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• W2026793457 abstract "Recombination is a major source of genetic heterogeneity in the human immunodeficiency virus type 1 (HIV-1) population. The main mechanism responsible for the generation of recombinant viruses is a process of copy choice between the two copies of genomic RNA during reverse transcription. We previously identified, after a single cycle of infection of cells in culture, a recombination hot spot within the gp120 gene, corresponding to the top portion of a RNA hairpin. Here, we determine that the hot region is circumscribed to 18 nucleotides located in the descending strand of the stem, following the sense of reverse transcription. Three factors appeared to be important, albeit at different extents, for the high rate of recombination observed in this region. The position of the hot sequence in the context of the RNA structure appears crucial, because changing its location within this structure triggered differences in recombination up to 20-fold. Another pivotal factor is the presence of a perfectly identical sequence between donor and acceptor RNA in the region of transfer, because single or double nucleotide differences in the hot spot were sufficient to almost completely abolish recombination in the region. Last, the primary structure of the hot region also influenced recombination, although with effects only in the 2-3-fold range. Altogether, these results provide the first molecular dissection of a hot spot in infected cells and indicate that several factors contribute to the generation of a site of preferential copy choice. Recombination is a major source of genetic heterogeneity in the human immunodeficiency virus type 1 (HIV-1) population. The main mechanism responsible for the generation of recombinant viruses is a process of copy choice between the two copies of genomic RNA during reverse transcription. We previously identified, after a single cycle of infection of cells in culture, a recombination hot spot within the gp120 gene, corresponding to the top portion of a RNA hairpin. Here, we determine that the hot region is circumscribed to 18 nucleotides located in the descending strand of the stem, following the sense of reverse transcription. Three factors appeared to be important, albeit at different extents, for the high rate of recombination observed in this region. The position of the hot sequence in the context of the RNA structure appears crucial, because changing its location within this structure triggered differences in recombination up to 20-fold. Another pivotal factor is the presence of a perfectly identical sequence between donor and acceptor RNA in the region of transfer, because single or double nucleotide differences in the hot spot were sufficient to almost completely abolish recombination in the region. Last, the primary structure of the hot region also influenced recombination, although with effects only in the 2-3-fold range. Altogether, these results provide the first molecular dissection of a hot spot in infected cells and indicate that several factors contribute to the generation of a site of preferential copy choice. The genome of retroviruses consists of a single-stranded RNA molecule of positive polarity, present in two copies within retroviral particles in the form of a dimer (1Vogt V.M. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 27-69Google Scholar). After infection of a target cell, reverse transcription will generate a double-stranded DNA molecule that will be integrated into the genome of the host. A process that has been shown to be extremely frequent in the human immunodeficiency virus type 1 (HIV-1) 3The abbreviations used are: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; RTP, reverse transcription products; nt, nucleotides; NC, nucleocapsid protein. is template switching between the two copies of genomic RNA during the reverse transcription step (2Galetto R. Negroni M. AIDS Rev. 2005; 7: 92-102PubMed Google Scholar). According to recent estimates, from 3 to 30 switching events can occur per genome for each infectious cycle, depending on the type of infected cell (3Jetzt A.E. Yu H. Klarmann G.J. Ron Y. Preston B.D. Dougherty J.P. J. Virol. 2000; 74: 1234-1240Crossref PubMed Scopus (297) Google Scholar, 4Levy D.N. Aldrovandi G.M. Kutsch O. Shaw G.