1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Institute of Cardiovascular Research, University of Leeds, Leeds LS2 9JT, UK
3 Department of Pathology and Microbiology, University of Bristol, Bristol BS8 1TD, UK
Correspondence
Adrian Whitehouse
a.whitehouse{at}leeds.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
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., 1999
; Cotter & Robertson, 1999
). LANA has been shown to associate with host-cell mitotic chromosomes via a region at its N terminus encompassing aa 522 (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 performed and the resulting pattern of expression observed. Cos-7 cells were transfected using Lipofectamine 2000 (Invitrogen) and 2 µg of construct DNA and incubated 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 microscope (Leica TCS SP) and a PlanApo 100x UV oil-immersion lens.
|
In order to further characterize the ORF73 CBD, a range of 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-73C
1, 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-73C
2 and pEGFP-NLS-73C
3, which contain small deletions from the C or N terminus of pEGFP-NLS-73C
1 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-73C1, sequence analysis was performed and identified a motif at aa 291293 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. 2
a). 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 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.
|
|
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 self-association and this region is conserved in other LANA-like proteins. Schwam et al. (2000)
also suggest that this C-terminal 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 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 522 (Piolot et al., 2001
); however deletion of aa 11291143 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 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 host-cell chromosomes is dependent upon its ability to form homo-multimers.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Ballestas, M. E. & Kaye, K. M. (2001). Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen 1 mediates episome persistence through cis-acting terminal repeat (TR) sequence and specifically binds TR DNA. J Virol 75, 32503258.
Ballestas, M. E., Chatis, P. A. & Kaye, K. M. (1999). Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284, 641644.
Chang, Y., Moore, P. S., Talbot, S. J., Boshoff, C. H., Zarkowska, T., Godden, K., Paterson, H., Weiss, R. A. & Mittnacht, S. (1996). Cyclin encoded by KS herpesvirus. Nature 382, 410.[CrossRef][Medline]
Churchill, M. E. & Suzuki, M. (1989). SPKK motifs prefer to bind to DNA at A/T-rich sites. EMBO J 8, 41894195.[Abstract]
Cotter, M. A. & Robertson, E. S. (1999). The latency-associated nuclear antigen tethers the Kaposi's sarcoma-associated herpesvirus genome to host chromosomes in body cavity-based lymphoma cells. Virology 264, 254264.[CrossRef][Medline]
Dittmer, D., Lagunoff, M., Renne, R., Staskus, K., Haase, A. & Ganem, D. (1998). A cluster of latently expressed genes in Kaposi's sarcoma-associated herpesvirus. J Virol 72, 83098315.
Hall, K. T., Giles, M. S., Goodwin, D. J., Calderwood, M. A., Carr, I. M., Stevenson, A. J., Markham, A. F. & Whitehouse, A. (2000a). Analysis of gene expression in a human cell line stably transduced with herpesvirus saimiri. J Virol 74, 73317337.
Hall, K. T., Giles, M. S., Goodwin, D. J., Calderwood, M. A., Markham, A. F. & Whitehouse, A. (2000b). Characterization of the herpesvirus saimiri ORF73 gene product. J Gen Virol 81, 26532658.
Jung, J. U., Stager, M. & Desrosiers, R. C. (1994). Virus-encoded cyclin. Mol Cell Biol 14, 72357244.[Abstract]
Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499509.[Medline]
Kapoor, P. & Frappier, L. (2003). EBNA1 partitions EpsteinBarr virus plasmids in yeast cells by attaching to human EBNA1-binding protein 2 on mitotic chromosomes. J Virol 77, 69466956.
Kapoor, P., Shire, K. & Frappier, L. (2001). Reconstitution of EpsteinBarr virus-based plasmid partitioning in budding yeast. EMBO J 20, 222230.
Kedes, D. H., Lagunoff, M., Renne, R. & Ganem, D. (1997). Identification of the gene encoding the major latency-associated nuclear antigen of the Kaposi's sarcoma-associated herpesvirus. J Clin Invest 100, 26062610.
