Reconstitution of Concerted DNA Integration with Purified Components

ADVANCES IN VIRUS RESEARCH, VOL.52

RECONSTITUTION OF CONCERTED DNA INTEGRATION WITH PURIFIED COMPONENTS Patrick Hindmarsh and Jonathan Leis Department of Biochemistry Case Western Reserve University Cleveland, Ohio 441 06-4935

I. Characteristics of Retrovirus DNA Integration in Vzvo 11. Oligodeoxynucleotides as Integration Substrates 111. Early in Vztro Reconstitution Integration Systems Iv. Reconstitution of ASV Concerted DNA Integration V. Mutations in U5 or U3 LTRs That Influence Integration VI. Reconstitution of HIV-1 Concerted DNA Integration VII. HMG Proteins VIII. Role of HMG Proteins in Integration IX. Conclusion References

In order to understand more fully the mechanism of integration and the role of viral and cell proteins-in the process, it is important t o develop and characterize in uitro reconstituted model systems that mimic as closely as possible integration in uiuo. Such systems will also be invaluable as screening tools to search for new inhibitors directed IN) and at the human immunodeficiency virus type 1integrase (HlV-1 the development of unique multidrug strategies for controlling acquired immunodeficiency syndrome (AIDS). In this article, recent advances in the development of reconstituted concerted DNA integration systems are outlined. I. CHARACTERISTICS OF RETROVIRUS DNA INTEGRATION IN Vwo All retroviruses replicate their RNA genomes through a doublestranded DNA intermediate. As part of the replication mechanism, the viral DNA is integrated into the host chromosome through the action of the virus-encoded IN. Integration occurs randomly in the target DNA (26). During the process of integration, 2 bp are removed from the ends of the viral DNA, and there are small base pair duplications of host DNA at the site of insertion (8-10,19,22,29,36,37,41).The size of the duplication depends on the virus; as examples, HIV-1IN causes a 5-bp duplication and avian sarcoma virus (ASV) IN a 6-bp duplication 397

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of flanking host DNA sequences. The IN recognition sequences have been mapped genetically in viral DNA to the ends of the long terminal repeats (LTRs) (5,6,30,31,36).Moreover, Murphy and Goff (30) demonstrated that when deletions were placed in the U3 LTR, 5’ to the conserved CA dinucleotide, end processing in both U3 and U5 LTRs was adversely affected. This result implies that integration in v i m occurs by a concerted mechanism in which the two LTR ends of the viral DNA are inserted into a single target site such that a mutation in one LTR can influence the processing of the other (see Fig. 12.1 for a representation of an IN-DNA complex). 11. OLIGODEOXYNUCLEOTIDES AS INTEGRATION SUBSTRATES The use of blunt-ended oligodeoxynucleotide duplexes that correspond to the LTR termini constituted the first simplified IN-reconstituted system. With the use of 15-bp duplexes representing the ASV U3 or U5 termini, it was demonstrated that ASV IN catalyzed the specific cleavage of the 3’ end of the minus strands adjacent to the conserved CA dinucleotide, thereby producing 5’ overhangs (24) (Fig. 12.1).Subsequent to these studies, duplex oligodeoxynucleotides were shown to undergo joining reactions in which one duplex integrated into another (7,23). The overall efficiency of the reaction was low compared to that of integration catalyzed by preintegration complexes isolated from cells,

FIG12.1. Diagrammatic representation of a concerted integration complex with IN, donor DNA, an acceptor DNA, and HMG-I(Y ). An IN dimer is depicted by the large open circles, interacting with both the processed LTR ends of the donor and the acceptor DNA. HMG-I(Y) is shown interacting with the donor DNA.

