for PCR and a pair of synthetic oligonucleotides (Table 1; restriction sites are underlined).
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Plasmids encoding pUL56 NLS fused to the reporter protein -galactosidasegreen fluorescent protein (
-GalGFP).
Plasmids encoding -GalpUL56NLSGFP chimeras were constructed by using plasmid pHM829, which encodes a
-GalGFP fusion protein (Sorg & Stamminger, 1999
). In order to generate the chimeras pHM829pUL56NLS, pHM829pUL56nlsAA1 to pHM829pUL56nlsAA6 and pHM829pUL56revNLS, plasmid pHM829 was digested with restriction endonucleases BamHI and XbaI. Gene fragments generated using pHM829 as template with a 3' primer containing a BamHI site and 5' oligonucleotides containing XbaI sites (Table 1
; restriction sites underlined) were inserted into the digested plasmid.
Generation of glutathione S-transferase (GST)pUL56 fusion constructs.
To obtain the GSTpUL56NLS chimeras GSTpUL56NLS, GSTpUL56revNLS and GSTpUL56nlsAA4, plasmid pGEX-5X-1 (Amersham Pharmacia Biotech) was digested with restriction endonucleases EcoRI and PstI. Gene fragments generated using pGEX-5X-1 as template with the antisense oligonucleotide and sense oligonucleotides shown in Table 1 (restriction sites underlined) were inserted into the digested plasmid.
Plasmid GSTpUL56C was generated by digestion of pRC/CMV-UL56 with EcoRI and pGEX-5X-1 (Amersham Pharmacia Biotech) with NotI. To create blunt ends, linearized plasmids were incubated with Klenow enzyme (Roche Diagnostics) as recommended by the supplier. pRC/CMV-pUL56 was digested sequentially with XhoI and EcoRV and pGEX-5X-1 with XhoI. The 1·5 kb fragment encoding the carboxy-terminal half of HCMV pUL56 was inserted into pGEX-5X-1.
Transient transfection.
For transient expression, COS-7 cells were seeded on 60 mm Petri dishes with glass cover slips. COS-7 cells at 60% confluence were transfected with the appropriate DNA (10 µg per well) by the lipofectin method (Life Technologies). Antigen expression was analysed by indirect immunofluorescence at 40 h post-transfection.
Immunofluorescence analysis and antibodies.
For immunofluorescence, mock-infected and infected HFF or transfected COS-7 cells grown on glass cover slips were fixed in 4% paraformaldehyde as described previously (Smuda et al., 1997 ). After fixation, incubation with the following primary antibodies was carried out for 60 min at room temperature: HCMV pUL56-specific human polyclonal antibody pabUL56 (Bogner et al., 1993
), which was purified from high-titre human serum by column affinity chromatography (Affigel 10/15pUL56), mouse anti-NOR MAb (Novus Molecular), directed against the nucleolus-organizing region, diluted 1:100 in 3% BSA/PBS, MAb Anti-Xpress (Invitrogen), diluted 1:500 in 3% BSA/PBS, and
-Gal-specific MAb (Promega), diluted 1:1000 in 3% BSA/PBS. After extensive rinsing with PBS, further incubation was carried out for 45 min with FITC-labelled goat anti-human F(ab')2 fragments, FITC-labelled goat anti-mouse F(ab')2 fragments or, for double immunofluorescence analysis, Texas red-labelled goat anti-mouse F(ab')2 fragments (Dianova), diluted 1:100 in 3% BSA/PBS. In the case of double immunofluorescence staining of proteins, the primary antibodies were incubated together whereas secondary antibodies were added consecutively. A Zeiss microscope with digital photographic equipment (Spot camera system, version 2.1.2, Diagnostic Instruments) was used for analysis of fluorescence signals and for taking phase-contrast images.
In vitro binding assays.
