Vol. 64, No. 11
JOURNAL OF VIROLOGY, Nov. 1990, p. 5626-5627 0022-538X/90/115626-02$02.00/0
Copyright © 1990, American Society for Microbiology
NOTES
Analysis of the Junctions between Human Immunodeficiency Virus Type 1 Proviral DNA and Human DNA CORNELIS VINK,1 MARTIJN GROENINK,2 YPE ELGERSMA,1 RON A. M. FOUCHIER,2 MATTHIJS TERSMETTE,2 AND RONALD H. A. PLASTERK1* Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam,1 and Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam,2 The Netherlands Received 29 May 1990/Accepted 27 July 1990 Integrated retroviral DNA is flanked by short direct repeats of the target DNA. The length of these repeats is specific for the provirus that is integrated (H. E. Varmus, in J. A. Shapiro, ed., Mobile Genetic Elements, 1983). For the human immunodeficiency virus type I (HIV-1), the length of the direct repeats in the target DNA was shown to be 5 bp in one case (Muesing et al., Nature [London] 313:450-458, 1985) and 7 bp in another (Starcich et al., Science 227:538-540, 1985). One possible explanation for this discrepancy is that the direct repeats flanking HIV-1 proviruses are variable. To investigate this, we analyzed the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. In each clone the provirus was flanked by a 5-bp direct repeat of human DNA. Analysis of the proviral clone previously described as being flanked by a 7-bp direct repeat of target DNA (Starcich et al., op. cit.) revealed that this clone was flanked by a 5-bp repeat instead. Therefore, we conclude that HIV-1 proviruses are flanked by 5-bp direct repeats of human DNA. The sequences of the 5-bp duplications from the different proviral clones do not have any apparent similarity to each other or to HIV-1 DNA.
Integration of a double-stranded DNA copy of the viral RNA into the genome of the infected cell is an essential step for efficient retroviral replication. The integrated provirus can then be expressed to form the RNAs and proteins needed for the assembly of new virions. These virions bud from the cell surface and infect other permissive cells. Early in infection, a double-stranded DNA copy of the retroviral genome is synthesized by the viral reverse transcriptase. This linear DNA molecule can then be integrated into the genome of the host. Integrated proviruses are colinear with the unintegrated double-stranded DNA that is present in the cytoplasm of infected cells. Upon integration, most retroviruses lose 2 bp from each end of the double-stranded DNA copy of the viral genome (for a review, see Varmus and Brown [13]). Proviruses are flanked by short direct repeats of the target DNA (4 to 6 bp) and terminate with 5'-TG and CA-3'. The length of the target DNA duplication is virus specific (12). For human immunodeficiency virus (HIV), the length of the direct repeats flanking the proviruses was shown to be 5 bp in one case (5) and 7 bp in another (10). We wanted to reinvestigate the number of base pairs duplicated in the target DNA, since it seemed unlikely that it would be variable. Here we report the analysis of the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. The molecular cloning of HIV-1 proviral DNA will be described in detail elsewhere (M. Groenink et al., unpublished data). The viruses we analyzed were different isolates from the same patient. High-molecular-weight genomic DNA was isolated from peripheral blood lymphocytes in*
fected with HIV-1 in tissue culture (11). Genomic DNA fragments were cloned into bacteriophage lambda DNA. HIV-1 DNA-containing lambda phages were identified by plaque hybridization. The inserts from these phages were subcloned into vector pUC18 (4). The junctions between human DNA and HIV-1 proviral DNA within the resulting plasmids were sequenced. The junction sequences from nine clones are shown in Fig. 1. All proviral clones were flanked by a 5-bp direct repeat of the target DNA. The flanking sequences from two other HIV-1 proviruses have been published (5, 10). One of the proviruses was flanked by a 5-bp direct repeat of human DNA (5), in accordance with our results. The other provirus, however, clone HXB-2, was shown to be flanked by a 7-bp target DNA duplication (10). The sequence of this duplication is 5'-TAGTAGT-3'. To investigate this discrepancy, we sequenced the junctions between HIV-1 proviral DNA and human DNA from clone HXB-2. In contrast to the results of Starcich et al. (10), we found a 5-bp direct repeat sequence flanking the HIV-1 provirus (Fig. 1). The sequence of this repeat was 5'-GTAGT-3'. An explanation for this inconsistency could be that the G nucleotide that flanks the 3' end (CA-3') of the proviral U5 sequence (Fig. 1) was absent from or not observed in clone HXB-2 sequenced by Starcich et al. (in which case the provirus would be flanked by a 7-bp target DNA duplication). The sequences of the 5-bp direct repeats were different in each proviral clone. Although the repeats from clones 1, 7, 8, and 9 did show some similarity (5'-GTPuNN-3', where Pu is a purine and N is any nucleotide), there does not seem to be a specific target sequence for integration. This lack of any sequence motif for integration sites is consistent with the results found for other retroviruses (1, 8, 9). We cannot
Corresponding author. 5626
NOTES
VOL. 64, 1990 U3 end
1. 2. 5. 6. 7. 8. 9.