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where as much as 80%of the donor substrate DNA underwent integration (2,14,29,32,36). Nevertheless, studies with oligodeoxynucleotidederived model systems have elucidated a number of important mechanistic properties of the integration reaction (26). Unfortunately, oligodeoxynucleotide substrates do not exhibit the concerted properties characteristic of in uivo integration. For example, when duplexes representing the U5 and U3 ends were mixed, the activity observed was the sum of the activity seen with the individual U5 and U3 duplexes by themselves (24). There is one exception. Kukolj and Skalka (25) designed short duplex substrates whose sequences matched those of U3 and U5 ends of ASV and HIV-1 DNA but were covalently synapsed across the termini by short single-stranded linkers (Fig. 12.2). These substrates were used more efficiently than oligodeoxynucleotide duplexes that were not linked. Moreover, substrates with a paired wild-type and mutated terminus were cleaved poorly at both ends, indicating that when termini were juxtaposed, the processing of both ends displayed concerted behavior. With the use of tethered donor molecules, the optimum spacing for the ASV system was shown to be two nucleotides. This placed the two-conserved CA dinucleotide processing sites six nucleotides apart, a separation equal to the staggered break introduced into the target DNA. The optimum separation of the HIV-1 conserved CA dinucleotides was five nucleotides, again matching the staggered break introduced into the target DNA in viuo. If both strands were tethered, the efficiency of the reaction was considerably decreased, due in part to a loss of torsion flexibility imparted by the gap in one strand. These studies provided the first insights into the relative organization of DNA ends during a concerted processing reaction. They also provided biochemical evidence that molecular communication must take place between IN monomers bound to both viral DNA ends. 111. EARLY IN VITRO RECONSTITUTION INTEGRATION SYSTEMS Fujiwara and Craigie (13) were the first to describe a cell-free integration system that used extracts of Moloney murine leukemia virus U5LTR 5' 3'-44

U3LTR

CTT CATT TA AAT ' 3 -A L T T C

5'

FIG12.2. Diagrammatic representation of a two-ended ASV substrate with an extra TA dinucleotide between the U5 and U3 LTR junctions and a 2-bp gap on the viral minus strand that is provided by annealing of the two separate oligodeoxynucleotides.

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(MoMLV), a large linear donor DNA containing modified LTR termini with 5' overhangs and lambda DNA as the target. The donor DNA was constructed by placing an NdeI cleavage site between tandemly linked U5 and U3 LTR termini inserted in a plasmid. After restriction endonuclease digestion, a linear donor was produced with LTR termini that differed from wild type by 1bp and contained 5' two-base AT overhangs rather than blunt ends (Fig. 12.3). Katz et al. (23) used purified ASV IN to reconstitute integration with a similar NdeI-constructed ASV donor DNA. Integrants in both systems were analyzed by packaging the lambda acceptor DNA, introducing the phage into cells, plaque purifjmg, and sequencing (Fig. 12.3). Integrants displayed properties

h

PhageDNA

FIG12.3. Lambda DNA-based integration assay. The donor DNA contains an NdeI restriction site. After restriction digestion, a linear DNA is produced with ends that differ from the wild-type termini by one deoxynucleotide. The donor DNA is then integrated into lambda DNA in the presence of IN, the lambda DNA is packaged into phage, and plaques are isolated which represent individual integration events.

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characteristic of i n vivo retroviral integration, including the loss of 2 bp from the ends of both LTR termini, short duplications of the lambda DNA at the site of integration, and random distribution of integration sites.

IV.RECONSTITUTION OF ASV CONCERTED DNA INTEGRATION The first system using purified IN was reported by Fitzgerald et al. (12). The system included purified avian myeloblastosis virus (AMV) IN, lambda as the linear acceptor DNA, and a donor DNA, 3.4 kb in length, containing 30 bp from the ends of the LTRs with preprocessed 5’AT overhangs, and an supF suppressor gene as a selectable marker. The linear donor was again constructed using the NdeI restriction enzyme site (12). A small percentage of the integrants isolated from the reaction exhibited concerted integration and displayed properties expected of ASV DNA integration in vivo. However, the remainder arose through nonconcerted integration events, which produced deletions in the acceptor DNA. Vora et al. (38) established a similar system using a 487-bp donor DNA also with precleaved NdeI ends. Here again, products arose from both concerted and nonconcerted reactions. Further analysis of the concerted integration products showed that they resulted from two one-ended integration events by different donor DNAs into the same acceptor rather than both ends being provided by the same donor. Before sequence analysis, the ends of the two donor molecules had to be ligated. As a consequence, U3-U5, U3-U3, or U5-U5 donor combinations were detected, complicating analysis of integration product (14,38). In subsequent reports, changing of the buffer conditions (38,39), improved the overall efficiency of integration but with varying percentages of concerted products detected. The ASV reconstituted system that appears to approximate concerted integration i n vivo most closely uses purified recombinant ASV IN, a 3.4-kb supercoiled or linear target DNA, and a blunt-ended linear minidonor DNA substrate only 294 bp in length (1,lB). This small size was chosen to maximize the probability that ends from the same donor would come in contact. Also, the donor contains only 15 bp from the ASV LTR termini sequences flanking a supF suppressor tRNA gene (Fig. 12.4A).More important, a host cell protein from the High Mobility Group (HMG) family was added to the reaction so that its ability to bend DNA would assist in juxtaposing the U3 and U5 LTR termini in an integration complex. This could favor concerted DNA integration (1)(Fig. 12.1).Although only a small percentage of the donor DNA was

