Evidence for a Mechanism of Recombination during Reverse Transcription Dependent on the Structure of the Acceptor RNA

Abstract

Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination. Genetic recombination is a major force driving retroviral evolution. In retroviruses, recombination proceeds mostly through copy choice during reverse transcription. Using a reconstituted in vitro system, we have studied the mechanism of strand transfer on a major recombination hot spot we previously identified within the genome of HIV-1. We show that on this model sequence the frequency of copy choice is strongly influenced by the folding of the RNA template, namely by the presence of a stable hairpin. This structure must be specifically present on the acceptor template. We previously proposed that strand transfer follows a two-step process: docking of the nascent DNA onto the acceptor RNA and strand invasion. The frequency of recombination under copy choice conditions was not dependent on the concentration of the acceptor RNA, in contrast with strand transfer occurring at strong arrests of reverse transcription. During copy choice strand transfer, the docking step is not rate limiting. We propose that the hairpin present on the acceptor RNA could mediate strand transfer following a mechanism reminiscent of branch migration during DNA recombination. human immunodeficiency virus nucleotides reverse transcription nucleocapsid protein stem-loop By reshuffling large regions of genetically distinct genomes, recombination speeds up the rate of evolution (1Coffin J.M. Curr. Top. Microbiol. Immunol. 1992; 176: 143-164Crossref PubMed Scopus (180) Google Scholar, 2Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6900-6903Crossref PubMed Scopus (268) Google Scholar). Recombination constitutes the most frequent genomic aberration in retroviruses; its frequency of occurrence is equal to the cumulative frequency of all the other types of mutations (3Negroni M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google Scholar). The most intensively studied member of this group of viruses, the human immunodeficiency virus type 1 (HIV-1),1 illustrates the impact of recombination on the dynamics of retroviral evolution. In this case, at the very least 10% of the circulating strains have been generated by genetic recombination among different HIV-1 subtypes (4Sharp P.M. Bailes E. Robertson D.L. Gao F. Hahn B.H. Biol. Bull. 1999; 196: 338-342Crossref PubMed Scopus (71) Google Scholar). In retroviruses, recombination occurs mainly during reverse transcription (5Hu W.S. Temin H.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1556-1560Crossref PubMed Scopus (382) Google Scholar). Each viral particle contains two copies of single-stranded positive genomic RNA (6Vogt V.M. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). If two different variants of a virus infect the same cell, as recently documented for HIV-1 (7Jung A. Maier R. Vartanian J.P. Bocharov G. Jung V. Fischer U. Meese E. Wain-Hobson S. Meyerhans A. Nature. 2002; 418: 144Crossref PubMed Scopus (337) Google Scholar), the viral progeny will be constituted by homozygous and heterozygous virions. Recombination can then occur when a heterozygous virus infects a new cell. Indeed, during synthesis of the (−) DNA strand the reverse transcriptase (RT) can switch template and, guided by the local sequence homology, transfer DNA synthesis from one genomic RNA molecule (the donor) onto the other (acceptor RNA). In a heterozygous virion this process, known as copy choice, leads to genetic recombination (8Vogt P.K. Silvestri L. Possible Episomes in Eukaryotes. North Holland, 1973: 35-41Google Scholar). Despite the dramatic impact of recombination on the evolution of retroviruses, the underlying mechanisms are not yet understood. Based on the observation that the purification of viral RNA from retroviral particles led to the isolation of fragmented molecules, it was suggested that the genomic RNA is not intact within the viral particle. It was therefore proposed that the switch would be a consequence of a block of reverse transcription caused by a break on the RNA, a model called “forced copy choice” (9Coffin J.M. J. Gen. Virol. 1979; 42: 1-26Crossref PubMed Scopus (314) Google Scholar). In this case the stalling of the RT would constitute the crucial step of the process by allowing an extensive degradation of the RNA template by the RNase H activity carried by the RT itself, as demonstrated for (−)DNA strong stop strand transfer (10Telesnitsky A. Goff S.P. EMBO J. 1993; 12: 4433-4438Crossref PubMed Scopus (57) Google Scholar). The resulting single-stranded DNA would then be available for annealing onto the complementary sequence provided by the acceptor RNA. A similar situation can be encountered if stalling is generated by strong pause occurring during reverse transcription of an intact template. Indeed, a prominent pause site detected duringin vitro reverse transcription of a stretch of the HIV-1nef gene was shown to increase significantly the local frequency of strand transfer (11DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 12Wu W. Blumberg B.M. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 325-332Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In these instances the stalling of reverse transcription is regarded as the limiting step for strand transfer. This idea has, however, recently been challenged by increasing evidence demonstrating that pausing during reverse transcription and strand transfer are not necessarily coupled (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 14Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar, 15Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In parallel, studies carried on RNA hairpin regions have suggested that could template by (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, H. RNA. PubMed Scopus Google M. Fay P.J. Bambara R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In these it was proposed that the hairpin the of strand transfer by an and acceptor RNA that (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, H. RNA. PubMed Scopus Google M. Fay P.J. Bambara R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for the occurrence of recombination hot during in vitro reverse transcription by HIV-1 RT the the of these hot and the presence of hairpin regions in the RNA template M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar, A. L. Roques Buc H. M. PubMed Scopus Google Scholar). Based on this it was proposed that template proceeds through a two-step docking of the acceptor RNA onto the nascent DNA and of the RNA by the acceptor RNA M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). The step would be guided by the folding of the RNA. A on the site of the virus shown that the hairpin present in that a strong pause of reverse transcription that the of the docking step R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In in vitro studies on the on strand transfer of the nucleocapsid protein a major of the reverse transcription R. H. U. C. J. Virol. 