HIV-1 proviruses integrated in a cell-free system; they found a 5-bp duplication. This corresponds to the length of the duplications we observed resulting from HIV-1 integrations -__ ---A---AA G---A in vivo. ---A- G----
AC TGGAA GGGCT AATTC ACTCC CAACG AAGAC AAGAT
Consensus:
3. 4.
ACTTA GAGGG CCTTA ACTGT TTTTT TTTTT
GTAGT GCATG TTTAC GATCA ATAAG AAGAG TCGAG GTGTT AACAA GTATT TTGAG GTAAG
----- --------- --- T----- --------- --------- --------- --------- --------- --------- -----
-----
----T ----T ---- T ----T ----T ---- T ---- T ---- T
-
-----
-----
-----
-----
---A- G----
- ---A-----__ - ---A-----__ - ---A-----__ -----__ ---A1. Brown, - ---A-----__
U5 end Consensus: 1
CAGAC CCTTT TAGTC AGTGT GGAAA ATCTC TAGCA GT
.
2. 3. 4. 5. 6. 7. 8. 9
.
5627
--T--
- -
__ __ __ __ __ __
---A
-- -- -
-----A
----
----- ----- -
-A-- -A-- -
_- ---- - -A-- -
AGTTC CCCTG CTAAA TACTA AAATA GGAGT TTATA CACTG
-----
-----
-----
-----
GTAGT GCATG TTTAC GATCA ATAAG AAGAG GTGTT GTATT
-----
-----
GTAAG GATTA
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
FIG. 1. Junctions between HIV-1 proviral DNA and human DNA. The consensus for the HIV-1 long terminal repeat terminal sequences is from reference 14. The terminal nucleotides of this consensus are shown in boldface letters. These nucleotides are removed during proviral integration. The names of the clones are: 1, pHXB-2D (isolation of this clone has been described before; this plasmid is a subclone of lambda clone HXB-2 [3]); 2, 168.7 1; 3, 320.2A 1.1; 4, 320.2A 1.2; 5, 320.2A 2.1; 6, 320.3 4; 7, 320.3 7; 8, 320.3 1; 9, 320.3 6. The 5-bp direct repeats of human DNA that flank the proviral DNAs are underlined. The junctions were sequenced by the dideoxy chain termination method (6) with the Sequenase system (U.S. Biochemical Corp.). The sequence primers used were 5'-GTGTGCCCGTCTGTTGT-3' and 5'-TAGCCTTGTGTGTGGTA-3'. The first primer hybridizes to sequences present in the long terminal repeat so that its 3' end is 60 nucleotides from the U5 end of the long terminal repeat. The second primer hybridizes to U3 sequences so that its 3' end is 53 nucleotides from the U3 end.