The TNT T7-coupled reticulocyte lysate system (Promega) was used to synthesize [35S]methionine-labelled (Amersham) importin in a coupled transcription/translation reaction by using 1 µg plasmid pRSETB-hSRP1
(Weis et al., 1995
) as template according to the protocol of the supplier (Promega). For in vitro binding analysis, GSTpUL56 fusion proteins were expressed in E. coli BL21 and protein purification was carried out according to the manufacturer's instructions (Pharmacia). Equal amounts of GSTpUL56 fusion proteins loaded on glutathioneSepharose 4B (Amersham Pharmacia Biotech) were incubated overnight at 4 °C with in vitro-translated importin
in 500 µl binding buffer (0·05% NP-40, 50 mM HEPESNaOH pH 7·3, 10% glycerol, 0·1% BSA, 300 mM NaCl). Samples were washed with binding buffer and subsequently subjected to SDSPAGE, consecutive fixation and autoradiography (Bonner & Laskey, 1974
). A BioImager (Raytest) was used for quantification of radioactive signals.
Immunoprecipitation.
Immunoprecipitation of in vitro-translated importin with MAb Anti-Xpress (Invitrogen) was carried out as described previously (Radsak et al., 1990
). All incubation and washing cycles were performed with immunoprecipitation buffer [0·02 M TrisHCl pH 9·0, 0·3 M NaCl, 10% (v/v) glycerol, 0·001 M CaCl2, 0·5 mM MgCl2, 0·002 M EDTA, 0·5% (v/v) NP-40] as described by Blanton & Tevethia (1981)
. Immunoprecipitates were analysed by SDSPAGE and autoradiography.
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Results |
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Nuclear translocation of full-length HCMV pUL56 is mediated by a carboxy-terminal stretch of basic amino acids
In order to identify specific domains responsible for nuclear import, the amino acid sequence of pUL56 (850 aa) was scanned by computer analysis for the presence of putative NLS motifs. By using criteria such as predicted surface exposure (Roberts et al., 1987 ; Rost & Sander, 1993
) and comparison of the pUL56 amino acid sequence with known monopartite and bipartite NLS motifs (Boulikas, 1993
; Dingwall & Laskey, 1991
; Robbins et al., 1991
), a putative monopartite NLS motif was identified at the extreme carboxy terminus of pUL56. The sequence consisted of a stretch of 12 amino acids, eight of which had a basic character (aa 816827; Fig. 2A
). The motif exhibited similarity to the polyomavirus large T NLS (Richardson et al., 1986
) and contained the putative NLS consensus sequence K(K/R)X(K/R) (Chelsky et al., 1989
).
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Amino acid residues 816827 of HCMV pUL56 mediate nuclear targetting of a cytosolic reporter protein
In order to determine further the signal character of the pUL56 12 amino acid motif, it was fused to an adequate reporter by using vector pHM829 (Sorg & Stamminger, 1999 ), which enables expression of protein fragments that are fused to
-Gal and GFP. After subcloning of the coding sequence for aa 816827 of pUL56 into the multiple cloning site of vector pHM829, the fusion construct was expressed transiently in COS-7 cells (Fig. 3
). In order to demonstrate that the inserted amino acid sequence was expressed correctly, the subcellular localization of the chimeric proteins was examined by GFP signals and immunofluorescence staining with an anti-
-Gal MAb and a Texas red-conjugated secondary antibody. Nuclei and nucleoli were visualized by taking phase-contrast micrographs of the cells. This approach revealed exclusively intranuclear detection of pUL56 NLS fusion proteins (Fig. 3A
). In addition to homogeneous distribution of fluorescence signals, bright intranuclear patches were detected (Fig. 3A
;
-Gal, GFP) representing nucleoli, which were seen as darker regions in phase-contrast images (Fig. 3A
; phase-c). These patches appeared not to be identical to the inclusion bodies observed in cells at late times after infection (Fig. 1A
; 48 h p.i.). The order of basic and neutral amino acids within the external parts of the pUL56 NLS sequence (RRVR or RPRR) is similar, implying that the signal could also be functional in the reverse orientation. In order to determine the subcellular distribution of the reversed pUL56 NLS, the respective residues were fused to the reporter protein
-GalGFP. Chimeric protein constructs were transfected transiently into COS-7 cells prior to immunofluorescence analysis at 40 h after transfection, as described above. This approach revealed retention of fusion proteins in the cytoplasm (Fig. 3B
;
-Gal, GFP). As a control, expression of vector pHM829 alone revealed clear retention of the reporter proteins in the cytoplasm (Fig. 3C
). These results demonstrated that the newly identified monopartite NLS (aa 816827) of HCMV pUL56, but not the reversed NLS sequence, can serve as an efficient nuclear-targetting signal in mammalian cells.
