Identification of a short amino acid sequence essential for efficient nuclear targeting of the Kaposi’s sarcoma-associated herpesvirus/human herpesvirus-8 K8 protein

Stéphanie Portes-Sentis1, Evelyne Manet1, Géraldine Gourru1, Alain Sergeant1 and Henri Gruffat1

Laboratoire de virologie humaine, U412 INSERM, ENS-Lyon, 46 allée d’Italie, 69364 Lyon cedex 07, France1

Author for correspondence: Henri Gruffat. Fax +33 4 72 72 87 77. e-mail hgruffat{at}cri.ens-lyon.fr


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The K8 protein of Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 is a member of the bZIP family of transcription factors, which has homology with the Epstein–Barr virus transcription and replication factor, EB1. In this report, we have analysed the subcellular localization of the K8 protein and characterized a 12 amino acid sequence rich in basic residues which is responsible for targeting the protein to the cell nucleus. Furthermore, we show that a K8 mutant lacking the nuclear localization sequence can be directed to the nucleus by co-expression with an intact K8 protein, suggesting that K8 homodimerizes in the cytoplasm of the cell in vivo.


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Kaposi’s sarcoma-associated herpesvirus (KSHV), also called human herpesvirus-8, was first identified and characterized from a Kaposi’s sarcoma patient’s skin lesion in 1994 (Chang et al., 1994 ). Although KSHV DNA is present in KS lesions, KS cell lines established in vitro do not usually harbour viral DNA (Flamand et al., 1996 ). However, various KSHV-infected human B-cell lines derived from primary effusion lymphomas (PEL), also known as ‘body cavity based lymphoma’ (BCBL), are available for molecular studies (Cesarman et al., 1995 , 1996 ; Renne et al., 1996 ). In the PEL cell lines, few viral genes are expressed, suggesting that the virus is predominantly in a latent state (Miller et al., 1997 ; Sarid et al., 1998 ). Following completion of the entire KSHV genomic sequence (Neipel et al., 1997 ; Russo et al., 1996 ), KSHV was classified in the genus Rhadinovirus of the lymphotropic Gammaherpesvirinae subfamily.

KSHV has significant homologies with herpesvirus saimiri (HVS) and Epstein–Barr virus (EBV) (Moore et al., 1996 ). In EBV, two immediate early gene products, R (also called Rta) and EB1 (also called ZEBRA or Zta), are required for the switch from latency to the lytic cycle. R and EB1 are both transcriptional activators which directly bind specific sites on the DNA and activate expression of the EBV early genes. Furthermore, EB1 is required for transactivation of the EBV origin of replication (OriLyt), which is functional during the productive cycle. The KSHV orf50 gene has been shown to encode a protein structurally and functionally homologous to the EBV R transcription factor, which is encoded by the BRLF1 gene (Lukac et al., 1999 ; Sun et al., 1998 ). Downstream of the KSHV orf50, the orfK8 gene encodes the KSHV K8 protein, which shares some structural homology with EB1 (Gruffat et al., 1999 ; Lin et al., 1999 ).

We and others (Gruffat et al., 1999 ; Lin et al., 1999 ) have shown that K8 is a new member of the bZIP family of transcription factors. K8, like its EBV homologue, EB1, contains a leucine-zipper (ZIP) motif in its C-terminal region (Fig. 1A) and is able to form homodimers in solution. Adjacent to the leucine-zipper motif, K8 and EB1 both possess a basic region (Fig. 1A) which, in the case of EB1, is responsible for its binding to DNA. No function, however, has as yet been ascribed to the K8 protein, neither in the activation of KSHV genes nor in the activation of KSHV replication. In order to further characterize the biological functions of the K8 protein, we investigated its subcellular localization: we show that K8 is a nuclear protein which forms homodimers in the cell and have precisely characterized a 12 amino acid sequence which serves as a nuclear localization signal (NLS).