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PATRICK HINDMARSH AND JONATHAN LEIS u5

u3

B

C RF 1 Acceptor DNA (3.4 Kbp )

ASV IN HMG I(Y)

(-j/&b Donor DNA (294 bp)

1 I

-

self integration

concerted integration

non-concerted integration

FIG12.4. Reconstitution of Integration in uitro. (A) A diagrammatic representation of the ASV minidonor DNA substrate. The 15 bp of the ASV U3 and U5 LTR termini sequences are shown. The conserved CA dinucleotide is underlined. (B)Diagrammatic representation of reconstituted integration with a minidonor DNA, purified IN, acceptor DNA, and HMG proteins. Possible product DNAs are shown resulting from either concerted or nonconcerted integration via one or two donor DNA molecule one-ended integration events. (C) Donor DNA self-integration products.

integrated into the acceptor in this system, more than 90% of the integrants detected utilized a concerted mechanism employing a single donor molecule (1).A representation of this product is shown in Fig. 12.4B. Removal of either the U3 or the U5 LTR sequences from the donor substrate resulted in a substantial reduction in total integration products detected. Depicted in Fig. 12.4B and Fig. 12.4C are one-ended donor insertion events, which could arise through a nonconcerted DNA integration mechanism, and self-integrant products of the donor DNA. The amount of self-integrant reaction products may be suppressed in favor of interintegration products with the addition of a cell protein referred t o as “barrier to autointegration factor (BAFY (28).

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The initial HMG protein family member added to the system was HMG-1. It stimulated the integration reaction about fourfold greater than the activity observed in the presence of IN alone. Subsequently, Farnet and Bushman (11) reported that an HMG protein family member, HMG-UY), was detected in HIV-1 preintegration complexes isolated from cells. Moreover, integration was dependent on the continued presence of HMG-UY). The addition of HMG-I(Y) t o the ASV minidonor DNA reconstituted system stimulated integration by more than 10-fold, with the substrate minidonor DNA being integrated via a concerted mechanism during the course of the reaction (18). The presence of the supF tRNA gene in the minidonor DNA (Fig. 12.4A) provides genetic selection for individual integrants in cells containing an expression vector with antibiotic resistance genes with amber mutations in the coding sequences. Individual integrants, isolated from reactions reconstituted in the presence of HMG-1 or HMG-I(Y1, showed end processing, concerted insertion with base pair duplication of acceptor DNA flanking the integrated donor DNA, and random integration of the donor into the target, all characteristic of in uiuo integration (1,181. V. MUTATIONS IN U5 OR U3 LTRs THAT INFLUENCE INTEGRATION The value of reconstituted systems is their ability to analyze rapidly mutations that influence integration. In the case of ASV IN, the percentage of nonconcerted integration events can be increased by introducing base changes into the LTR sequences (1,17,39), that presumably alter the binding affinity of IN for the LTR recognition sequences. If the reaction conditions are changed to favor IN-DNA contacts, the concerted nature of the integration can be rescued and individual integrants sequenced (17). When a 4-bp substitution was placed in the ASV U5 LTR, changing CTTCATT to GAAGATT, it resulted in a slight decrease in the efficiency of integration activity compared to a donor with a wild-type U5 LTR (1). However, one out of every seven integrants sequenced contained deletions in the LTRs. In one case, 10 bp were removed from the U5 LTR so that IN used the first internal CA dinucleotide for the nucleophilic attack. In a second integrant, IN left the mutation in the U5 LTR but deleted sequences in the wild-type U3 LTR utilizing the first internal GA dinucleotide to drive the integration reaction (1).This latter result reproduced genetic changes observed in uiuo (30)when a mutation placed at one LTR altered the processing of the other. The 15bases of the U5 and U3 LTR IN recognition sequences

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are related to one another in that they are nearly perfect inverted repeat sequences with 3-bp differences. Mutations placed in U3 and U5 have similar effects on integration in terms of specificity. However, mutations placed in U3 have a three times more deleterious effect on the efficiency of integration in uitro than the same mutation introduced into the U5 LTR (17,24,39). Thus, analysis of mutations in purified reconstituted systems have provided new insights into the mechanism of IN recognition of the terminal LTR sequences.

