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Structural Organization of Avian Retrovirus Integrase in Assembled Intasomes Mediating Full-site Integration

2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês

10.1074/jbc.m314270200

1083-351X

Ajaykumar C. Vora, Sibes Bera, Duane P. Grandgenett,

RNA Interference and Gene Delivery

Retrovirus preintegration complexes (PIC) purified from virus-infected cells are competent for efficient concerted integration of the linear viral DNA ends by integrase (IN) into target DNA (full-site integration). In this report, we have shown that the assembled complexes (intasomes) formed in vitro with linear 3.6-kbp DNA donors possessing 3′-OH-recessed attachment (att) site sequences and avian myeloblastosis virus IN (4 nm) were as competent for full-site integration as isolated retrovirus PIC. The att sites on DNA with 3′-OH-recessed ends were protected by IN in assembled intasomes from DNase I digestion up to ∼20 bp from the terminus. Several DNA donors containing either normal blunt-ended att sites or different end mutations did not allow assembly of complexes that exhibit the ∼20-bp DNase I footprint at 14 °C. At 50 and 100 mm NaCl, the ∼20-bp DNase I footprints were produced with wild type (wt) U3 and gain-of-function att site donors for full-site integration as previously observed at 320 mm NaCl. Although the wt U5 att site donors were fully competent for full-site integration at 37 °C, the ∼20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 °C. Under suboptimal assembly conditions for intasomes using U3 att DNA, DNase I probing demonstrated an enhanced cleavage site 9 bp from the end of U3 suggesting that a transient structural intasome intermediate was identified. Using a single nucleotide change at position 7 from the end and a series of small size deletions of wt U3 att site sequences, we determined that sequences upstream of the 11th nucleotide position were not required by IN to produce the ∼20-bp DNase I footprint and full-site integration. The results suggest the structural organization of IN at the att sites in reconstituted intasomes was similar to that observed in PIC. Retrovirus preintegration complexes (PIC) purified from virus-infected cells are competent for efficient concerted integration of the linear viral DNA ends by integrase (IN) into target DNA (full-site integration). In this report, we have shown that the assembled complexes (intasomes) formed in vitro with linear 3.6-kbp DNA donors possessing 3′-OH-recessed attachment (att) site sequences and avian myeloblastosis virus IN (4 nm) were as competent for full-site integration as isolated retrovirus PIC. The att sites on DNA with 3′-OH-recessed ends were protected by IN in assembled intasomes from DNase I digestion up to ∼20 bp from the terminus. Several DNA donors containing either normal blunt-ended att sites or different end mutations did not allow assembly of complexes that exhibit the ∼20-bp DNase I footprint at 14 °C. At 50 and 100 mm NaCl, the ∼20-bp DNase I footprints were produced with wild type (wt) U3 and gain-of-function att site donors for full-site integration as previously observed at 320 mm NaCl. Although the wt U5 att site donors were fully competent for full-site integration at 37 °C, the ∼20-bp DNase I footprint was not observed under a variety of assembly conditions including low NaCl concentrations at 14 °C. Under suboptimal assembly conditions for intasomes using U3 att DNA, DNase I probing demonstrated an enhanced cleavage site 9 bp from the end of U3 suggesting that a transient structural intasome intermediate was identified. Using a single nucleotide change at position 7 from the end and a series of small size deletions of wt U3 att site sequences, we determined that sequences upstream of the 11th nucleotide position were not required by IN to produce the ∼20-bp DNase I footprint and full-site integration. The results suggest the structural organization of IN at the att sites in reconstituted intasomes was similar to that observed in PIC. Integration of the retroviral DNA genome into host chromosomes is an essential step in the life cycle of retroviruses. Significant progress has been made in understanding the organization of cytoplasmic PIC 1The abbreviations used are: PIC, preintegration complexes; IN, integrase; att, attachment site; bp, base pairs; wt, wild type; AMV, avian myeloblastosis virus; MLV, murine leukemia virus; HIV-1, human immunodeficiency virus, type 1; LTR, long terminal repeats; MM, bacteriophage Mu-mediated; kbp, kilobase pair(s). containing the viral DNA genome produced by reverse transcription in retrovirus-infected cells. Within the PIC, the viral IN removes the two terminal nucleotides from the 3′-OH ends of the linear blunt-ended viral DNA (1Brown P.O. Coffin J. Hughes S. Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 161-203Google Scholar, 2Fujiwara T. Mizuuchi K. Cell. 1988; 54: 497-504Google Scholar, 3Brown P.O. Bowerman B. Varmus H.E. Bishop J.M. Cell. 1987; 49: 347-356Google Scholar). The two viral DNA ends in the PIC are held together by a protein bridge (4Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Google Scholar). The PIC are capable of performing the concerted insertion of the viral DNA ends into host DNA in vivo and into exogenously supplied target DNA in vitro, here termed full-site integration (3Brown P.O. Bowerman B. Varmus H.E. Bishop J.M. Cell. 1987; 49: 347-356Google Scholar, 5Lee Y.M. Coffin J.M. Mol. Cell. Biol. 1991; 11: 1419-1430Google Scholar, 6Wei S.Q. Mizuuchi K. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10535-10540Google Scholar, 7Brown H.E. Chen H. Engelman A. J. Virol. 1999; 73: 9011-9020Google Scholar, 8Chen H. Wei S.Q. Engelman A. J. Biol. Chem. 1999; 274: 17358-17364Google Scholar). The PIC containing the recessed termini are called intasomes (9Chen H. Engelman A. Mol. Cell. Biol. 2001; 21: 6758-6767Google Scholar), which appear analogous to type 1 transpososomes involved in Mu transposition (10Mizuuchi M. Baker T.A. Mizuuchi K. Cell. 1992; 70: 303-311Google Scholar, 11Williams T.L. Baker T.A. Science. 2000; 289: 73-74Google Scholar, 12Chaconas G. Lavoie B.D. Watson M.A. Curr. Biol. 1996; 6: 817-820Google Scholar). The retrovirus IN specifically interacts with the viral att sites (∼20 bp) located at the DNA termini (1Brown P.O. Coffin J. Hughes S. Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 161-203Google Scholar, 6Wei S.Q. Mizuuchi K. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10535-10540Google Scholar, 7Brown H.E. Chen H. Engelman A. J. Virol. 1999; 73: 9011-9020Google Scholar, 8Chen H. Wei S.Q. Engelman A. J. Biol. Chem. 1999; 274: 17358-17364Google Scholar). Characterization of IN interactions with the viral DNA in purified intasomes by MM-PCR footprinting revealed protection and enhancements near the termini (∼20 bp from the ends) with an extended region of protection mapping several hundred bp from the ends (6Wei S.Q. Mizuuchi K. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10535-10540Google Scholar, 8Chen H. Wei S.Q. Engelman A. J. Biol. Chem. 1999; 274: 17358-17364Google Scholar). The requirement for a large footprint in relationship to the requirement of having a 30 min) with the blunt-ended donor primarily resulted in half-site products with a minor population of full-site integration products (23Vora A.C. Grandgenett D.P. J. Virol. 1995; 69: 7483-7488Google Scholar). Third, no DNase I footprint protection by IN was observed on the blunt-ended DNA at 14 °C suggesting the lack of a stable association by IN under these assay conditions (Fig. 3C). Similarly, negative DNase I footprint protection by IN was obtained using a 3.6-kbp DNA containing either wt U3 or wt U5 blunt-ends that were extended by one nucleotide (Table I). In contrast, IN at the same concentrations and assay conditions using a 3′-OH-recessed donor was fully capable of significant full-site integration activity (Fig. 3B, lanes 1–5; lane 2 at 3 nm IN had 15% donor incorporated into full-site products at 37 °C in 5 min). The results suggest that IN does not form a stable complex with G U5 DNA with blunt-ends at 14 °C although these IN-DNA complexes mediate efficient 3′-OH processing at 37 °C. Taken together, the ∼20-bp DNase I footprints observed with 3′-OH-recessed viral DNA ends at 14 °C (13Vora A. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Google Scholar, 14Chiu R. Grandgenett D.P. J. Virol. 