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Fig. 1. Expression profiles of the K8 protein in PEL cells and characterization of its subcellular localization. (A) Schematic representation of the EB1 and K8 proteins. The different domains characterized in the two proteins are indicated. (B) Total cell extracts of the KSHV- and EBV-negative B-cell lymphoma line DG75 (lanes 1 and 2), the EBV-positive (KHSV-negative) B-cell lymphoma line Raji (lanes 5 and 6), the KSHV-positive (EBV-negative) PEL cell lines BCBL1 (lanes 3 and 4) and BGB1 (lanes 9 and 10), the KSHV- and EBV-positive PEL cell line BC-3 (lanes 7 and 8) and the epithelial cell line HeLa, transfected with an expression plasmid for the Flag–K8 protein (lane 12) or not transfected (lane 11), were separated by SDS–10% PAGE. Protein expression was analysed by immunoblotting using an anti-K8 rabbit polyclonal serum. Cells were pretreated (+) or not (-) with TPA (20 ng/ml) for 48 h. (C) Expression kinetics of the K8 protein in BCBL1 cells after TPA treatment. BCBL1 cells were treated with 20 ng/ml TPA and collected at 0, 4, 8, 12, 24, 48 and 72 h post-treatment. Total cell extracts were separated by SDS–10% PAGE. Protein expression was analysed by immunoblotting using an anti-K8 rabbit polyclonal serum. (D) Subcellular localization of the K8 protein in BCBL1 cells treated with TPA (panels a/b) and in HeLa cells transfected with an expression plasmid for the K8 protein (panels c/d). For panels a and b, the cytoplasm of the cells was labelled with fluorescein–phalloidin (Molecular Probes) and the K8 protein was visualized (panel b) by indirect immunofluorescence using the anti-K8 rabbit serum as primary antibody and a cyanine 5-conjugated goat anti-rabbit IgG as secondary antibody. For panels c and d, the nuclei of the cells were stained with propidium iodide and the K8 protein was visualized by indirect immunofluorescence using the anti-K8 rabbit serum as primary antibody and a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG as secondary antibody (panel d).

 
In order to characterize the K8 protein, we first generated a specific antiserum. The K8 protein was produced in E. coli as a histidine-tagged fusion protein (His–K8) following cloning of the K8 cDNA in the pET15b vector (Novagen). The His–K8 protein was purified on a Ni–NTA column (Qiagen) by standard techniques and then used as the immunogen to produce a rabbit polyclonal antiserum. Protein extracts from a panel of KSHV-positive or -negative B-cell lines were analysed by Western blotting using the anti-K8 serum. A 35 kDa protein was specifically detected in all the KSHV-positive cell lines (Fig. 1B, lanes 3, 4, 7, 8, 9 and 10). This signal was not obtained with KSHV-negative cell lines (Fig. 1B, lanes 1, 2, 5 and 6). This result clearly shows that the anti-K8 serum reacted with a specific KSHV product. The amount of protein increased strongly following treatment of the KSHV-positive cells with TPA (phorbol 12-myristate 13-acetate) (Fig. 1B, lanes 4, 8 and 10). To confirm that the 35 kDa protein detected with the anti-K8 serum corresponded to the K8 protein, protein extracts from HeLa cells transfected or not with a Flag–K8 expression plasmid were analysed by Western blotting using the anti-K8 serum. As expected, a specific signal of 35 kDa was detected only with the extracts from the transfected cells (Fig. 1B, lanes 11 and 12). These results demonstrate that the rabbit polyclonal anti-K8 serum is indeed specifically directed against the K8 protein.

To characterize the expression kinetics of the K8 protein, BCBL1 cells treated with TPA were collected at 4, 8, 15, 24, 48 and 72 h post-treatment. Protein extracts were analysed by Western blotting using the anti-K8 serum. The K8 protein was detected at a low level in the KSHV-infected cell population (Fig. 1 C, lane 1), but 8 h after TPA treatment the level of K8 increased to reach a maximum of expression at 48 h (Fig. 1 C, lanes 2 to 7).The expression kinetics of the K8 protein followed the kinetics of K8 mRNA expression (Gruffat et al., 1999 ).

Using an indirect immunofluorescence assay, we observed that the polyclonal antibodies reacted with antigens present in the nucleus of BCBL1 cells (Fig. 1D, panels a and b). The staining was specific for KSHV-infected cells as it was not observed in Raji and DG75 cells (data not shown). HeLa cells transfected with an expression plasmid for the K8 protein also showed a distinct nuclear staining with the anti-K8 polyclonal antibody (Fig. 1D, panels c and d).

