Chromosome Structure and Human Immunodeficiency Virus Type 1 cDNA Integration: Centromeric Alphoid Repeats Are a Disfavored Target

ABSTRACT Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

[1]  M. Bukrinsky,et al.  Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  F. Bushman,et al.  Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro , 1995, Journal of virology.

[3]  W. Haseltine,et al.  Integration of human immunodeficiency virus type 1 DNA in vitro. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  F. Bushman,et al.  Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M F Singer,et al.  LINE-1: a mammalian transposable element. , 1987, Biochimica et biophysica acta.

[6]  C. Schmid,et al.  A transposon-like element in human DNA , 1985, Nature.

[7]  C. Pauza.  Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination. , 1990, Virology.

[8]  T. Heidmann,et al.  Isolation of novel human endogenous retrovirus-like elements with foamy virus-related pol sequence , 1995, Journal of virology.

[9]  F. Bushman,et al.  HIV cDNA integration: molecular biology and inhibitor development , 1996, AIDS.

[10]  F. Bushman,et al.  Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites , 1997, Journal of virology.

[11]  A. Abo,et al.  The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix , 1997, Journal of virology.

[12]  D. Grandgenett,et al.  Retroviral integration: in vitro host site selection by avian integrase , 1994, Journal of virology.

[13]  D. Voytas,et al.  The Saccharomyces Ty5 retrotransposon family is associated with origins of DNA replication at the telomeres and the silent mating locus HMR. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  L E Orgel,et al.  In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[15]  H L Robinson,et al.  Acceptor sites for retroviral integrations map near DNase I-hypersensitive sites in chromatin , 1986, Journal of virology.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  H. Varmus,et al.  Retroviral integration into minichromosomes in vitro. , 1992, The EMBO journal.

[18]  F. Bushman,et al.  Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Brown,et al.  Host sequences flanking the HIV provirus. , 1990, Nucleic acids research.

[20]  J. Griffith,et al.  Human immunodeficiency virus type 1 may preferentially integrate into chromatin occupied by L1Hs repetitive elements. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  E. Rassart,et al.  Association of a host DNA structure with retroviral integration sites in chromosomal DNA. , 1994, Virology.

[22]  P. Brown,et al.  Correct integration of retroviral DNA in vitro , 1987, Cell.

[23]  D. Chalker,et al.  Ty3 integrates within the region of RNA polymerase III transcription initiation. , 1992, Genes & development.

[24]  H. Willard,et al.  Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome. , 1985, Nucleic acids research.

[25]  S. Lukyanov,et al.  An improved PCR method for walking in uncloned genomic DNA. , 1995, Nucleic acids research.

[26]  A. Wolffe.  Centromeric Chromatin: Histone deviants , 1995, Current Biology.

[27]  Lucio Luzzatto,et al.  Somatic Mutations in Paroxysmal Nocturnal Hemoglobinuria: A Blessing in Disguise? , 1997, Cell.

[28]  R. Britten,et al.  Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Emerman,et al.  Human immunodeficiency virus infection of cells arrested in the cell cycle. , 1992, The EMBO journal.

[30]  H. Varmus,et al.  Simian virus 40 minichromosomes as targets for retroviral integration in vivo. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[31]  S. Sandmeyer,et al.  Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element , 1995, Science.

[32]  W. Haseltine,et al.  Determination of viral proteins present in the human immunodeficiency virus type 1 preintegration complex , 1991, Journal of virology.

[33]  J. Griffith,et al.  A cluster of strong topoisomerase II cleavage sites is located near an integrated human immunodeficiency virus. , 1993, Journal of molecular biology.

[34]  R. Plasterk,et al.  Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA , 1990, Journal of virology.

[35]  M. Batzer,et al.  Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution , 1995, Molecular and cellular biology.

[36]  P. Brown,et al.  Human immunodeficiency virus integration in a cell-free system , 1990, Journal of virology.

[37]  F. Bushman,et al.  Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition , 1997, Journal of virology.

[38]  P. Brown,et al.  Disruption of the terminal base pairs of retroviral DNA during integration. , 1997, Genes & development.

[39]  J. Coffin,et al.  Nonrandom integration of retroviral DNA in vitro: effect of CpG methylation. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Coffin,et al.  Highly preferred targets for retrovirus integration , 1988, Cell.

[41]  F. Bushman,et al.  HIV nuclear import is governed by the phosphotyrosine-mediated binding of matrix to the core domain of integrase , 1995, Cell.

[42]  M. Emerman,et al.  Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene , 1992, Journal of virology.

[43]  M. Breindl,et al.  Transcriptionally active genome regions are preferred targets for retrovirus integration , 1990, Journal of virology.

[44]  F. Bushman,et al.  Target-sequence preferences of HIV-1 integration complexes in vitro. , 1996, Virology.

[45]  H. Varmus,et al.  DNA bending creates favored sites for retroviral integration: an explanation for preferred insertion sites in nucleosomes. , 1994, The EMBO journal.

[46]  W. Earnshaw,et al.  The Centromere: Hub of Chromosomal Activities , 1995, Science.

[47]  R. Jaenisch,et al.  Retrovirus insertion inactivates mouse α 1(I) collagen gene by blocking initiation of transcription , 1986, Nature.

[48]  A. Smit,et al.  The origin of interspersed repeats in the human genome. , 1996, Current opinion in genetics & development.

[49]  D. Voytas,et al.  Silent chromatin determines target preference of the Saccharomyces retrotransposon Ty5. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[50]  A. Smit,et al.  Identification of a new, abundant superfamily of mammalian LTR-transposons. , 1993, Nucleic acids research.

[51]  Harold E. Varmus,et al.  Nucleosomes, DNA-binding proteins, and DNA sequence modulate retroviral integration target site selection , 1992, Cell.

[52]  J. Coffin,et al.  Distribution of targets for avian retrovirus DNA integration in vivo. , 1994, Genes & development.

[53]  G. Natsoulis,et al.  Hotspots for unselected Ty1 transposition events on yeast chromosome III are near tRNA genes and LTR sequences , 1993, Cell.

[54]  J. Griffith,et al.  Sequence analysis of the human DNA flanking sites of human immunodeficiency virus type 1 integration , 1996, Journal of virology.

[55]  F. Bushman,et al.  Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[56]  F. Bushman,et al.  HIV-1 cDNA Integration: Requirement of HMG I(Y) Protein for Function of Preintegration Complexes In Vitro , 1997, Cell.

[57]  R. Jaenisch,et al.  Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites , 1987, Journal of virology.

[58]  F. Bushman,et al.  The influence of DNA and nucleosome structure on integration events directed by HIV integrase. , 1994, The Journal of biological chemistry.

[59]  Wentao Song,et al.  Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family , 1993 .

[60]  F. Bushman,et al.  Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[61]  S. Brady.  Motor neurons and neurofilaments in sickness and in health , 1993, Cell.