2003; 77: 6482-6492Google Scholar) and its correlation with full-site integration suggests that the stable strand transfer conformational structure of IN is different from what is associated with blunt-end viral DNA at 14 °C. DNase I Footprint Is Observed on G U3 att Sites but Not on Wild Type U5 at Low IN and NaCl Concentrations—We had previously shown that AMV IN was not able to produce a ∼20-bp DNase I footprint on wt U5 DNA possessing 3′-OH-recessed ends at 0.32 m NaCl in the assembly mixture at 14 °C even though the strand transfer activities appeared normal at 37 °C (13Vora A. Grandgenett D.P. J. Virol. 2001; 75: 3556-3567Google Scholar). The possibility existed that IN was not stably associated with U5 at 0.32 m NaCl but could be at lower NaCl concentrations. A variety of new conditions were tested to determine whether IN was able to produce the ∼20-bp footprint on U5 at 14 °C. The NaCl concentrations were varied from 50 to 320 mm using different concentrations of IN (5–42 nm). The ∼20-bp footprints were not observed at the termini (data not shown). We also tested whether the presence of target facilitated the formation of stable IN-DNA complexes at 14 °C (33Hazuda D.J. Felock P. Witmer M. Wolfe A. Stillmock K. Grobler J.A. Espeseth A. Gabryelski L. Schleif W. Blau C. Miller M.D. Science. 2000; 287: 646-650Google Scholar, 34Espeseth A.S. Felock P. Wolfe A. Witmer M. Grobler J. Anthony N. Egbertson M. Melamed J.Y. Young S. Hamill T. Cole J.L. Hazuda D.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11244-11249Google Scholar). We observed no DNase I footprints if, target DNA was added after 30 min of assembly time with U5 att DNA followed by 15 min of further incubation at 14 °C, prior to DNase I digestion. In contrast, we were able to demonstrate that IN at low concentrations produced the normally observed ∼20-bp DNase I footprint using G U3 att DNA containing 3′-OH-recessed ends at 50 and 100 mm NaCl (Fig. 4A). When IN was increased to 30 nm under these low salt conditions, the entire length of the DNA was essentially protected from DNase I digestion suggesting nonspecific multimerization of IN subunits on the DNA (data not shown). Aliquots of the same above samples (Fig. 4A) were analyzed for strand transfer activities at 37 °C for 5 min (Fig. 4B). Relatively higher quantities of all half-site products at 50 mm (Fig. 4B, lanes 2–4) or 100 mm NaCl (Fig. 4B, lanes 5–7) were observed than with IN at 0.32 m NaCl (Fig. 4B, lanes 8–10). The quantities of full-site products produced with 4 nm IN at 50, 100, and 320 mm NaCl were 22, 32, and 15% of the total donor incorporated (Fig. 4B, lanes 2, 5, and 8, respectively). The ratio of full-site to half-site integration products produced with the U3 att donor were effected by both IN and NaCl concentrations. In summary, the lack of the ∼20-bp footprint on the U5 att donor by IN was not apparently because of higher salt concentrations. DNase I Cleaves at Sensitive att Site Nine Nucleotides from the End Terminus in Suboptimal Assembled Intasomes—Physical interactions of IN with att site sequences suggest that the first ∼12 nucleotides from the end are functionally important for 3′-OH processing and strand transfer activities both in vivo and in vitro (31Craigie R. Craig N. Craigie R. Gellert M. Lambowitz A. Mobile DNA II. ASM Press, Bethesda, MD2002: 613-630Google Scholar, 35Esposito D. Craigie R. EMBO J. 1998; 17: 5832-5843Google Scholar). Detailed MM-PCR footprinting of HIV-1 intasomes revealed the major area of enhancements at nucleotides 9 and 10 from the end on U3 and, nucleotides 11 and 13 nucleotides from the end on U5 (7Brown H.E. Chen H. Engelman A. J. Virol. 1999; 73: 9011-9020Google Scholar). We investigated whether probing of suboptimal assembled avian intasomes by DNase I would provide molecular insights into the contacts between IN and the viral att site near the terminus. Under normal assembly conditions at 14 °C and 45 min, complete DNase I protection by IN mapping up to ∼20 bp of att terminal sequences is observed with wt U3, G U3, or G U5 donors containing 3′-OH-recessed ends (Fig. 4) (Table I). The same footprints were observed on the 32P 5′-labeled wt U3 att donor in the presence of 2-fo