2000; PubMed Scopus Google Scholar), have suggested a mechanism of recombination by the of the RNA by pausing of reverse transcription in M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google and M. Buc H. Biol. PubMed Scopus Google Scholar). Indeed, strand transfer in vitro in M. Buc H. Campbell A. Annual Review of Genetics. 35. Annual Reviews, Palo Alto, CA2001: 275-302Google it not to a increase of pausing during reverse as for a mechanism of template by pausing of DNA synthesis M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). the is a RNA E. R. EMBO J. 1999; PubMed Scopus (71) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), it was suggested that the of strand transfer in its presence was of its to the of the RNA M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). the HIV-1 strains to a case is constituted by A and generated by recombination on the for the of the D.L. P.M. Hahn B.H. Nature. 1995; PubMed Scopus Google Scholar). Recombination on this of genome reshuffling of the of for the regions and to regions through The of regions and with to the regions of the protein been shown to be for allowing the virus to by the of the J.P. J. J. Virol. 2000; PubMed Scopus Google Scholar). In a we RNA from the HIV-1 genome to the mechanism of template by HIV-1 reverse We that the genomic sequence for the the most hot spot we during that A. L. Roques Buc H. M. PubMed Scopus Google Scholar). a on recombination during of in with different HIV-1 subtypes shown the occurrence of frequent recombination in the same Gao A. J. Virol. 2002; PubMed Scopus Google Scholar). In the of that constituted the hot spot within the was called and was in a model template it was by the that it on the viral genome 1 It was that by the RNA the frequency of strand transfer on was by a to A. L. Roques Buc H. M. PubMed Scopus Google Scholar). We to this as In this we of the to the of the RNA structure in the transfer process and to the of the mechanism for copy choice by HIV-1 the RNA at as A with a sequence complementary to the of the RNA to was at a of and in a 1 by the for at by a to The at its a of nucleotides constituted by the sequence The RNA was then for at in a of in a of of and of on and by at in a and 1 The RNA was with and in the was under at of the structure of the RNA, of the RNA in the reverse transcription at for to and on The RNA was then with and and for at and single-stranded RNA with for for and for C. F. M. J.P. PubMed Scopus Google Scholar). The was by by The by on was The of identified by to a generated by extensive of the same RNA The identified as single-stranded in as in the structure by the M. PubMed Scopus Google Scholar). The for RNA synthesis generated following J. A Laboratory Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Scholar). Each was its in RNA RNA synthesis was as previously M. M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). transcription of the RNA was carried in the presence of the acceptor RNA a concentration of annealing an specifically onto the template was at a of to RNA of in at for by a to the and for on the with as in H. C. A. Roques Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar), the protein was at this step at a of 1 molecule of of RNA and for at transcription was by the of HIV-1 RT at a concentration of carried for The was by with The for 1 at with and The then to RNase of the reverse transcription and synthesis of the DNA strand as previously M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). and and carried as previously A. L. Roques Buc H. M. 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The reverse transcription are then as for the copy choice and in E. the and and the reverse transcription that have the of the acceptor RNA will be molecules, this specifically of the of strand transfer the The of generated is therefore to the of that strand transfer. to a frequency of occurrence of strand transfer under these conditions one to the of that would have been if strand transfer this and with the of these with under copy choice reverse transcription was in on the same RNA for the copy choice The resulting reverse transcription then as and in E. the of RNA in this was the same as the copy choice RNA in the strand transfer the of in the strand transfer to in the frequency of strand transfer under copy choice conditions This a with the for copy choice the same and molecules, are generated in the two The of the concentration of the acceptor RNA on the strand transfer was then studied in under the copy choice and the copy choice This was carried not on RNA on two other we previously studied for to strand transfer in the and a of the gene we called A. L. Roques Buc H. M. PubMed Scopus Google Scholar). two as of recombination hot spot (the sequence and of a strand transfer is a (the The recombination at a concentration of template the concentration of acceptor RNA from to 1 the copy choice for all the studied the frequency of strand transfer for This is in contrast with the one under copy choice conditions for the the frequency of transfer was dependent on the concentration of acceptor template in the of We have studied the mechanism of copy choice recombination during in vitro reverse transcription by HIV-1 In a we a for recombination hot among of the HIV-1 