Laboratoire de virologie humaine, U412 INSERM, ENS-Lyon, 46 allée dItalie, 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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Abstract |
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Main text |
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KSHV has significant homologies with herpesvirus saimiri (HVS) and EpsteinBarr 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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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
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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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 (FlagK8) and the K8 protein deleted of its nuclear localization signal, fused to the HA epitope (HAK8nls) (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 FlagK8 fusion protein was localized in the cell nucleus (Fig. 3B
, panel a). This localization was not altered by co-expression of the HAK8
nls fusion protein (Fig. 3B
, panel b). When the HAK8
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 FlagK8 protein, HAK8
nls was found to be partially nuclear (Fig. 3B
, panel d). This nuclear targeting of HAK8
nls occurred in a FlagK8 dose-dependent manner (data not shown), consistent with the hypothesis that K8/K8
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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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.
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Acknowledgments |
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References |
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Received 12 September 2000;
accepted 24 November 2000.