In order to characterize the K8 NLS we generated a series of K8 deletion mutants (Fig. 2A) and analysed their subcellular localization in HeLa cells by indirect immunofluorescence, using the polyclonal antibody directed against K8. The K8 protein, which has been shown to be a structural homologue of the EBV EB1 protein, was detected by a distinct nuclear staining (Fig. 2A, panels a/a') and the immunofluorescence pattern was very similar to that observed for EB1 when using a specific anti-EB1 monoclonal antibody (Fig. 2A, panels g/g'). Deletion of the N-terminal part of the K8 protein (up to residue 95) did not affect the subcellular localization of the protein (Fig. 2A, panels b/b'). A K8 protein with its C-terminal part (from residue 159) deleted was also detected in the nucleus (Fig. 2A, panels c/c'), but when the C-terminal domain was expressed alone, it was detected only in the cytoplasm of the transfected cells (Fig. 2A, panels d/d'). These results suggest that an NLS is present between amino acids 96 and 158 of the K8 protein. The majority of the NLS characterized so far are composed of stretches of basic residues such as that characterized for the simian virus 40 large T antigen (PKKKRKV). Analysis of the K8 protein primary sequence reveals the presence of a region rich in basic amino acids between residues 124 and 135. This region could thus contribute to the nuclear localization of the K8 protein either alone or in conjunction with another region of the protein. In order to test this hypothesis we deleted this motif from the K8 protein to generate the K8{Delta}nls mutant. When expressed in HeLa cells, this mutant protein was localized in the cell cytoplasm (Fig. 2A, panels e/e'). The basic-rich motif TRRSKRRLHRKF between residues 124 and 135 in the K8 protein thus appears to be crucial for nuclear localization of the protein. In order to confirm that the basic residues in this region are important for nuclear signalling, we exchanged the three basic residues KRR for the neutral residues AFA in the mutant K8AFA. This mutated protein was not imported into the nucleus of transfected cells (Fig. 2A, panels f/f').



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Fig. 2. Mapping of the K8 NLS. (A) Subcellular localization of K8 variants visualized by indirect immunofluorescence. After transfection, cells were fixed and the nuclei of the cells were stained with propidium iodide. The subcellular localization of K8 and the K8 variants as well as EB1 was visualized by indirect immunofluorescence using either the polyclonal rabbit anti-K8 antibody, or a monoclonal antibody specific for EB1 (AZ125), as first antibodies. The second antibodies were, respectively, a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody and a fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody. Schematic representation of EB1, K8 and each K8 mutant is shown above the corresponding panels. (B) Subcellular localization of GFP–K8 chimeras. The GFP part of the chimera is represented in light green and the K8 part in grey. HeLa cells were transfected with expression plasmids for the different proteins. After transfection, cells were fixed and the nuclei of the cells were stained with propidium iodide. The GFP fluorescence was revealed under UV light.

 
In order to determine whether the motif characterized above was sufficient to localize an heterologous protein to the cell nucleus, we constructed chimeras containing the K8 domain, between amino acids 112 to 158 (both unmodified and mutated), fused in-frame to the C terminus of green fluorescent protein (GFP) (Fig. 2B). After transfection of HeLa cells with expression plasmids for these various chimeras, their subcellular localization was determined by GFP fluorescence. As expected, GFP was localized equally throughout the cytoplasm and the nucleus of the cells (Fig. 2B, panels a/a'). However, the chimera containing K8 residues 112 to 158 (GFP K8 112–158) was wholly localized in the cell nucleus (Fig. 2B, panels b/b'). Deletion of amino acids 125 to 134 (GFP K8 112–158{Delta}nls) or point mutations of the three KRR basic residues in this region (GFP K8 112–158AFA) were sufficient to impair the nuclear localization of these fusion proteins. Indeed, these mutants show the same staining as GFP alone (Fig. 2B, compare panels a/a' to panels c/c' and d/d'). Taken together these results demonstrate that a basic-rich region of the K8 protein localized between amino acids 112 and 158 contains a functional NLS which alone is sufficient to direct a protein to the cell nucleus.