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4204-4209Crossref PubMed Scopus (370) Google Scholar). When the two genomic RNAs are not identical (heterozygous viruses), this process can generate chimeric DNA molecules that will result in the production of recombinant viruses in the subsequent generation. In HIV-1, the high genetic heterogeneity and the frequent occurrence of coinfection of a cell by genetically divergent viruses (5Jost S. Bernard M.C. Kaiser L. Yerly S. Hirschel B. Samri A. Autran B. Goh L.E. Perrin L. N. Engl. J. Med. 2002; 347: 731-736Crossref PubMed Scopus (212) Google Scholar, 6Ramos A. Hu D.J. Nguyen L. Phan K.O. Vanichseni S. Promadej N. Choopanya K. Callahan M. Young N.L. McNicholl J. Mastro T.D. Folks T.M. Subbarao S. J. Virol. 2002; 76: 7444-7452Crossref PubMed Scopus (152) Google Scholar, 7Fang G. Weiser B. Kuiken C. Philpott S.M. Rowland-Jones S. Plummer F. Kimani J. Shi B. Kaul R. Bwayo J. Anzala O. Burger H. AIDS. 2004; 18: 153-159Crossref PubMed Scopus (108) Google Scholar, 8Chohan B. Lavreys L. Rainwater S.M. Overbaugh J. J. Virol. 2005; 79: 10701-10708Crossref PubMed Scopus (91) Google Scholar) favor the generation of viral particles carrying two non-identical copies of genomic RNA. As a consequence, recombination is extremely frequent in this virus and is considered nowadays as the main source of HIV-1 genetic variability worldwide (9Rambaut A. Posada D. Crandall K.A. Holmes E.C. Nat. Rev. Genet. 2004; 5: 52-61Crossref PubMed Scopus (399) Google Scholar). The mechanisms underlying recombination in HIV-1 have been intensively studied during the last decade. Most recombination events have been shown to occur by template switching during synthesis of the first DNA strand, when the reverse transcriptase (RT) uses the genomic RNA as a template (3Jetzt A.E. Yu H. Klarmann G.J. Ron Y. Preston B.D. Dougherty J.P. J. Virol. 2000; 74: 1234-1240Crossref PubMed Scopus (297) Google Scholar, 10Anderson J.A. Bowman E.H. Hu W.S. J. Virol. 1998; 72: 1195-1202Crossref PubMed Google Scholar, 11Yu H. Jetzt A.E. Ron Y. Preston B.D. Dougherty J.P. J. Biol. Chem. 1998; 273: 28384-28391Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 12Zhang J. Tang L.Y. Li T. Ma Y. Sapp C.M. J. Virol. 2000; 74: 2313-2322Crossref PubMed Scopus (62) Google Scholar). In retroviruses, reverse transcription is coupled to the degradation of the RNA template by the RT-encoded RNase H activity, a process that is mandatory for template switching (13DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 14Telesnitsky A. Goff S.P. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 121-160Google Scholar, 15Negroni M. Ricchetti M. Nouvel P. Buc H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6971-6975Crossref PubMed Scopus (37) Google Scholar). The degradation of the template RNA (donor RNA) is required to leave the trailing nascent DNA in a single-stranded form, available for annealing onto the other copy of genomic RNA present within the viral particle (acceptor RNA). This interaction would then drive the ultimate transfer of the growing 3′ end of the nascent DNA onto the second RNA moiety, leading to the generation of a chimeric DNA carrying genetic information from both copies of genomic RNA. Different causes have been proposed to be responsible for template switching (16Negroni M. Buc H. Annu. Rev. Genet. 2001; 35: 275-302Crossref PubMed Scopus (118) Google Scholar), such as breaks on the genomic RNA (17Coffin J.M. J. Gen. Virol. 1979; 42: 1-26Crossref PubMed Scopus (314) Google Scholar, 18DeStefano J.J. Raja A. Cristofaro J.V. Virology. 2000; 276: 7-15Crossref PubMed Scopus (13) Google Scholar) or strong pause sites during reverse transcription (13DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 19DeStefano J.J. Bambara R.A. Fay P.J. J. Biol. Chem. 1994; 269: 161-168Abstract Full Text PDF PubMed Google Scholar, 20Roda 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). The role of stalling of reverse transcription in response to these obstacles to DNA synthesis would be to allow a more extensive degradation of the RNA template by the RT-encoded RNase H activity (13DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 19DeStefano J.J. Bambara R.A. Fay P.J. J. Biol. Chem. 1994; 269: 161-168Abstract Full Text PDF PubMed Google Scholar, 20Roda 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, 21Peliska J.A. Benkovic S.J. Science. 1992; 258: 1112-1118Crossref PubMed Scopus (289) Google Scholar). The idea that template switching could occur preferentially at strong pause sites of reverse transcription has been supported by the correlation between a high rate of template switching, and the nearby presence of a strong pause site observed in cell-free reconstituted systems with several templates (13DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 19DeStefano J.J. Bambara R.A. Fay P.J. J. Biol. Chem. 1994; 269: 161-168Abstract Full Text PDF PubMed Google Scholar, 22Wu 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, 23Derebail S.S. DeStefano J.J. J. Biol. Chem. 2004; 279: 47446-47454Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Stalling of reverse transcription is indeed supposed to enhance the efficiency of template switching by allotting more time to the RNase H activity to degrade the donor RNA (24Wisniewski M. Balakrishnan M. Palaniappan C. Fay P.J. Bambara R.A. J. Biol. Chem. 2000; 275: 37664-37671Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 25Svarovskaia E.S. Delviks K.A. Hwang C.K. Pathak V.K. J. Virol. 