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Discussion |
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Furthermore, we demonstrated nuclear translocation of pUL56 in the absence of other viral proteins (Fig. 1B, C
). After transfection of authentic pUL56 or epitope-tagged HispUL56, homogeneous intranuclear immunofluorescence signals were obtained, whereas nucleolar regions remained unstained. As expected, bright intranuclear inclusion bodies were not detectable under these conditions, suggesting that additional virus products, e.g. viral DNA and/or other viral proteins, may be needed for this particular localization.
Intranuclear transport of pUL56 in the absence of other viral proteins implied the existence of an endogenous NLS. Two structurally different types of NLS have been described: the monopartite SV40 large T antigen NLS (Kalderon et al., 1984 ) and the bipartite NLS of Xenopus laevis nucleoplasmin (Robbins et al., 1991
). A universal NLS consensus sequence for all nuclear proteins has not yet been established, other than an accumulation of basic residues within a short stretch of amino acids (Dingwall & Laskey, 1991
), often containing a glycine or proline residue as a helix-breaker' (Dang & Lee, 1989
). HCMV pUL56 contains an obvious stretch of basic amino acids at the extreme carboxy terminus (RRVRATRKRPRR; aa 816827), with a proline residue at position 825. Computer analysis suggests that residues 822827 are exposed at the surface of the protein. The sequence motif contains two clusters of basic amino acids (RRVR and RKRPRR) and is comparable to that of the NLS of polyomavirus large T (Richardson et al., 1986
) as well as to the proposed consensus sequence, K(K/R)X(K/R), for monopartite NLSs (Chelsky et al., 1989
; Fig. 2A
). Deletion of the putative NLS sequence indeed resulted in retention of pUL56 in the cytoplasm (Fig. 2B
). Moreover, the 12 amino acid sequence mediated nuclear transport when fused to the cytosolic reporter protein
-GalGFP (Fig. 3A
). Therefore, the NLS of HCMV pUL56 identified here meets the criteria of a nuclear targetting signal. These observations are in line with findings regarding nuclear transport of the homologous HSV-1 protein, ICP18.5 (Pellett et al., 1986
), which is also independent of other viral proteins (J. D. Baines, personal communication); however, an NLS was not identified for this HSV-1 protein. Regarding the PrV homologue ICP18.5, on the other hand, a sequence motif with a putative signal character consisting of four arginine residues appears to be located at positions 461464 (Pederson & Enquist, 1991
).
As demonstrated for the NLS of SV40 and other NLS sequences such as the polyomavirus major capsid protein VP1 NLS (Chang et al., 1992 ; Kalderon et al., 1984
), a single amino acid substitution largely eliminated nuclear targetting. In the case of the HCMV pUL56, we were able to demonstrate that residues 7 and 8 (R and K) of the 12 amino acid sequence are apparently essential for nuclear translocation (Fig. 4D
). Substitution of residues 9 and 10 (R and P) of the NLS resulted in nuclear as well as cytoplasmic localization (Fig. 4E
); the functionality of the NLS was not eliminated in this case but seemed to be diminished. We suggest that residues 9 and 10, including the helix-breaker proline, may stabilize a loop structure of the amino acid chain at the region of the NLS that is probably exposed at the surface and therefore more accessible to transport factors that interact with NLS sequences, such as importin
(Conti et al., 1998
; Görlich et al., 1995
). Both amino acids, arginine at position 822 and lysine at position 823, also appear to play a pivotal role in nuclear targetting of full-length HispUL56 (Fig. 5
).