VI. RECONSTITUTION OF HIV-1 CONCERTED DNA INTEGRATION Goodarzi et al. (15)have reported a concerted, reconstituted, HIV-1 IN-dependent integration system that uses a 469-bp donor DNA constructed with the NdeI preprocessed ends. This system still exhibited insertion of two donor DNA molecules into a target, and only about half of the integrants resulted from a concerted DNA mechanism. An HIV-1 minidonor DNA integration system comparable to ASV described by Aiyar et al. (1)has also been developed with the use of recombinant HIV-1 IN (18).This system appears to approximate integration in uiuo more closely. The H N - 1 donor DNA contains only 20 bp of the HIV-1 LTR termini flanking thesupFsuppressor gene and uses the same target DNA and HMG protein family members as the ASV system. In contrast to the ASV reconstituted system, where 60%of the sequenced integrants had 6-bp acceptor DNA duplications characteristic of ASV integration in uiuo, the duplication of the acceptor DNA at the site of HIV-1 donor integration was almost exclusively 5 bp, characteristic of HIV-1 integration in uiuo (18). The differences in base pair duplications in ASV and HIV-1 reconstituted systems may reflect differences in the stability or conformational characteristics of protein-protein interactions among the respective ASV and HIV-1IN multimers, that form complexes with the ends of the donor and acceptor DNA (Fig. 12.1). Such differences could influence the spacing of staggered breaks introduced into the acceptor DNA, thereby altering the size of the duplications. The HIV-1 IN-dependent reactions also differ from ASV in that less than half or approximately 75% of the HMG-2 and HMG-I(Y) integrants examined, respectively, resulted from a concerted mechanism (18). The remainder resulted from multiple independent one-ended donor integration events that produced deletions in the target DNA (18). Although all of the HMG proteins tested stimulated integration in uitro, HMGI(Y) yielded the most concerted DNA integration products,

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consistent with the finding of this protein in HIV-1 preintegration complexes (11).

VII. HMG PROTEINS HMG proteins represent a large family of nonhistone DNA binding proteins that are localized primarily in the nucleus of eukaryotic cells and can be extracted in the presence of acid and salts. The proteins are classified into several families including HMGl/-2, HMG-14/-17, and HMG-I(Y). The HMG proteins are relatively small, ranging from approximately 11 kDa for HMG-I(Y) and HMG14/-17 to 25 kDa for HMG-1/-2, and are known to modulate chromatin structure and function (3).Because of their many functional similarities, two subgroups of HMG proteins, the HMG-11-2 family and the HMG-I(Y) family, have been included as founding members of a new category of nuclear proteins called “architectural transcription factors”(4,3,16).Their common functional features include (a) binding to the minor groove of doublestranded DNA, (b) recognizing DNA structure rather than sequence, (c) preferentially interacting with bent, supercoiled, or distorted DNA structures, (d) binding to non-B-form DNA structures such as four-way junctions and cisplatin adducts, (e) unwinding, bending, and supercoiling DNA substrates in the absence ofATP hydrolysis, and (f) selectively interacting with other sequence-specific transcription factors as part of gene transcription regulatory complexes. The DNA binding domains of the HMG-l/-2 and HMG-I(Y) proteins have markedly different three-dimensional structures and somewhat different substrate binding properties (3)(Fig. 12.5). For example, the HMG-1/-2 proteins interact in a sequence-independent manner with the minor groove of DNA. The interaction occurs through two DNA binding domains known as “HMG1 boxes” (271, which are a conserved set of amino acids folded into three helices forming an L-shaped structure (34,40).In contrast, the HMGI(Y) proteins preferentially bind to the minor groove of A-T-rich regions of B-form DNA through their DNA binding domains known as “A-T hooks” (35). When bound to DNA, these domains assume an extended planar crescent-shaped structure similar to that of the A-T minor groove binding drugs netropsin and distamycin (20).Nevertheless, both the structure and critical DNA-binding amino acids of the A-T hook or the HMG-1 box motifs are highly conserved (3). This suggests thab the DNA-binding domains of the HMGI(Y) and HMGl/-2 proteins