Kellam, P., Boshoff, C., Whitby, D., Matthews, S., Weiss, R. A. & Talbot, S. J. (1997). Identification of a major latent nuclear antigen, LNA-1, in the human herpesvirus 8 genome. J Hum Virol 1, 1929.[Medline]
Lee, M.-A., Diamond, M. E. & Yates, J. L. (1999). Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by EpsteinBarr virus. J Virol 73, 29742982.
Leight, E. R. & Sugden, B. (2000). EBNA-1: a protein pivotal to latent infection by EpsteinBarr virus. Rev Med Virol 10, 83100.[CrossRef][Medline]
Lupton, S. & Levine, A. J. (1985). Mapping genetic elements of EpsteinBarr virus that facilitate extrachromosomal persistence of EpsteinBarr virus-derived plasmids in human cells. Mol Cell Biol 5, 25332542.[Medline]
Piolot, T., Tramier, M., Coppey, M., Nicolas, J.-C. & Marechal, V. (2001). Close but distinct regions of human herpesvirus 8 latency-associated nuclear antigen 1 are responsible for nuclear targeting and binding to human mitotic chromosomes. J Virol 75, 39483959.
Rainbow, L., Platt, G., Simpson, G., Sarid, R., Gao, S., Stoiber, H., Herrington, C., Moore, P. & Schulz, T. (1997). The 222- to 234-kilodalton latent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) is encoded by orf73 and is a component of the latency-associated nuclear antigen. J Virol 71, 59155921.[Abstract]
Russo, J. J., Bohenzky, R. A., Chien, M.-C. & 8 other authors (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc Natl Acad Sci U S A 93, 1486214867.
Schwam, D. R., Luciano, R. L., Mahajan, S. S., Wong, L. & Wilson, A. C. (2000). Carboxy terminus of human herpesvirus 8 latency-associated nuclear antigen mediates dimerization, transcriptional repression, and targeting to nuclear bodies. J Virol 74, 85328540.
Shire, K., Ceccarelli, D. F. J., Avolio-Hunter, T. M. & Frappier, L. (1999). EBP2, a human protein that interacts with sequences of the EpsteinBarr virus nuclear antigen 1 important for plasmid maintenance. J Virol 73, 25872595.
Suzuki, M. (1989). SPKK, a new nucleic acid-binding unit of protein found in histone. EMBO J 8, 797804.[Abstract]
Talbot, S. J., Weiss, R. A., Kellam, P. & Boshoff, C. (1999). Transcriptional analysis of human herpesvirus-8 open reading frames 71, 72, 73, K14, and 74 in a primary effusion lymphoma cell line. Virology 257, 8494.[CrossRef][Medline]
Thome, M., Schneider, P., Hofmann, K. & 11 other authors (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517521.[CrossRef][Medline]
Viejo-Borbolla, A., Kati, E., Sheldon, J. A., Nathan, K., Mattsson, K., Szekely, L. & Schulz, T. F. (2003). A domain in the C-terminal region of latency-associated nuclear antigen 1 of Kaposi's sarcoma-associated herpesvirus affects transcriptional activation and binding to nuclear heterochromatin. J Virol 77, 70937100.
Virgin, H., IV, Latreille, P., Wamsley, P., Hallsworth, K., Weck, K., Dal Canto, A. & Speck, S. (1997). Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71, 58945904.[Abstract]
Wu, H., Kapoor, P. & Frappier, L. (2002). Separation of the DNA replication, segregation, and transcriptional activation functions of EpsteinBarr nuclear antigen 1. J Virol 76, 24802490.
Yates, J., Warren, N., Reisman, D. & Sugden, B. (1984). A cis-acting element from the EpsteinBarr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc Natl Acad Sci U S A 81, 38063810.[Abstract]
Yates, J. L., Warren, N. & Sugden, B. (1985). Stable replication of plasmids derived from EpsteinBarr virus in various mammalian cells. Nature 313, 812815.[Medline]
Received 18 June 2003;
accepted 3 October 2003.