exclude the possibility that there are highly preferred host sites for integration of HIV-1 in human DNA because, as was shown for Rous sarcoma virus, identification of these highly preferred sites requires analysis of several thousand proviral clones (7). We conclude that integrated HIV-1 proviruses are flanked by 5-bp direct repeats of target DNA. We have begun to analyze hybrid Moloney murine leukemia virus-HIV integration systems (13a), and these may point to the factor(s) that is responsible for the length of the target DNA duplication (4 bp for Moloney virus, 5 bp for HIV) and therefore is probably responsible for the staggered cut in the target DNA that precedes the actual integration reaction. Although there does not seem to be a target sequence specificity for integration by HIV-1, it cannot be excluded that factors such as DNA supercoiling, chromatin structure, and transcription play a role in exposing certain regions as potential targets for integration. Recently, Ellison et al. (2) analyzed target sites of three
LITERATURE CITED P. O., B. Bowerman, H. E. Varmus, and J. M. Bishop. 1987. Correct integration of retroviral DNA in vitro. Cell 49:347-356. 2. Ellison, V., H. Abrams, T. Roe, J. Lifson, and P. Brown. 1990. Human immunodeficiency virus integration in a cell-free system. J. Virol. 64:2711-2715. 3. Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Science 316:262-265. 4. Messing, J., R. Crea, and P. H. Seeberg. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309-321. 5. Muesing, M. A., D. H. Smith, C. D. Cabradilla, C. V. Benton, L. A. Lasky, and D. J. Capon. 1985. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature (London) 313:450-458. 6. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 7. Shih, C.-C., J. P. Stoye, and J. M. Coffin. 1988. Highly preferred targets for retrovirus integration. Cell 53:531-537. 8. Shimotohno, K., and H. M. Temin. 1980. No apparent nucleotide sequence specificity in cellular DNA juxtaposed to retrovirus proviruses. Proc. Natl. Acad. Sci. USA 77:7357-7361. 9. Shoemaker, C., S. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore. 1980. Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc. Natl. Acad. Sci. USA 77:3932-3936. 10. Starcich, B., L. Ratner, S. F. Josephs, T. Okamoto, R. C. Gallo, and F. Wong-Staal. 1985. Characterization of long terminal repeat sequences of HTLV-III. Science 227:538-540. 11. Tersmette, M., R. E. Y. de Goede, B. J. M. Al, I. N. Winkel, R. A. Gruters, H. T. Cuypers, H. G. Huisman, and F. Miedema. 1988. Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytiuminducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. J. Virol. 62:20262032. 12. Varmus, H. E. 1983. Retroviruses, p. 411-503. In J. Shapiro (ed.), Mobile genetic elements. Academic Press, Inc., New York. 13. Varmus, H. E., and P. 0. Brown. 1989. Retroviruses, p. 53-108. In M. M. Howe and D. E. Berg (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 13a.Vink, C., D. C. van Gent, and R. H. A. Plasterk. 1990. Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells. J. Virol. 64:52195222. 14. Wain-Hobson, S., P. Sonigo, 0. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17.
JOURNAL OF VIROLOGY, Nov. 1990, p. 5626-5627 0022-538X/90/115626-02$02.00/0
Copyright © 1990, American Society for Microbiology
NOTES
Analysis of the Junctions between Human Immunodeficiency Virus Type 1 Proviral DNA and Human DNA CORNELIS VINK,1 MARTIJN GROENINK,2 YPE ELGERSMA,1 RON A. M. FOUCHIER,2 MATTHIJS TERSMETTE,2 AND RONALD H. A. PLASTERK1* Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam,1 and Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam,2 The Netherlands Received 29 May 1990/Accepted 27 July 1990 Integrated retroviral DNA is flanked by short direct repeats of the target DNA. The length of these repeats is specific for the provirus that is integrated (H. E. Varmus, in J. A. Shapiro, ed., Mobile Genetic Elements, 1983). For the human immunodeficiency virus type I (HIV-1), the length of the direct repeats in the target DNA was shown to be 5 bp in one case (Muesing et al., Nature [London] 313:450-458, 1985) and 7 bp in another (Starcich et al., Science 227:538-540, 1985). One possible explanation for this discrepancy is that the direct repeats flanking HIV-1 proviruses are variable. To investigate this, we analyzed the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. In each clone the provirus was flanked by a 5-bp direct repeat of human DNA. Analysis of the proviral clone previously described as being flanked by a 7-bp direct repeat of target DNA (Starcich et al., op. cit.) revealed that this clone was flanked by a 5-bp repeat instead. Therefore, we conclude that HIV-1 proviruses are flanked by 5-bp direct repeats of human DNA. The sequences of the 5-bp duplications from the different proviral clones do not have any apparent similarity to each other or to HIV-1 DNA.
