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J Virol, May 1998, p. 4005-4014, Vol. 72, No. 5
Infectious Disease Laboratory, The Salk
Institute for Biological Studies, La Jolla, California 92037
Received 22 August 1997/Accepted 19 January 1998
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.
To replicate, a retrovirus must
integrate a cDNA copy of its RNA genome into a chromosome of the host.
The host integration acceptor sites are not expected to be present as
naked DNA but rather associated with histones and other DNA-binding
proteins in chromatin. DNA packaging in vivo is expected to influence
integration site selection, and the choice of integration site may have
profound effects on both the virus and the host (13, 57).
The determinants of integration efficiency in vivo remain incompletely
defined, despite their importance.
Previous surveys of in vivo integration sites have led to several
proposals for factors influencing site selection. Studies of Moloney
murine leukemia virus have supported a model in which open chromatin
regions at transcription units were favored, since associated features
such as DNase I-hypersensitive sites (45, 58) or CpG islands
(47) were apparently enriched near integration sites.
Another study proposed that unusual host DNA structures were common
near integration sites (34). A recent study of avian leukosis virus integration frequencies at several chromosomal sites
failed to show any major differences among the regions studied (62), contrary to an earlier report (50). For
human immunodeficiency virus type 1 (HIV-1), it has been proposed that
integration may be favored near repetitive elements (including LINE-1
elements [54] or Alu islands
[55]) or topoisomerase cleavage sites (24).
Assays of integration in vitro have revealed several effects of
proteins bound to target DNA. Simple DNA-binding proteins can block
access of integration complexes to target DNA, creating regions
refractory for integration (3, 9, 44). In contrast, wrapping
DNA on nucleosomes can create hot spots for integration at sites of
probable DNA distortion (40-42, 44). Distortion of DNA in
several other protein-DNA complexes can also favor integration (3,
35), consistent with the possibility that DNA distortion is
involved in the integrase mechanism (11, 48).
Here we present two experiments designed to address some of the
questions surrounding integration site selection in vivo. We have (i)
sequenced 61 integration junctions made after experimental infection of
cultured human T cells and compared them with 104 control DNA fragments
from uninfected human cells and (ii) used a region-specific PCR assay
to assess the frequency of integration near several repeated-sequence
families. In addition, we have identified a weakly conserved sequence
at in vivo integration sites and determined that it is favored for
integration when tested in vitro.
DNA manipulation.
Plasmids containing synthetic integration
target sites were prepared by annealing pairs of oligonucleotides
(CH10-1-CH10-2, CH11-1-CH11-2, and CH13-1-CH13-2) (Table
1) and ligating them with pUC19 DNA that
had been cleaved with EcoRI and HindIII. The standard cloning methods used were as described previously
(46). Integration target DNAs were prepared by cleaving the
plasmids mentioned above with PvuII, which releases the
oligonucleotide insert together with flanking plasmid DNA.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Chromosome Structure and Human Immunodeficiency
Virus Type 1 cDNA Integration: Centromeric Alphoid Repeats Are a
Disfavored Target
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Oligonucleotides used in this study
Construction of DNA libraries. To generate a large pool of independent integration events, SupT1 cells (2 × 107 cells) were infected with the HXB2 or R9 (56) (referred to as R8 in reference 22) HIV-1 strain. Viral stocks were assayed by measuring the concentration of p24, and the infectivity was scored by the MAGI assay (28). Cells were infected at a multiplicity of 1 to 10 and harvested 12 to 14 h later. The cellular genomic DNA was depleted of low-molecular-weight DNA prior to cloning as described previously (39).
