A naturally occurring C-terminal truncated isoform of the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus does not associate with viral episomal DNA Free

Abstract

The latency-associated nuclear antigen (LANA) encoded by orf73 of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to viral episomal DNA and nuclear heterochromatin in infected cells. A 3·2 kb transcript in KSHV-positive primary effusion lymphoma (PEL) cells (BCP-1 and BC-3) encoding a C-terminal truncated form of LANA (LANA-Δ76) has been identified. This transcript has the addition of a poly(A) tail at nt 3264 of orf73 resulting in an in-frame stop codon (TAA) effectively truncating LANA by 76 aa (∼8 kDa). Examination of the coding region revealed the presence of a non-canonical polyadenylation signal (AGTAAA) 17 nt upstream of the poly(A) tail. The protein expressed from this transcript is representative of the faster migration of the LANA doublet bands observed by SDS-PAGE and Western blot. Mutation of the poly(A) signal from AGTAAA to TGTACA produced a protein that co-migrated with the larger LANA isoform. A C-terminal LANA-Δ76 EGFP fusion protein localized to the nucleus but did not co-localize with endogenous LANA in BCP-1 cells, or heterochromatin in HEK293 cells. Using an electrophoretic mobility shift assay (EMSA), the authors were able to show that LANA-Δ76 does not bind to the KSHV terminal repeat motif known to interact with LANA. These data provide evidence for the presence of an isoform of LANA that may perform alternative functions in KSHV-infected cells.

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2004-06-01
2024-03-29
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References