2000; 74: 7171-7178Crossref PubMed Scopus (74) Google Scholar, 26Wisniewski M. Chen Y. Balakrishnan M. Palaniappan C. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 28400-28410Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and by increasing the time of residence of the reverse transcriptase in the given sequence interval (23Derebail S.S. DeStefano J.J. J. Biol. Chem. 2004; 279: 47446-47454Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 27Moumen A. Polomack L. Unge T. Veron M. Buc H. Negroni M. J. Biol. Chem. 2003; 278: 15973-15982Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, mounting evidence indicates that preferential sites for recombination do not necessarily correlate with stalling of reverse transcription. A particularly well documented case is the occurrence of preferential transfer in structured regions of the RNA template (20Roda 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, 23Derebail S.S. DeStefano J.J. J. Biol. Chem. 2004; 279: 47446-47454Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 27Moumen A. Polomack L. Unge T. Veron M. Buc H. Negroni M. J. Biol. Chem. 2003; 278: 15973-15982Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 28Kim 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, 29Negroni M. Buc H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6385-6390Crossref PubMed Scopus (75) Google Scholar, 30Balakrishnan M. Fay P.J. Bambara R.A. J. Biol. Chem. 2001; 276: 36482-36492Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 31Moumen A. Polomack L. Roques B. Buc H. Negroni M. Nucleic Acids Res. 2001; 29: 3814-3821Crossref PubMed Scopus (51) Google Scholar, 32Balakrishnan M. Roques B.P. Fay P.J. Bambara R.A. J. Virol. 2003; 77: 4710-4721Crossref PubMed Scopus (53) Google Scholar, 33Chen Y. Balakrishnan M. Roques B.P. Bambara R.A. J. Biol. Chem. 2005; 280: 14443-14452Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In some of these cases the hairpin structures were suggested to induce stalling of reverse transcription at their base favoring the annealing of the acceptor RNA on the nascent DNA (20Roda 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). Using an original system to study recombination after a single cycle of infection of cells in culture, we recently identified a hot spot within the coding portion of the gp120 gene from the LAI strain of HIV-1, where the recombination rate per nucleotide was up to 10 times higher than those observed in the surrounding regions (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In that work, which provided the first demonstration of the existence of a hot spot for recombination in infected cells, copy choice was studied on a 400-nt long region that was subdivided into five subregions named R1 to R5 following the sense of (-)-DNA synthesis. The hot region corresponded to region R2, which spanned the 58 nt that make the upper part of a hairpin (C2 hairpin), whose structure was deduced by enzymatic probing in vitro (27Moumen A. Polomack L. Unge T. Veron M. Buc H. Negroni M. J. Biol. Chem. 2003; 278: 15973-15982Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In addition, by introducing mutations in the lower part of the stem in such a way as to alter the stability of the hairpin, we were able to affect the efficiency of recombination in R2. In this study, we have addressed the question of which determinants, within R2 itself, are responsible for the high degree of transfer observed. DNA Constructs—The construction of the genomic plasmids and their description are fully detailed in Ref. 34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar. Mutagenesis of the hairpin structure was done using standard molecular biology and cloning techniques. All constructions were verified by sequencing. The transcomplementation plasmids used were pCMVΔR8.2 (35Naldini L. Blomer U. Gallay P. Ory D. Mulligan R. Gage F.H. Verma I.M. Trono D. Science. 