For construction of library 1 (Fig. 1, method 1), DNA from infected cells was cleaved with HindIII and circularized by ligation (31). Sixty-six nanograms of DNA was used as the template for PCR. HUA and HUB, divergently oriented primers complementary to the HIV long terminal repeats (LTRs), were used for the first amplification. Amplification was carried out for 35 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 3 min. The products were purified by using the Qiaquick PCR purification kit (Qiagen, Santa Clarita, Calif.). One microliter from the 50-µl column eluate was used as the template for the second-round PCR (20 cycles; program as described above) with nested primers det3b and IP3. For construction of library 2 (Fig. 1, method 2) DNA fragments sheared by sonication (average length, about 1.5 kb) were made blunt-ended by treatment with Bal 31 followed by T4 DNA polymerase and deoxynucleoside triphosphates. Ligation of adapters, amplification, and cloning were carried out as described previously (51), except that primers HUB and IP3 were used as viral end primers for the first and second amplifications, respectively. PCR products were cloned by using the pCR II TA cloning vector from Invitrogen (San Diego, Calif.). The products of PCRs contained two contaminants in addition to the desired integration junctions, one derived from a circular form of the viral DNA (2-LTR circle) and the second from the 3' internal part of the viral DNA (for a discussion, see reference 31). Colonies containing host-virus junctions were distinguished from colonies containing contaminating sequences by PCR. Bacterial colonies containing plasmids were resuspended in PCR buffer and amplified with Taq polymerase for 20 cycles of 1 min at 94°C, 30 s at 60°C, and 1 min at 72°C. The circle junctions were detected using primers det3a and sc8. The internal fragment was detected using primers sc10 and IP3. The inserts were sequenced by using primers TA6 and TA7, which are complementary to the vector (pCR II; Invitrogen). Sequences of integration junctions and controls were determined by the dideoxy sequencing method. Each sequence was determined at least twice. For each integration site clone, the sequence of 34 bases of viral DNA at the LTR tip was determined, in addition to the flanking host DNA. For most integration site clones (59 of 61), all of the cloned human DNA adjacent to the proviral DNA was sequenced. A control experiment was carried out to exclude a possible artifact. Since DNA samples were treated with DNA ligase, free HIV genomes might have become joined to host DNA fragments by DNA ligase instead of integration. This is unlikely in the case of library 1, however, since the blunt-ended or 3' cleaved forms of the HIV cDNA would not be expected to become ligated to the protruding 5' ends generated by cleavage with HindIII. However, to document this expectation, a control experiment was performed in which purified unintegrated HIV cDNA was incubated in the presence of DNA ligase with HindIII-cleaved sequences and possible ligation was assayed by PCR across the ligation junction (one primer complementary to the HIV DNA and the other complementary to the HindIII-cleaved test DNA). No ligation was detected (data not shown). In the case of library 2, hypothetical ligation of unintegrated HIV cDNA should have yielded predominantly the vectorette linker joined directly to HIV cDNA, since DNA ends from the linkers were present in vast excess over ends from viral or human DNA. However, no such forms were detected (data not shown). Internal evidence also argues against this class of artifacts. For example, the 5-bp consensus host sequence flanking integration sites identified here closely resembles that found in a previous study employing conventional cloning and sequencing (55), an observation that helps validate each study.DNA sequence analysis. Sequences were analyzed by comparison to the nonredundant human sequence (nr) database, the human cDNA (dbEST) database, and the MONTH (November 1997) database by using BLASTN with Search Launcher and Repeat Masker. Default parameters were used. For comparisons between integration sites and control libraries, only a subset of the available sequence was considered (see Table 2), with either an average length of 144 bp or a length of exactly 50 bp (see Table 3). A total of 8,809 bp of human DNA flanking 61 integration sites was sequenced and analyzed for the integration site libraries (see Tables 2 and 3). The lengths of flanking human DNA sequences analyzed ranged from 37 to 430 bp. For the control human DNA fragments, a total of 14,989 bp in a total of 104 DNA clones were sequenced. Lengths of sequences analyzed ranged from 51 to 264 bp. Links to integration site and control sequences can be found at http://www.salk.edu/faculty/bushman.html.
Similarities to repeated sequences were ranked in accordance with the Smith-Waterman parameter (SW) generated by Repeat Masker (see A. F. A. Smit and P. Green, RepeatMasker at http://ftp.genome.washington.edu/RM/RepeatMasker.html) or by the probability of matching by chance generated by BLASTN (1) (P value) (see http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-blast?Jform=0). Minimum similarities for each sequence class considered to be significant matches are as follows: cDNA, P = 4.6 × 10
6; LINE 1, SW = 217; Alu repeat,
SW = 195; alphoid repeat, SW = 218; other repeats, SW = 190. Most regions of sequence similarity extended over at least 50 bp,
although in the case of the lowest scoring cDNA, a 31-bp perfect match
was judged to be significant.
Integration in vitro. Preintegration complexes (PICs) were extracted from a 6-h coculture of SupT1 cells grown in RPMI 1640 medium containing 10% fetal calf serum and chronically infected MoltIIIB cells stimulated with phorbol 12-myristate 13-acetate as previously described by Farnet and Haseltine (19). In vitro integration was achieved by incubating 400 µl of PIC extract with 1.2 µg of DNA from uninfected SupT1 cells for 45 min. The integration product was recovered by incubating it with proteinase K in 0.5% sodium dodecyl sulfate followed by extraction with phenol-chloroform. The same procedure was followed for the inactive PICs after first incubating the concentrated PICs in 15 mM EDTA for 5 min prior to adding target DNA. Integration assays with recombinant HIV-1 integrase were carried out essentially as described previously (4, 10).