Integration of a double-stranded DNA copy of the viral RNA into the genome of the infected cell is an essential step for efficient retroviral replication. The integrated provirus can then be expressed to form the RNAs and proteins needed for the assembly of new virions. These virions bud from the cell surface and infect other permissive cells. Early in infection, a double-stranded DNA copy of the retroviral genome is synthesized by the viral reverse transcriptase. This linear DNA molecule can then be integrated into the genome of the host. Integrated proviruses are colinear with the unintegrated double-stranded DNA that is present in the cytoplasm of infected cells. Upon integration, most retroviruses lose 2 bp from each end of the double-stranded DNA copy of the viral genome (for a review, see Varmus and Brown [13]). Proviruses are flanked by short direct repeats of the target DNA (4 to 6 bp) and terminate with 5'-TG and CA-3'. The length of the target DNA duplication is virus specific (12). For human immunodeficiency virus (HIV), the length of the direct repeats flanking the proviruses was shown to be 5 bp in one case (5) and 7 bp in another (10). We wanted to reinvestigate the number of base pairs duplicated in the target DNA, since it seemed unlikely that it would be variable. Here we report the analysis of the junctions between HIV-1 proviral DNA and human DNA from nine individual clones. The molecular cloning of HIV-1 proviral DNA will be described in detail elsewhere (M. Groenink et al., unpublished data). The viruses we analyzed were different isolates from the same patient. High-molecular-weight genomic DNA was isolated from peripheral blood lymphocytes in*
fected with HIV-1 in tissue culture (11). Genomic DNA fragments were cloned into bacteriophage lambda DNA. HIV-1 DNA-containing lambda phages were identified by plaque hybridization. The inserts from these phages were subcloned into vector pUC18 (4). The junctions between human DNA and HIV-1 proviral DNA within the resulting plasmids were sequenced. The junction sequences from nine clones are shown in Fig. 1. All proviral clones were flanked by a 5-bp direct repeat of the target DNA. The flanking sequences from two other HIV-1 proviruses have been published (5, 10). One of the proviruses was flanked by a 5-bp direct repeat of human DNA (5), in accordance with our results. The other provirus, however, clone HXB-2, was shown to be flanked by a 7-bp target DNA duplication (10). The sequence of this duplication is 5'-TAGTAGT-3'. To investigate this discrepancy, we sequenced the junctions between HIV-1 proviral DNA and human DNA from clone HXB-2. In contrast to the results of Starcich et al. (10), we found a 5-bp direct repeat sequence flanking the HIV-1 provirus (Fig. 1). The sequence of this repeat was 5'-GTAGT-3'. An explanation for this inconsistency could be that the G nucleotide that flanks the 3' end (CA-3') of the proviral U5 sequence (Fig. 1) was absent from or not observed in clone HXB-2 sequenced by Starcich et al. (in which case the provirus would be flanked by a 7-bp target DNA duplication). The sequences of the 5-bp direct repeats were different in each proviral clone. Although the repeats from clones 1, 7, 8, and 9 did show some similarity (5'-GTPuNN-3', where Pu is a purine and N is any nucleotide), there does not seem to be a specific target sequence for integration. This lack of any sequence motif for integration sites is consistent with the results found for other retroviruses (1, 8, 9). We cannot
Corresponding author. 5626
NOTES
VOL. 64, 1990 U3 end
1. 2. 5. 6. 7. 8. 9.
HIV-1 proviruses integrated in a cell-free system; they found a 5-bp duplication. This corresponds to the length of the duplications we observed resulting from HIV-1 integrations -__ ---A---AA G---A in vivo. ---A- G----
AC TGGAA GGGCT AATTC ACTCC CAACG AAGAC AAGAT
Consensus:
3. 4.
ACTTA GAGGG CCTTA ACTGT TTTTT TTTTT
GTAGT GCATG TTTAC GATCA ATAAG AAGAG TCGAG GTGTT AACAA GTATT TTGAG GTAAG
----- --------- --- T----- --------- --------- --------- --------- --------- --------- -----
-----
----T ----T ---- T ----T ----T ---- T ---- T ---- T
-
-----
-----
-----
-----
---A- G----
- ---A-----__ - ---A-----__ - ---A-----__ -----__ ---A1. Brown, - ---A-----__
U5 end Consensus: 1
CAGAC CCTTT TAGTC AGTGT GGAAA ATCTC TAGCA GT
.
2. 3. 4. 5. 6. 7. 8. 9
.
