Abstract
The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroviruses. The process by which the avian sarcoma virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.
Full Text
The Full Text of this article is available as a PDF (463.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andrake M. D., Skalka A. M. Multimerization determinants reside in both the catalytic core and C terminus of avian sarcoma virus integrase. J Biol Chem. 1995 Dec 8;270(49):29299–29306. doi: 10.1074/jbc.270.49.29299. [DOI] [PubMed] [Google Scholar]
- Andrake M. D., Skalka A. M. Retroviral integrase, putting the pieces together. J Biol Chem. 1996 Aug 16;271(33):19633–19636. doi: 10.1074/jbc.271.33.19633. [DOI] [PubMed] [Google Scholar]
- Bizub-Bender D., Kulkosky J., Skalka A. M. Monoclonal antibodies against HIV type 1 integrase: clues to molecular structure. AIDS Res Hum Retroviruses. 1994 Sep;10(9):1105–1115. doi: 10.1089/aid.1994.10.1105. [DOI] [PubMed] [Google Scholar]
- Boulikas T. Nuclear localization signals (NLS). Crit Rev Eukaryot Gene Expr. 1993;3(3):193–227. [PubMed] [Google Scholar]
- Bowerman B., Brown P. O., Bishop J. M., Varmus H. E. A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev. 1989 Apr;3(4):469–478. doi: 10.1101/gad.3.4.469. [DOI] [PubMed] [Google Scholar]
- Bujacz G., Jaskólski M., Alexandratos J., Wlodawer A., Merkel G., Katz R. A., Skalka A. M. High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J Mol Biol. 1995 Oct 20;253(2):333–346. doi: 10.1006/jmbi.1995.0556. [DOI] [PubMed] [Google Scholar]
- Bukrinsky M. I., Haggerty S., Dempsey M. P., Sharova N., Adzhubel A., Spitz L., Lewis P., Goldfarb D., Emerman M., Stevenson M. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993 Oct 14;365(6447):666–669. doi: 10.1038/365666a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bukrinsky M. I., Sharova N., Dempsey M. P., Stanwick T. L., Bukrinskaya A. G., Haggerty S., Stevenson M. Active nuclear import of human immunodeficiency virus type 1 preintegration complexes. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6580–6584. doi: 10.1073/pnas.89.14.6580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cannon P. M., Byles E. D., Kingsman S. M., Kingsman A. J. Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication. J Virol. 1996 Jan;70(1):651–657. doi: 10.1128/jvi.70.1.651-657.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
- Cochrane A. W., Jones K. S., Beidas S., Dillon P. J., Skalka A. M., Rosen C. A. Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J Virol. 1991 Oct;65(10):5305–5313. doi: 10.1128/jvi.65.10.5305-5313.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Di Marzio P., Choe S., Ebright M., Knoblauch R., Landau N. R. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol. 1995 Dec;69(12):7909–7916. doi: 10.1128/jvi.69.12.7909-7916.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
- Dyda F., Hickman A. B., Jenkins T. M., Engelman A., Craigie R., Davies D. R. Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science. 1994 Dec 23;266(5193):1981–1986. doi: 10.1126/science.7801124. [DOI] [PubMed] [Google Scholar]
- Eijkelenboom A. P., Lutzke R. A., Boelens R., Plasterk R. H., Kaptein R., Hård K. The DNA-binding domain of HIV-1 integrase has an SH3-like fold. Nat Struct Biol. 1995 Sep;2(9):807–810. doi: 10.1038/nsb0995-807. [DOI] [PubMed] [Google Scholar]
- Farnet C. M., Haseltine W. A. Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex. J Virol. 1991 Apr;65(4):1910–1915. doi: 10.1128/jvi.65.4.1910-1915.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gallay P., Swingler S., Aiken C., Trono D. HIV-1 infection of nondividing cells: C-terminal tyrosine phosphorylation of the viral matrix protein is a key regulator. Cell. 1995 Feb 10;80(3):379–388. doi: 10.1016/0092-8674(95)90488-3. [DOI] [PubMed] [Google Scholar]