Region-specific analysis of integration acceptor sites.
Integration junctions were amplified essentially as described
previously (9, 30, 44). Cellular DNA templates were prepared from infected and uninfected samples as described above. Integration products were visualized by nested PCR. Products were first amplified with viral primer HUB and a repeat primer. Products were then reamplified with the viral primer IP3 which had been end labeled by
treatment with [
-32P]ATP and kinase and a nested
repeat primer. The primers for repeated sequences were designed by
aligning multiple repeat copies and identifying conserved regions.
Primers for amplifying repeated sequences were as follows (see Table 1
for sequences; in each case, the second primer is the nested second
primer). Alu1, SC24 and CH12 (27); LINE-1, CH5
and CH6 (64); alphoid repeat, SC21 and SC23 (61);
and THE 1, CH15 and CH16 (52). The amounts of integration
products generated in vivo and in vitro that were used as templates for
PCR were adjusted to provide equal numbers of proviruses in each case.
The first round of PCR was carried out for 30 cycles of 94°C for
30 s, 55°C for 30 s, and 72°C for 1 min. For the second
round of PCR, 2 µl from the initial PCR was added to a 25-µl
reaction mixture and the mixture was amplified for 20 cycles of 94°C
for 30 s, 60°C for 30 s, and 72°C for 30 s. TaqStart
antibody (Clontech, Palo Alto, Calif.) was used in both amplifications
(hot-start PCR) in accordance with the manufacturer's recommendations.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Construction of integration site libraries. DNA for library construction was obtained from a human T-cell line (SupT1) acutely infected with cell-free stocks of HIV-1. Cellular DNA was harvested 12 to 14 h after initiation of infection, allowing initial integration to be studied separately from selection during subsequent growth of cells.
Libraries were constructed by two different methods in an effort to control for possible biases introduced in the DNA cloning steps (Fig. 1). For library 1, genomic DNA from infected cells was digested with HindIII, which cleaved the population of proviruses near the viral DNA ends and at numerous positions in flanking host DNA. HindIII-cleaved DNA was then circularized by treatment with DNA ligase, and virus-host DNA junctions were amplified with divergent primers complementary to viral end sequences (inverse PCR) (31, 49). For library 2, DNA fragments were made blunt ended by treatment with Bal 31 nuclease and T4 DNA polymerase and ligated to short linkers. DNA fragments were amplified with primers complementary to the linker and the HIV cDNA end (vectorette PCR) (51). PCR fragments were then cloned and sequenced. Sixty-one integration sites were analyzed by this means.
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Analysis of integration site libraries. Analysis of the sequencing data presented several challenges. Our raw sequence data contained different numbers of base pairs determined for each DNA clone analyzed. To compare the integration site and control data sets in a meaningful fashion, it was necessary to compare matching numbers of base pairs in each DNA clone and then compare the frequencies of appearance of different types of sequences in each data set. The average length of host DNA flanking integration sites was 144 bp, so sequences in the control library, which were slightly longer, were each truncated to yield test sequences with an average length of 144 bp (further parameters describing the data sets are presented in Materials and Methods).
Some copies of the human repeated DNA sequences are quite divergent from the family consensus sequence, presenting a challenge for identification. Repeated sequences were identified here by a two-step process. The program Repeat Masker, which compares unknown sequences to a set of consensus sequences derived from human repeat sequences (52), was used first. In a second step, all sequences were compared to the nr, dbEST, and MONTH (November 1997) databases by using BLASTN with default settings. In some cases, highly repeated sequences missed by Repeat Masker were identified by BLASTN and further analysis allowed them to be grouped into known sequence classes. The minimum degrees of similarity scored as matches are given in Materials and Methods. Analysis of cDNA matches presented another challenge. New sequences are being added to the dbEST database at a high rate, and even during the course of this work many anonymous sequences were found in later searches to match new cDNAs. The data presented here represent the number of matches to cDNAs as of November 1997, but new additions to the database will likely increase the number of matches in the future. For cDNAs, there was a natural partitioning of sequences into plausible and unlikely matches, since integration into a transcribed region should yield a near-perfect match over a discrete region. Integration sites sequenced and the matches to known sequences are summarized in Table 2 and 3. Sequences were classified as transcription units, Alu elements, LINE elements, alphoid repeats, other repeats, or anonymous. Transcription units were identified in database searches either as cDNAs or as sequences within the transcribed regions of known genes. Alu elements and LINE elements are the familiar interspersed nuclear repeats characteristic of human DNA. Alphoid repeats comprise the alpha satellite DNA, tandem arrays of 171-bp repeats associated with centromeric heterochromatin (38, 61). The "other repeat" class included several types, namely, SINE elements apart from Alu elements, low-complexity repeats, and retrovirus-related sequences such as THE 1 elements (36) and MLT1 sequences (14, 52) (for a recent summary of nomenclature, see reference 52). Anonymous sequences were defined as sequences contained in none of the classes.