1996; 272: 263-267Crossref PubMed Scopus (4016) Google Scholar), coding for HIV-1 gag, pol, and accessory proteins; and pHCMV-G (36Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Crossref PubMed Scopus (444) Google Scholar), which carries the gene for the G protein of the vesicular stomatitis virus envelope. For assays with vector particles lacking accessory proteins the pCMVΔR8.74 transcomplementation plasmid was used (37Zufferey R. Nagy D. Mandel R.J. Naldini L. Trono D. Nat. Biotechnol. 1997; 15: 871-875Crossref PubMed Scopus (1567) Google Scholar), together with the pHCMV-G plasmid. Cells—293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, and streptomycin (from Invitrogen), and maintained at 37 °C with 10% CO2. MT4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics at 37 °C with 5% CO2. Production of Vector Particles—HIV-1-based vectors were produced by transient transfection of 293T cells using the calcium phosphate method as described in Ref. 34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar. Briefly, cells were transfected with an HIV-1 encapsidation plasmid (pCMVΔR8.2) (35Naldini L. Blomer U. Gallay P. Ory D. Mulligan R. Gage F.H. Verma I.M. Trono D. Science. 1996; 272: 263-267Crossref PubMed Scopus (4016) Google Scholar) and a vesicular stomatitis virus envelope expression plasmid (pHCMV-G) (36Yee J.K. Miyanohara A. LaPorte P. Bouic K. Burns J.C. Friedmann T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9564-9568Crossref PubMed Scopus (444) Google Scholar), together with two plasmids that generate the defective genomic RNAs shown in Fig. 1A. For the generation of viral vectors devoid of accessory proteins, pCMVΔR8.2 was replaced by pCMVΔR8.74 (37Zufferey R. Nagy D. Mandel R.J. Naldini L. Trono D. Nat. Biotechnol. 1997; 15: 871-875Crossref PubMed Scopus (1567) Google Scholar). To eliminate non-internalized DNA from viral preparations, the supernatants from transfected cells were DNase I treated prior to concentration using Vivaspin Ultrafiltration Concentrators (molecular weight cut-off 50,000). The amount of p24 present in vector preparations was determined by using a HIV-1 p24 enzyme-linked immunosorbent assay kit (PerkinElmer Life Sciences). Single Cycle Infection Assays—MT4 cells were transduced with 200 ng of p24 antigen per 106 cells (corresponding to an approximate multiplicity of infection of 20) in a 500-μl volume in 35-mm dishes. Two-hours post-transduction the cells were diluted up to a 4-ml volume with supplemented RPMI medium and maintained at 37 °C in a 5% CO2 incubator for 40 h. The reverse transcription products (RTP) were purified from the cytoplasmic fraction of transduced cells using the method described by Hirt (38Hirt B. J. Mol. Biol. 1967; 26: 365-369Crossref PubMed Scopus (3351) Google Scholar), because most of the RTP remain in an unintegrated form and, additionally, the genomic vectors lack the FLAP sequence shown to enhance nuclear import of RTP (39Zennou V. Petit C. Guetard D. Nerhbass U. Montagnier L. Charneau P. Cell. 2000; 101: 173-185Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Briefly, cells were lysed, high molecular weight DNA was removed by precipitation, and the lysates were cleared by ultracentrifugation. After phenol chloroform extraction from the supernatant, low molecular weight DNA was ethanol-precipitated and purified using the NucleoSpin® Extract clean-up kit (Macherey-Nagel). The purified double-stranded DNA was digested with DpnI to eliminate possible contaminating DNA of bacterial origin prior to PCR amplification (20 cycles) with primers BH and SH, and cloning in Escherichia coli. Plating on isopropyl 1-thio-β-d-galactopyranoside/5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (IPTG/X-gal) containing dishes allowed blue/white screening of recombinant and parental colonies, respectively (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). To determine the regions where strand transfer occurred, 48 recombinant clones were analyzed for each triplicate assay. A full panel of controls run in parallel to ascertain that the recombinant molecules were generated during reverse transcription has been previously described (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Estimation of the Recombination Rates—The transfection of equal amounts of pLac+ and pLac- plasmids leads to the production of similar quantities of each of the genomic RNAs as previously reported (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), because the same promoter sequence is present in both genomic plasmids. Given that the RNAs also share the same sequences for dimerization and encapsidation, these processes are expected to yield 50% of heterozygous, and 50% of homozygous particles, with an equal amount of lac +/+ and lac-/- vectors, as usually assumed (40An W. Telesnitsky A. AIDS Rev. 2002; 4: 195-212PubMed Google Scholar). After transduction of MT4 