When the pUL56 NLS sequence was fused to reporter proteins, not only diffuse nuclear staining but also nucleolar localization of the chimeras was detected (Figs 3A and 4C
, G
). Transport into the nucleolus is probably dependent on the co-operation of several domains and/or on interactions with other macromolecules within the nucleolus (Nosaka et al., 1989
; Schmidt-Zachmann & Nigg, 1993
). In some cases, the signals for nucleolar transport have been shown to overlap with NLS sequences (Siomi et al., 1988
; Warner & Sloboda, 1999
). Therefore, one possible explanation for this particular distribution may be that the function of the pUL56 NLS is extended by adjacent residues of the reporter protein, creating an additional signal that mediates nucleolar transport including the interaction with nucleolar macromolecules. Interestingly, substitution of residues 1, 2, 3, 4, 11 and 12 (R, R, V, R, R and R) within the NLS motif by alanine did not influence nuclear import of the reporter proteins, but eliminated nucleolar localization (Fig. 4A
, B
, F
). Regarding the nuclear targetting function of the NLS of pUL56, the phenomenon of subnuclear compartmentation is intriguing, but of less interest.
Regarding the question of whether import of pUL56 into the nucleus is carrier mediated, the recombinant human NLS receptor subunit hSRP1 (Weis et al., 1995
) of the importin complex was used for in vitro binding experiments. We were able to demonstrate that the importin
homologue hSRP1
interacts specifically with the carboxy terminus of HCMV pUL56 in an NLS-dependent manner (Fig. 6A
). In order to support the significance of this interaction, GST constructs containing the reversed NLS of pUL56 (GSTpUL56revNLS) or the pUL56 NLS containing substitutions of residues 7 and 8 by alanine (GSTpUL56nlsAA4) were used for in vitro binding assays. Fusion of the reversed pUL56 NLS sequence to the cytoplasmic reporter protein
-GalGFP resulted in a predominantly cytoplasmic localization of the chimera (Fig. 3B
). The specific interaction between the reversed NLS fused to GST and hSRP1
was about 85% weaker than the binding of the GSTwild-type NLS chimera in in vitro binding assays (Fig. 6A
, B
; lanes 6 and 7). These results are in line with experiments described for the SV40 large T antigen NLS. By using chemical cross-linking analysis, a synthetic peptide containing the reversed NLS sequence did not compete for the binding of importin
to wild-type SV40 NLS (Adam et al., 1989
). These authors concluded that it is not only the charge density of NLS peptides that is responsible for binding and that there is a correspondence between the sequences directing nuclear transport in vivo and their ability to compete for NLS peptide binding of importin
in vitro. It has also been shown that the reversed peptide was incapable of directing nuclear transport in vivo (Lobl et al., 1990
). Therefore, the weaker interaction between the reversed NLS sequence of HCMV pUL56 and hSRP1
may be explained by the requirement for a specific conformation in the context of adjacent residues that depends on a specific order of amino acids in this region.
Regarding the interaction between importin and GSTpUL56nlsAA4, containing substitutions of residues 7 and 8 by alanine, a decrease of about 90% was observed when compared with the binding of wild-type pUL56 NLS fused to GST (Fig. 6B
). This result was in line with the finding that pUL56nlsAA4 fused to the reporter protein
-GalGFP also resulted in the predominant retention of the reporter protein chimera in the cytoplasm (Fig. 4D
).
Taken together, our results imply that HCMV pUL56 can be recognized via its NLS by importin , the adaptor protein of the heterodimeric importin complex, in transfected or infected cells and is subsequently transported into the nucleus by the importin-dependent pathway. Our observations thus indicate that a pUL56-mediated step in virus maturation is probably dependent on the functional nuclear import machinery of the host cell.
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Acknowledgments |
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References |
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Received 8 March 2000;
accepted 26 May 2000.