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A

HMG I (Y) A 50-90

C

HMG I (Y) ( I I , l l l ) + w ~ A T Hook

I

P57A

P61A

ATHook

P8JA

1

P87A

A T Hook

coo-

D HMG -11-2

%+

{

I

-

coo-

FIG12.5. HMG proteins and mutants. (A) HMG-I(Y). Basic DNA-binding domains referred to as “A-T hooks” are shown by the boxes. (B) HMG-I(Y)A50-90. A deletion mutant of HMG-I(Y) lacking the N-terminal DNA-binding domain. (C) HMG-I(Y) (11,111). HMG-I(Y) protein containing point amino acid substitutions in the central and C-terminal domains, as indicated. (D) HMG-1I-2. Basic DNA binding domains are shown by the boxes marked “HMG Box.” An acidic protein-protein interaction domain is represented by the open box.

are evolutionarily related and may account for many of their in uitro functional similarities.

VIII. ROLEOF HMG PROTEINS IN INTEGRATION HMG protein family members increase the efficiency of integration in uitro by acting on the donor DNA (l),but without forming stable complexes with IN or the LTR IN recognition sequences, as judged from gel shift and immunoprecipitation experiments (18).A truncated HMG-I(Y) protein (A50-90),which lacks one of the N-terminal DNAbinding domain motifs (A-T hook region) and its acidic C-terminal tail region (Fig. 12.5), is capable of stimulating integration as well as wildtype HMG-UY). This truncated protein preserves the region of HMGI(Y) which binds most tightly to substrate DNAs (24; C. S. Hill and

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R. Reeves, unpublished data, 1998). In contrast, another mutant of HMG-I(Y) (11,111)which has several point mutations in the A-T hook region preventing protein-DNA interactions while retaining proteinprotein interactions (Fig. 12.5) does not stimulate integration in vitro. These results imply that HMG-I(Y) needs to associate with the DNA t o stimulate integration. In addition, they indicate that only the last two A-T hook regions and the intervening peptide backbone of HMGI(Y) are necessary for the integration reaction. Among the common functional characteristics of HMG proteins is their ability to bend and unwind DNA in uitro. For example, DNA ligase-mediated ring closure (i.e., cyclization) assays have demonstrated that both HMG-1(34) and HMG-I(Y) (R. Reeves and G. Banks, unpublished data) are capable of bending short, rigid pieces of DNA (i.e., below the persistence length) into closed circles. This has led to the simple notion that both the HMG-1(1) and HMG-I(Y) (11,17) proteins promote concerted integration by bending the donor DNA to bring its LTR ends into close proximity (Fig. 12.1). However, as both HMG-1 and HMG-I(Y) proteins are capable of unwinding DNA substrates in uitro (21,33,40), it is also possible that these proteins function by modulating the helical twist at the ends of the donor DNA. Such modulation might enhance the affinity of IN for DNA ends, and this would improve the efficiency of its nucleophilic attack on the integration target DNA. IX. CONCLUSION In summary, totally reconstituted systems are now available that mimic concerted retroviral DNA integration in v i v a As a result, we should see significant advances in our knowledge of the mechanism of integration and the role that viral IN and cell proteins play in the reaction. Moreover, these systems provide the means to test the effectiveness of inhibitors directed at HIV-1 IN, which could add a new dimension to multidrug strategies now being used to treat AIDS.

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2. Brown, P. O., Bowerman, B., Varmus, H. E., and Bishop, J. M. (1987).Correct integration of retroviral DNA in vitro. Cell (Cambridge, Mass.) 49,347-356.

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40. Weir, H. M. Kraulis, P. J., Hill, C. S., Raine, A. R., Laue, E. D., and Thomas, J. 0. (1993). Structure of the HMG box motif in the P-domain of HMG1. EMEO J. 12, 1311-1319. 41. Yamamoto, T., Jay, G . , and Pastan, I. (1980). Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA. Proc. Natl. Acad. Sci. U.S.A. 77, 176-180.