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A weak consensus sequence at integration sites. Figure 2 presents an analysis of the 5 bp of host DNA at the junction between virus and host sequences expected to be duplicated upon integration. A weak consensus sequence can be derived from this data [5' GT(A/T)AC 3']. Only one end was sequenced for each integrant, so the duplicated nature of this sequence is inferred. The consensus sequence is rotationally symmetric, as expected, since each end of the HIV cDNA is joined to the 5' end of each strand of this sequence (Fig. 2). A closely related sequence was derived from a previous study of HIV integration sites by Stevens and Griffith [5' GTA(A/T)(T/C) 3'] (55). In this study, DNA from HIV-infected cells was cloned in lambda vectors, followed by isolation of provirus-containing clones by hybridization and sequencing of 29 proviral integration sites. The observation that our methods and that of Stevens and Griffith yielded similar integration site consensus sequences strongly validates each study.
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Region-specific assays of integration target sites. Several features of the sequencing data complicated interpretation. (i) The number of matching sequences detected was determined in part by the choice of parameters in the similarity search. (ii) In some clones the integration junctions were within the identified cDNA or repeated sequence, while in others the junctions were near but not within the identified sequence. In Tables 2 and 3, these were considered together. (iii) Although this study of HIV-1 integration site sequences is the largest yet reported, the differences between integration sites and controls were generally not clearly significant, as evaluated by the chi-square or Fisher's exact test. No finding was clearly significant in the analysis of both the 144-bp flanking sequences and the 50-bp sequence data. For these reasons, it was important to test some of the hypotheses generated by the sequence analysis by an independent method.
To this end, integration near repeated sequences was studied by using an assay based on PCR amplification of host-virus DNA junctions. In each reaction, one primer was complementary to an HIV-1 LTR end and the second primer was complementary to a repeated sequence (alphoid, Alu, LINE-1, or THE 1 repeats) (Fig. 3) (30, 44, 62). The first PCR amplification was followed by a second PCR with nested primers. The LTR primer in the second amplification was labeled at the 5' end with 32P. Amplification products were separated on DNA sequencing-type gels and analyzed by autoradiography. An integration event in or near the repeated sequence studied gave rise to a labeled band by amplification. Amplification of many such integration events gave rise to a ladder of labeled bands on the final autoradiogram.
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Primary DNA sequences favored for integration. Alignment of human DNA sequences at integration junctions yielded a consensus sequence (Fig. 2 and 4). A related sequence has been reported by Stevens and Griffith (55). To determine whether this sequence was favored for integration as naked DNA, several model sequences were synthesized and tested using integration in vitro. Target 1 contained the favored motif embedded in an arbitrary DNA sequence (Fig. 4A, target 1). Target 2 is identical to target 1 except for changes at the two most conserved positions (Fig. 4A, nucleotide positions 1 and 5) from the most favored nucleotide to the least favored. Target 3, like target 1, contained the favored target sequence but embedded in different arbitrary flanking DNA.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have used two methods to characterize chromosomal sites used by HIV-1 for integration in human SupT1 cells. We have sequenced a collection of integration sites and a collection of control sites and also analyzed integration near various repetitive sequences by using a PCR-based assay. DNA to be analyzed was prepared only 12 h after initiation of infection in an effort to obtain a population of sites unbiased by subsequent outgrowth of infected cells. In addition, the importance of a conserved host sequence at integration sites was tested by using integration in vitro. These studies clarify several factors influencing the selection of chromosomal sites for integration.
Comparison with integration site selection by yeast retrotransposons. Previous studies of Ty retrotransposons in yeast reveal that retroelement integration can be highly site specific. The yeast Ty retrotransposons replicate by transcription, reverse transcription, and integration by using reverse transcriptase and integrase enzymes similar in function and sequence to their retroviral counterparts (2). Ty elements differ from retroviruses in that all steps in replication take place in a single cell. For this reason, Ty retrotransposons must be fastidious in their selection of integration sites, since integration into a required cellular gene would be lethal for the host and suicidal for the transposon.