- Goff S. P. Genetics of retroviral integration. Annu Rev Genet. 1992;26:527–544. doi: 10.1146/annurev.ge.26.120192.002523. [DOI] [PubMed] [Google Scholar]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Hammarskjöld M. L., Li H., Rekosh D., Prasad S. Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron. J Virol. 1994 Feb;68(2):951–958. doi: 10.1128/jvi.68.2.951-958.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heinzinger N. K., Bukrinsky M. I., Haggerty S. A., Ragland A. M., Kewalramani V., Lee M. A., Gendelman H. E., Ratner L., Stevenson M., Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7311–7315. doi: 10.1073/pnas.91.15.7311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphries E. H., Coffin J. M. Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus. J Virol. 1976 Feb;17(2):393–401. doi: 10.1128/jvi.17.2.393-401.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphries E. H., Glover C., Reichmann M. E. Rous sarcoma virus infection of synchronized cells establishes provirus integration during S-phase DNA synthesis prior to cellular division. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2601–2605. doi: 10.1073/pnas.78.4.2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphries E. H., Temin H. M. Requirement for cell division for initiation of transcription of Rous sarcoma virus RNA. J Virol. 1974 Sep;14(3):531–546. doi: 10.1128/jvi.14.3.531-546.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
- Katz R. A., Skalka A. M. The retroviral enzymes. Annu Rev Biochem. 1994;63:133–173. doi: 10.1146/annurev.bi.63.070194.001025. [DOI] [PubMed] [Google Scholar]
- Khan E., Mack J. P., Katz R. A., Kulkosky J., Skalka A. M. Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 1991 Feb 25;19(4):851–860. doi: 10.1093/nar/19.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kulkosky J., Jones K. S., Katz R. A., Mack J. P., Skalka A. M. Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol. 1992 May;12(5):2331–2338. doi: 10.1128/mcb.12.5.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee Y. M., Coffin J. M. Efficient autointegration of avian retrovirus DNA in vitro. J Virol. 1990 Dec;64(12):5958–5965. doi: 10.1128/jvi.64.12.5958-5965.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis P. F., Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol. 1994 Jan;68(1):510–516. doi: 10.1128/jvi.68.1.510-516.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis P., Hensel M., Emerman M. Human immunodeficiency virus infection of cells arrested in the cell cycle. EMBO J. 1992 Aug;11(8):3053–3058. doi: 10.1002/j.1460-2075.1992.tb05376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lodi P. J., Ernst J. A., Kuszewski J., Hickman A. B., Engelman A., Craigie R., Clore G. M., Gronenborn A. M. Solution structure of the DNA binding domain of HIV-1 integrase. Biochemistry. 1995 Aug 8;34(31):9826–9833. doi: 10.1021/bi00031a002. [DOI] [PubMed] [Google Scholar]
- Morris-Vasios C., Kochan J. P., Skalka A. M. Avian sarcoma-leukosis virus pol-endo proteins expressed independently in mammalian cells accumulate in the nucleus but can be directed to other cellular compartments. J Virol. 1988 Jan;62(1):349–353. doi: 10.1128/jvi.62.1.349-353.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mumm S. R., Hippenmeyer P. J., Grandgenett D. P. Characterization of a stable eukaryotic cell line expressing the Rous sarcoma virus integrase. Virology. 1992 Aug;189(2):500–510. doi: 10.1016/0042-6822(92)90574-9. [DOI] [PubMed] [Google Scholar]
- Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
- Roe T., Reynolds T. C., Yu G., Brown P. O. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 1993 May;12(5):2099–2108. doi: 10.1002/j.1460-2075.1993.tb05858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwartz S., Felber B. K., Benko D. M., Fenyö E. M., Pavlakis G. N. Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J Virol. 1990 Jun;64(6):2519–2529. doi: 10.1128/jvi.64.6.2519-2529.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
- Stevenson M. Portals of entry: uncovering HIV nuclear transport pathways. Trends Cell Biol. 1996 Jan;6(1):9–15. doi: 10.1016/0962-8924(96)81032-4. [DOI] [PubMed] [Google Scholar]
- von Schwedler U., Kornbluth R. S., Trono D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6992–6996. doi: 10.1073/pnas.91.15.6992. [DOI] [PMC free article] [PubMed] [Google Scholar]