cells, the RTP are amplified by PCR and cloned in E. coli after digestion with BamHI and SacII. This procedure will allow cloning BamHI+/lac- and BamHI+/lac+ RTP, which can be generated by reverse transcription in homozygous lac-/- and in heterozygous particles (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). As a result, assuming that only one molecule of double-stranded DNA is generated from each viral particle, one-third of the total amount of colonies will correspond to RTP issued from lac-/- vectors. The total number of colonies is therefore multiplied by 2/3 to consider only the RTP issued from heterozygous particles. The possibility of cloning products of cellular origin was also ruled out as described in Ref. 34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar. The number of white colonies (N) is corrected by a factor given by n/48, where n is the number of colonies that resulted from cloning of RTP after analysis of 48 white colonies. The global frequency of recombination (F) is given by: F = b/{2/3[N(n/48) + b]}, where b is the number of blue colonies. The recombination rate per nucleotide (f) within a given interval (i) is given by: f = F(xi/X)/zi, where F is as above, xi is the number of colonies analyzed where recombination was identified to have occurred within the interval considered, X is the total number of colonies on which mapping was performed, and zi is the size in nucleotides of the interval. Primer Extension Assays—Reverse transcription was primed using a 5′-terminal-labeled deoxyoligonucleotide and, for each RNA analyzed, 3 pmol were annealed with the primer at a molar ratio of primer to RNA of 10:1. Annealing was performed at 65 °C for 5 min in a buffer containing 50 mm Tris-HCl (pH 7.8) and 75 mm KCl, followed by slow cooling to 40 °C. After incubation on ice for 2 min, dithiothreitol was added at a final concentration of 1 mm, together with 100 units of RNasin (Promega). The nucleocapsid protein (NC) was then added at a ratio of 1 molecule of NC for 8 nt of total RNA and incubated at 37 °C for 5 min. HIV-1 RT was added at a final concentration of 400 nm and incubated for 5 min at 37 °C to allow the formation of the reverse transcription complex. The reaction was started by addition of the four dNTPs (1 mm each, final concentration) and MgCl2 (at a final concentration of 7 mm), and stopped at various time intervals by addition of EDTA and SDS at a final concentration of 25 mm and 0.4%, respectively. The samples were incubated for 1 h at 56°C and ethanol precipitated after phenol and chloroform extraction. The products were analyzed by autoradiography using a PhosphorImager (Amersham Biosciences) after electrophoresis on 8% (w/v) polyacrylamide gels containing 8 m urea, using a loading buffer with formamide at a final concentration of 22.5%. Outline of the System for the Study of Recombination—Recombination was studied after a single cycle of infection using the procedure previously described (34Galetto R. Moumen A. Giacomoni V. Veron M. Charneau P. Negroni M. J. Biol. Chem. 2004; 279: 36625-36632Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Briefly, the system is based in the production of vesicular stomatitis virus envelope-pseudotyped HIV-1 particles by transient transfection of 293T cells with two transcomplementation and two genomic plasmids. The first two plasmids will allow the expression of all HIV-1 proteins except those encoded by the env gene in one case, and of the G protein from the vesicular stomatitis virus envelope in the other. The two genomic plasmids will, instead, direct the synthesis of two defective genomic RNAs that will be encapsidated in the viral particles, and that share a region of homology on which recombination is studied. The viral particles are collected and used to transduce MT4 cells in culture. The rationale is to study copy choice occurring in the region of homology in heterozygous virions by following the generation of BamHI+/lac+ RTP, as indicated in Fig. 1B. This is achieved by screening for the presence of a functional lacZ gene in E. coli after cloning these RTP, as detailed in the legend of Fig. 1. This will provide a snapshot of the products generated after a single infectious cycle in MT4 cells because, as they carry non-functional LTR sequences (Fig. 1B), they will not support transcription of new genomic RNAs. The proportion of lac+ bacterial clones over the total amount of colonies leads to an estimate of the frequency of recombination as detailed under “Experimental Procedures.” The presence of specific mutations on the donor or on the acceptor RNA along the region of homology allows it to divide into subregions (Fig. 1C) and, after sequencing recombinant RTP, to assess a recombination rate per nucleotide for each subregion as detailed under “Experimental Procedures.” Mapping the Hot Region in the Top Portion of the C2 Hairpin—Understanding whether the whole upper half of the hairpin, or if only a portion of this region, is a preferential site for recombination constitutes a crucial issue for dissecting the mechanism of copy choice ongoing in this sequence. To this end, we have introduced, in a first instance, additional mutations in R2 that allow distinguishing between recombination occurring in the upper part of the mounting stem (R2a), in the apical loop (R2b), or in the upper part of the descending stem (R2c) (Fig. 2A). In addition, a mutation introduced in R1 allowed it to split into two regions, one including the lower part of the mounting portion of the stem and the 20 nt that precede it (R1b), and the other region that constitutes the very beginning of the region of homology (R1a, Fig. 1C). The reason for subdividing R1 in two parts was to explore the possibility that the presence of the hairpin enhanced template switching by inducing stalling of the reverse transcription at its base. If this were the case, R1b should display a high degree of transfer. As shown in Fig. 3A (left panel), the frequency of recombination was quite homogeneous, with an average rate of 2.1 × 10-4 per nt (S.D. 0.6 × 10-4), except for region R2c that stood out with a rate five times higher than the average value (10.5 ± 2.3 × 10-4 per nt). R2c is a 18-nt long segment that constitutes the first part of the descending stem, reverse transcribed after the apical loop (Figs. 1C and 2A). The R1b region did not display a rate higher than the average, indicating that potential stalling at the base of the hairpin during reverse transcription does not result in increased strand transfer. This analysis indicates that the hot region is circumscribed (spanning 18 nt out of 400) and maps in a double-stranded portion of the RNA constituted by the descending limb of the stem. Effect of HIV-1 Accessory Proteins on the Distribution of Strand Transfer Events—Various viral accessory proteins have been shown to be present in HIV-1 cores (41Welker R. Hohenberg H. Tessmer U. Huckhagel C. Krausslich H.G. J. Virol. 2000; 74: 1168-1177Crossref PubMed Scopus (166) Google Scholar), possibly participating in the formation of the reverse transcription complex and affecting the mutation rate during reverse transcription (42Mansky L.M. Trends Genet. 1997; 13: 134-136Abstract Full Text PDF PubMed Scopus (8) Google Scholar). To determine whether these viral proteins have any role in the generation of the identified recombination hot spot, we carried out a recombination assay on C2 RNA after generating vector particles deprived of vif, vpr, vpu, and nef proteins, as described under “Experimental Procedures.” Template switching occurred at recombination rates similar to those found in the presence of the accessory proteins, as previously observed (43Rhodes T.D. Nikolaitchik O. Chen J. Powell D. Hu W.S. J. Virol. 2005; 79: 1666-1677Crossref PubMed Scopus (80) Google Scholar), and with preference for the same genomic region (Fig. 3A, right panel). Altogether, these observations suggest that the major determinants for the generation of the hot spot in R2c are not related to the presence of these cofactors and might therefore be determined by other parameters, as could be the primary sequence of this region, or its position in the C2 hairpin. Position Effect on Recombination—The observation that only the descending part of the hairpin constitutes a hot sequence for recombination raises the question of which determinants are responsible for this behavior. We first checked whether the location of R2c in the descending portion was essential for the high efficiency of transfer observed. To this end, we constructed a mutant hairpin where the portions of R2a and R2c engaged in the formation of the upper part of the hairpin were exchanged, as shown in Fig. 2B (SW RNA). The exchange was made maintaining the 3′ to 5′ polarity of the swapped sequences as in the C2 RNA, in such a way that the sequences the RT will come across w" @default.
• W2026793457 created "2016-06-24" @default.
• W2026793457 creator A5006646521 @default.
• W2026793457 creator A5020371132 @default.
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• W2026793457 date "2006-02-01" @default.
• W2026793457 modified "2023-10-18" @default.
• W2026793457 title "Dissection of a Circumscribed Recombination Hot Spot in HIV-1 after a Single Infectious Cycle" @default.
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