Ty elements integrate selectively in benign locations in host DNA. Ty1 integrates in a window of several hundred base pairs upstream of host polymerase III (Pol III)-transcribed genes (26). Ty3 is the most selective, integrating at the start site of transcription of Pol III-transcribed genes (12, 29). Ty5 shows a different specificity, integrating in telomeres and in the silent mating cassette DNA (65, 66). The potential for extreme integration site bias revealed in the Ty studies formed part of the motive for carrying out a large-scale investigation of integration site selection by HIV-1. In humans, integration in Pol III transcription units or telomeric repeats should have been detectable but no strong bias in favor of such sequences was found here or in previous studies with HIV or other retroviruses (23, 45, 47, 55, 58, 62). Evidently, HIV and Ty elements differ in this respect.Favored integration near active genes? Our data neither strengthen nor exclude the model that integration is favored in open chromatin near active genes (23, 45, 47, 58). Identifiable transcription units were present more frequently in the integration site libraries than in the control libraries. However, the difference was not statistically significant for the 144-bp sequence comparison, although it was significant for the 50-bp sequence comparison (Table 3).
Conclusions concerning integration site location will need to be reevaluated as new information becomes available. It will be particularly interesting to compile and analyze all the known integration site sequences (references 55, 59, and 60 and present study) when the sequence of the human genome is completed and cDNAs and regulatory regions are mapped onto the genomic DNA.Lack of evidence for favored integration near Alu or LINE elements. The data did not indicate that integration was favored near LINE elements or Alu elements as previously proposed (54, 55). Both the sequencing study and the region-specific PCR study failed to show any clear biases. One previous proposal was not directly tested. Stevens and Griffith proposed that integration might be favored near Alu islands, chromosomal regions containing clustered Alu repeats (55). Because our sequencing study examined relatively short flanking sequences (average length, 144 bp), clustering of Alu repeats near integration sites could not be assessed.
An effect of primary sequence. The data presented here also reveal a modest favoring of integration at a particular host DNA sequence. Previous studies of integration site sequences have revealed weakly conserved motifs for several retroviruses, including HIV (21, 43, 55). Two mechanisms might account for the observed sequence bias: the integration machinery might interact favorably with a factor bound at the conserved site, or the PIC itself might interact favorably with the conserved sequence as naked DNA. We found that the conserved sequence was favored in vitro as naked DNA, supporting the idea that the conserved sequence is favored in vivo due to interaction with the PIC itself.
Disfavored integration at centromeric alphoid repeats. The most striking feature of our data is the absence of integration in vivo into centromeric alphoid repeats. Alphoid repeats were absent in integration site sequences but present in controls, and alphoid sequences were selectively disfavored in the repeat-specific PCR integration assay. Several lines of evidence indicate that centromeric heterochromatin is organized differently than euchromatin. (i) Heterochromatic centromeres are seen to be more compact than euchromatin in fixed chromosome spreads (6). (ii) Alphoid sequences are more resistant to digestion with DNase I in isolated nuclei than are most DNAs (38, 63). (iii) Alphoid repeats are associated with the centromere-specific proteins CENP-A, CENP-B, and CENP-C (38, 63). On the basis of the data reported here, we propose that HIV-1 cDNA integration is obstructed by packaging DNA in centromeric heterochromatin. These data provide an unexpected demonstration of the long-standing possibility that certain types of chromatin may obstruct cDNA integration.
The mechanism of the integration block is unclear. The wrapping of DNA in heterochromatin may itself provide a steric block to integration, a possibility supported by the observation of condensed structures at centromeres. Other models are also possible. Since gene activity is probably reduced in heterochromatin, HIV may have evolved to avoid integration in heterochromatin to optimize gene expression. Alternatively, centromeric DNA might be sequestered at a nuclear location inaccessible to incoming PICs.| |
ACKNOWLEDGMENTS |
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S.C. and C.H. contributed equally to this work.
We thank Gary Karpen, Leslie Orgel, and members of the Bushman laboratory for suggestions and comments on the manuscript, Arian Smit for advice on identifying repeated sequences, and Leslie Barden and Allison Bocksruker for artwork and help in preparing the manuscript.
This work was supported by grants AI 34786 and AI 37489. S.C. was supported in part by the Rau Foundation. F.B. is a Scholar of the Leukemia Society of America.
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FOOTNOTES |
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* Corresponding author. Mailing address: Infectious Disease Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 453-4100, ext. 1630. Fax: (619) 554-0341. E-mail: rick_bushman{at}qm.salk.edu.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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