  1. An J., Lichtenstein A. K., Brent G., Rettig M. B. 2002; The Kaposi sarcoma-associated herpesvirus (KSHV) induces cellular interleukin 6 expression: role of the KSHV latency-associated nuclear antigen and the AP1 response element. Blood 99:649–654 [CrossRef]
    [Google Scholar]
  2. 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 75:3250–3258 [CrossRef]
    [Google Scholar]
  3. Ballestas M. E., Chatis P. A., Kaye K. M. 1999; Efficient persistence of extrachromosomal KSHV DNA mediated by latency-associated nuclear antigen. Science 284:641–644 [CrossRef]
    [Google Scholar]
  4. Boshoff C., Weiss R. A. 1998; Kaposi's sarcoma-associated herpesvirus. Adv Cancer Res 75:57–86
    [Google Scholar]
  5. Boshoff C., Gao S. J., Healy L. E. 10 other authors 1998; Establishing a KSHV+ cell line (BCP-1) from peripheral blood and characterizing its growth in Nod/SCID mice. Blood 91:1671–1679
    [Google Scholar]
  6. Caron H., van Schaik B., van der Mee M. 10 other authors 2001; The human transcriptome map: clustering of highly expressed genes in chromosomal domains. Science 291:1289–1292 [CrossRef]
    [Google Scholar]
  7. Cesarman E., Chang Y., Moore P. S., Said J. W., Knowles D. M. 1995a; Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med 332:1186–1191 [CrossRef]
    [Google Scholar]
  8. Cesarman E., Moore P. S., Rao P. H., Inghirami G., Knowles D. M., Chang Y. 1995b; In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences. Blood 86:2708–2714
    [Google Scholar]
  9. Cesarman E., Nador R. G., Aozasa K., Delsol G., Said J. W., Knowles D. M. 1996; Kaposi's sarcoma-associated herpesvirus in non-AIDS related lymphomas occurring in body cavities. Am J Pathol 149:53–57
    [Google Scholar]
  10. Chang Y., Cesarman E., Pessin M. S., Lee F., Culpepper J., Knowles D. M., Moore P. S. 1994; Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266:1865–1869 [CrossRef]
    [Google Scholar]
  11. Dittmer D. P. 2003; Transcription profile of Kaposi's sarcoma-associated herpesvirus in primary Kaposi's sarcoma lesions as determined by real-time PCR arrays. Cancer Res 63:2010–2015
    [Google Scholar]
  12. Friborg J. Jr, Kong W., Hottiger M. O., Nabel G. J. 1999; p53 inhibition by the LANA protein of KSHV protects against cell death. Nature 402:889–894
    [Google Scholar]
  13. Fujimuro M., Wu F. Y., ApRhys C., Kajumbula H., Young D. B., Hayward G. S., Hayward S. D. 2003; A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency. Nat Med 9:300–306 [CrossRef]
    [Google Scholar]
  14. Gao S. J., Kingsley L., Hoover D. R. 8 other authors 1996a; Seroconversion to antibodies against Kaposi's sarcoma-associated herpesvirus-related latent nuclear antigens before the development of Kaposi's sarcoma. N Engl J Med 335:233–241 [CrossRef]
    [Google Scholar]
  15. Gao S. J., Kingsley L., Li M. 10 other authors 1996b; KSHV antibodies among Americans, Italians and Ugandans with and without Kaposi's sarcoma. Nat Med 2:925–928 [CrossRef]
    [Google Scholar]
  16. Gao S. J., Zhang Y. J., Deng J. H., Rabkin C. S., Flore O., Jenson H. B. 1999; Molecular polymorphism of Kaposi's sarcoma-associated herpesvirus (Human herpesvirus 8) latent nuclear antigen: evidence for a large repertoire of viral genotypes and dual infection with different viral genotypes. J Infect Dis 180:1466–1476 [CrossRef]
    [Google Scholar]
  17. Garber A. C., Shu M. A., Hu J., Renne R. 2001; DNA binding and modulation of gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus. J Virol 75:7882–7892 [CrossRef]
    [Google Scholar]
  18. Garber A. C., Hu J., Renne R. 2002; Latency-associated nuclear antigen (LANA) cooperatively binds to two sites within the terminal repeat, and both sites contribute to the ability of LANA to suppress transcription and to facilitate DNA replication. J Biol Chem 277:27401–27411 [CrossRef]
    [Google Scholar]
  19. Graham F. L., Smiley J., Russell W. C., Nairn R. 1977; Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59–74 [CrossRef]
    [Google Scholar]
  20. Husain S. M., Usherwood E. J., Dyson H., Coleclough C., Coppola M. A., Woodland D. L., Blackman M. A., Stewart J. P., Sample J. T. 1999; Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8(+) T lymphocytes. Proc Natl Acad Sci U S A 96:7508–7513 [CrossRef]
    [Google Scholar]
  21. Kedes D. H., Operskalski E., Busch M., Kohn R., Flood J., Ganem D. 1996; The seroepidemiology of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus): distribution of infection in KS risk groups and evidence for sexual transmission. Nat Med 2:918–924 [CrossRef]
    [Google Scholar]
  22. 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 100:2606–2610 [CrossRef]
    [Google Scholar]
  23. 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 1:19–29
    [Google Scholar]
  24. Kellam P., Bourboulia D., Dupin N., Shotton C., Fisher C., Talbot S., Boshoff C., Weiss R. A. 1999; Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8. J Virol 73:5149–5155
    [Google Scholar]
  25. Krithivas A., Young D. B., Liao G., Greene D., Hayward S. D. 2000; Human herpesvirus 8 LANA interacts with proteins of the mSin3 corepressor complex and negatively regulates Epstein–Barr virus gene expression in dually infected PEL cells. J Virol 74:9637–9645 [CrossRef]
    [Google Scholar]
  26. Lennette E. T., Blackbourn D. J., Levy J. A. 1996; Antibodies to human herpesvirus type 8 in the general population and in Kaposi's sarcoma patients. Lancet 348:858–861 [CrossRef]
    [Google Scholar]
  27. Lim C., Sohn H., Gwack Y., Choe J. 2000; Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) binds ATF4/CREB2 and inhibits its transcriptional activation activity. J Gen Virol 81:2645–2652
    [Google Scholar]
  28. Mattsson K., Kiss C., Platt G. M., Simpson G. R., Kashuba E., Klein G., Schulz T. F., Szekely L. 2002; Latent nuclear antigen of Kaposi's sarcoma herpesvirus/human herpesvirus-8 induces and relocates RING3 to nuclear heterochromatin regions. J Gen Virol 83:179–188
    [Google Scholar]
  29. 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 75:3948–3959 [CrossRef]
    [Google Scholar]
  30. Platt G. M., Simpson G. R., Mittnacht S., Schulz T. F. 1999; Latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with RING3, a homolog of the Drosophila female sterile homeotic (fsh) gene. J Virol 73:9789–9795
    [Google Scholar]
  31. Radkov S. A., Kellam P., Boshoff C. 2000; The latent nuclear antigen of Kaposi sarcoma-associated herpesvirus targets the retinoblastoma-E2F pathway and with the oncogene Hras transforms primary rat cells. Nat Med 6:1121–1127 [CrossRef]
    [Google Scholar]
  32. Rainbow L., Platt G. M., Simpson G. R., Sarid R., Gao S. J., Stoiber H., Herrington C. S., Moore P. S., Schulz T. F. 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 71:5915–5921
    [Google Scholar]
  33. Renne R., Zhong W., Herndier B., McGrath M., Abbey N., Kedes D., Ganem D. 1996; Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture. Nat Med 2:342–346 [CrossRef]
    [Google Scholar]
  34. 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 93:14862–14867 [CrossRef]
    [Google Scholar]
  35. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. 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 74:8532–8540 [CrossRef]
    [Google Scholar]
  37. Soulier J., Grollet L., Oksenhendler E. 8 other authors 1995; Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 86:1276–1280
    [Google Scholar]
  38. 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 257:84–94 [CrossRef]
    [Google Scholar]
  39. 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 77:7093–7100 [CrossRef]
    [Google Scholar]
  40. Wakeling M. N., Roy D. J., Nash A. A., Stewart J. P. 2001; Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor. J Gen Virol 82:1187–1197
    [Google Scholar]
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