the presence of two strong hot one constituted by the hairpin the other by a for the of the we have mostly on the We have the folding of this hot spot in and shown the the frequency of recombination on this and the presence of a stable hairpin The of the same sequence to recombination at different on the structure provided the to for the to the by stem-loop on the and on the acceptor Indeed, the of the in the and that on this RNA sequence the presence of the stem-loop structure on the acceptor RNA to be the most for this to constitute a recombination hot transfer at and when the hairpin was present on the acceptor RNA of its presence on the template and It is that this observation for RNA conditions under we have the folding of the RNA in as as in the presence of the conditions to the situation in The that the folding of the acceptor RNA constitutes the crucial for strand transfer is in with on the of in the In it was shown that the the folding of the RNA E. R. EMBO J. 1999; PubMed Scopus (71) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), copy choice through its onto the acceptor RNA M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). We previously proposed that the transfer process follows two docking of the nascent DNA onto the acceptor template and by the acceptor RNA of the RNA from the of the nascent DNA M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar). The step is to on the of of the acceptor RNA and of the nascent is a of the concentration of these two in The in would an acceptor RNA onto the reverse transcription and would therefore not on the concentration of the acceptor RNA in these two we have the of recombination as a of the concentration of the acceptor RNA on different of acceptor to increase in the frequency of copy choice was The that recombination on the for of acceptor RNA, the for that the of the process on of the RNA on the concentration of the acceptor RNA. docking is to be to the concentration of RNA in we that the different of strand transfer are not of the of The in this that the frequency of recombination on the same sequence as a of its folding strongly that the structure of the RNA template is for the of copy The of hairpin in strand transfer been the of It was suggested that the by these regions could template by increasing the the and the acceptor through an occurring within the hairpin (13Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google H. RNA. PubMed Scopus Google Scholar, M. Fay P.J. Bambara R.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). not an the hairpin structure is not on the RNA and, by an with the acceptor RNA. the in A for copy choice that the of and acceptor is not rate limiting. A on strand transfer on a hairpin of the genomic RNA of the virus suggested an for hairpin in the two-step model R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In this new model and the docking of the nascent DNA on the acceptor RNA constitutes the limiting In this case stalling of reverse transcriptase at the of the hairpin on the RNA its degradation by the RNase H increasing the of the nascent DNA to the annealing for the docking In the present the hairpin in a similar if this the case, its presence would be on the RNA and not on the the of the pausing during reverse transcription of the hairpin the presence of a strong pause site in the of the This pause in a similar by the annealing of the nascent DNA onto the acceptor RNA. The of reverse transcription in the of strand transfer vitro and in has, in been previously J.J. Mallaber L.M. Fay P.J. Bambara R.A. 1992; PubMed Scopus Google Scholar, J. Virol. 2000; PubMed Scopus (75) Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the presence of this pause site most strand transfer, it is that it constitutes the crucial step for the In the observation that the of pausing on a RNA is not by the type of acceptor the frequency of recombination is different this if stalling of reverse transcription constituted the for the transfer during copy choice, the as a of the concentration of acceptor RNA would be to be similar to under copy choice a strong pause site is that this is not the case for of the We therefore that a pause site is not to recombination hot and that it is to recombination hot on the of the pausing during reverse transcription on the RNA. In of these a mechanism for template on hairpin regions of the template is proposed in strand this model is on the most conditions under constituted a hot the case the hairpin is present on the acceptor RNA it when and acceptor a hairpin the conditions of the and acceptor in the of the of the docking must necessarily occur within under these conditions the frequency of strand transfer within was as as when an and acceptor was present reverse transcription the hairpin frequency in the hairpin in and This observation that the is to this step could be by stalling of the at the pause site in the of the docking is the of the nascent DNA to be onto the acceptor RNA. the hairpin on the acceptor template is to the strand of the nascent DNA constitutes the in the mechanism of template in hairpin in the and model recently it is to the hairpin on the acceptor RNA could be and, be within hairpin regions on In this in model the of a structure to the that branch migration during DNA recombination a to this in that case Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), the of the of the hairpin on the acceptor would be by the of a when a hairpin is present on the acceptor RNA. In this evidence for the recombination hot and the structure of the viral genomic RNA. of the hairpin structure are crucial an the model proposed could for recombination occurring on most hairpin strand transfer within the hairpin. this not that template follows as for by the frequency of recombination within when are of the hairpin the observation that the recombination hot from to hairpin regions M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google A. L. Roques Buc H. M. PubMed Scopus Google that constitute for frequent template This a for the of the mechanism of template at the HIV-1 We and for during the of the model proposed in this We are to Roques for the of the with