5627
--T--
- -
__ __ __ __ __ __
---A
-- -- -
-----A
----
----- ----- -
-A-- -A-- -
_- ---- - -A-- -
AGTTC CCCTG CTAAA TACTA AAATA GGAGT TTATA CACTG
-----
-----
-----
-----
GTAGT GCATG TTTAC GATCA ATAAG AAGAG GTGTT GTATT
-----
-----
GTAAG GATTA
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
-----
FIG. 1. Junctions between HIV-1 proviral DNA and human DNA. The consensus for the HIV-1 long terminal repeat terminal sequences is from reference 14. The terminal nucleotides of this consensus are shown in boldface letters. These nucleotides are removed during proviral integration. The names of the clones are: 1, pHXB-2D (isolation of this clone has been described before; this plasmid is a subclone of lambda clone HXB-2 [3]); 2, 168.7 1; 3, 320.2A 1.1; 4, 320.2A 1.2; 5, 320.2A 2.1; 6, 320.3 4; 7, 320.3 7; 8, 320.3 1; 9, 320.3 6. The 5-bp direct repeats of human DNA that flank the proviral DNAs are underlined. The junctions were sequenced by the dideoxy chain termination method (6) with the Sequenase system (U.S. Biochemical Corp.). The sequence primers used were 5'-GTGTGCCCGTCTGTTGT-3' and 5'-TAGCCTTGTGTGTGGTA-3'. The first primer hybridizes to sequences present in the long terminal repeat so that its 3' end is 60 nucleotides from the U5 end of the long terminal repeat. The second primer hybridizes to U3 sequences so that its 3' end is 53 nucleotides from the U3 end.
exclude the possibility that there are highly preferred host sites for integration of HIV-1 in human DNA because, as was shown for Rous sarcoma virus, identification of these highly preferred sites requires analysis of several thousand proviral clones (7). We conclude that integrated HIV-1 proviruses are flanked by 5-bp direct repeats of target DNA. We have begun to analyze hybrid Moloney murine leukemia virus-HIV integration systems (13a), and these may point to the factor(s) that is responsible for the length of the target DNA duplication (4 bp for Moloney virus, 5 bp for HIV) and therefore is probably responsible for the staggered cut in the target DNA that precedes the actual integration reaction. Although there does not seem to be a target sequence specificity for integration by HIV-1, it cannot be excluded that factors such as DNA supercoiling, chromatin structure, and transcription play a role in exposing certain regions as potential targets for integration. Recently, Ellison et al. (2) analyzed target sites of three
LITERATURE CITED P. O., B. Bowerman, H. E. Varmus, and J. M. Bishop. 1987. Correct integration of retroviral DNA in vitro. Cell 49:347-356. 2. Ellison, V., H. Abrams, T. Roe, J. Lifson, and P. Brown. 1990. Human immunodeficiency virus integration in a cell-free system. J. Virol. 64:2711-2715. 3. Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Science 316:262-265. 4. Messing, J., R. Crea, and P. H. Seeberg. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309-321. 5. Muesing, M. A., D. H. Smith, C. D. Cabradilla, C. V. Benton, L. A. Lasky, and D. J. Capon. 1985. Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature (London) 313:450-458. 6. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467. 7. Shih, C.-C., J. P. Stoye, and J. M. Coffin. 1988. Highly preferred targets for retrovirus integration. Cell 53:531-537. 8. Shimotohno, K., and H. M. Temin. 1980. No apparent nucleotide sequence specificity in cellular DNA juxtaposed to retrovirus proviruses. Proc. Natl. Acad. Sci. USA 77:7357-7361. 9. Shoemaker, C., S. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore. 1980. Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc. Natl. Acad. Sci. USA 77:3932-3936. 10. Starcich, B., L. Ratner, S. F. Josephs, T. Okamoto, R. C. Gallo, and F. Wong-Staal. 1985. Characterization of long terminal repeat sequences of HTLV-III. Science 227:538-540. 11. Tersmette, M., R. E. Y. de Goede, B. J. M. Al, I. N. Winkel, R. A. Gruters, H. T. Cuypers, H. G. Huisman, and F. Miedema. 1988. Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytiuminducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. J. Virol. 62:20262032. 12. Varmus, H. E. 1983. Retroviruses, p. 411-503. In J. Shapiro (ed.), Mobile genetic elements. Academic Press, Inc., New York. 13. Varmus, H. E., and P. 0. Brown. 1989. Retroviruses, p. 53-108. In M. M. Howe and D. E. Berg (ed.), Mobile DNA. American Society for Microbiology, Washington, D.C. 13a.Vink, C., D. C. van Gent, and R. H. A. Plasterk. 1990. Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells. J. Virol. 64:52195222. 14. Wain-Hobson, S., P. Sonigo, 0. Danos, S. Cole, and M. Alizon. 1985. Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17.