We have previously shown that the K8 protein can form homodimers in vitro. For this reason, we investigated whether co-expression of wild-type K8 protein could drive the nuclear transport of K8 protein deleted of its NLS, by heterodimerization in the cytoplasm and subsequent localization of the complex to the nucleus. To achieve this, we constructed two plasmids expressing proteins with different tags: the K8 protein fused to the Flag epitope (Flag–K8) and the K8 protein deleted of its nuclear localization signal, fused to the HA epitope (HA–K8{Delta}nls) (Fig. 3A). These expression plasmids were transfected into HeLa cells either alone or together. As expected, indirect immunofluorescence with a monoclonal antibody directed against the Flag epitope showed that the Flag–K8 fusion protein was localized in the cell nucleus (Fig. 3B, panel a). This localization was not altered by co-expression of the HA–K8{Delta}nls fusion protein (Fig. 3B, panel b). When the HA–K8{Delta}nls fusion protein was expressed alone, it was detected (with a monoclonal antibody directed against the HA epitope) in the cell cytoplasm (Fig. 3B, panel c). However, when co-expressed with the Flag–K8 protein, HA–K8{Delta}nls was found to be partially nuclear (Fig. 3B, panel d). This nuclear targeting of HA–K8{Delta}nls occurred in a Flag–K8 dose-dependent manner (data not shown), consistent with the hypothesis that K8/K8{Delta}nls heterodimers are transported to the nucleus. These results strongly suggest that K8 forms homodimers in the cell and that a single NLS present on one of the two subunits of the homodimer is sufficient to effect nuclear localization of the complex.



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Fig. 3. Nuclear subcellular localization of a K8 variant lacking an NLS, through heterodimerization with an intact K8 protein. (A) Schematic representation of the wild-type K8 protein and a K8 variant with its NLS deleted. The two proteins were tagged at their N terminus by a Flag epitope and an HA epitope respectively. (B) Expression plasmids for the Flag–K8 protein or for the HA–K8{Delta}nls were transfected either alone or together into HeLa cells. The subcellular localization of the two proteins was visualized by indirect immunofluorescence using either an anti-Flag monoclonal antibody (panels a and b) or an anti-HA monoclonal antibody (panels c and d).

 
In conclusion, we have identified the sequence TRRSKRRLHRKF in the K8 protein as being sufficient to localize K8, or a heterologous protein such as GFP, in the cell nucleus. Mutation of the three central basic amino acids, KRR, in this sequence is completely detrimental to the function of the signal sequence. The K8 NLS displays the usual characteristic of classical NLSs: a short sequence rich in basic residues.

The K8 protein has been suggested to be a KSHV homologue of the EBV transcription/replication factor, EB1 (Gruffat et al., 1999 ; Lin et al., 1999 ). The EB1 NLS was previously characterized as a bipartite motif composed of two clusters (called BRA and BRB) of positively charged amino acids (Mikaelian et al., 1993 ). Furthermore, this bipartite signal overlaps with the DNA-binding domain of the protein. These two basic clusters are conserved among all the bZIP proteins and have been also shown to be responsible for the nuclear localization of the Jun protein (Chida & Vogt, 1992 ; Mikaelian et al., 1993 ). Our results show that the K8 protein has particular characteristics that distinguish it from both EB1 and the other members of the bZIP family of proteins: its NLS is composed of a single sequence and furthermore is completely separated from the putative DNA-binding domain of the protein. Alignment of the bZIP K8 domain with those of EB1 and c-Jun reveals several mismatches, particularly in the two motifs determined as important for nuclear signalling (Gruffat et al., 1999 ). The difference in amino acid sequence in the basic region of EB1 and the c-Jun bZIP domain compared to K8, reflecting the functional differences observed between them in terms of nuclear localization signalling, may suggest that the K8 bZIP domain has substantially diverged from those of other bZIP proteins. This begs the question of whether the K8 bZIP domain retains the capacity to bind DNA. Answering this question is of primary importance to understanding the function of K8.


   Acknowledgments
 
The BCBL1 cell line was obtained through the AIDS Research and Reference Program (Division of AIDS, NIAID, NIH) from Drs M. McGrath and Don Ganem. We thank Dr Stephane Ory for his precious advice for the confocal microscopy and Dr Robin Buckland for reading the manuscript. Research in the laboratory is financially supported by INSERM, the Association pour la Recherche contre le Cancer (ARC, grants no. 9271). S.P.S. was a recipient of an ARC fellowship. A. Sergeant and E. Manet are CNRS scientists.


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Received 12 September 2000; accepted 24 November 2000.