HIV-1 Preintegration Complexes Preferentially Integrate into Longer Target DNA Molecules in Solution as Detected by a Sensitive, Polymerase Chain Reaction-based Integration Assay
2001; Elsevier BV; Volume: 276; Issue: 50 Linguagem: Inglês
10.1074/jbc.m108000200
ISSN1083-351X
AutoresAlexei Brooun, Douglas D. Richman, Richard S. Kornbluth,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoAfter entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using λ exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs. After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using λ exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs. preintegration complexe integrase-directed PIC inhibitor long terminal repeat nonintegrase-directed PIC inhibitor polymerase chain reaction human immunodeficiency virus base pair(s) The integration of HIV into the host cell genome requires the integrase enzyme (1Hansen M.S. Carteau S. Hoffmann C. Li L. Bushman F. Genet. Eng. (N. Y.). 1998; 20: 41-61Crossref PubMed Scopus (36) Google Scholar, 2Asante-Appiah E. Skalka A.M. Adv. Virus Res. 1999; 52: 351-369Crossref PubMed Google Scholar) and mutations which destroy integrase activity block viral replication (3Wiskerchen M. Muesing M.A. J. Virol. 1995; 69: 376-386Crossref PubMed Google Scholar). These observations established integration as an important target for the development of new antiretroviral drugs (4Moore J.P. Stevenson M. Nat. Rev. Mol. Cell. Biol. 2000; 1: 40-49Crossref PubMed Scopus (172) Google Scholar). Recently, Hazuda et al. (5Hazuda 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: 646-650Crossref PubMed Scopus (1077) Google Scholar) found a new class of integrase inhibitors by screening a library of 250,000 compounds with a strand-transfer assay. Several di-keto compounds were found which inhibited HIV infection in CD4+ T cells in vitro, igniting the hope that integration inhibitors could become a new treatment modality (5Hazuda 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: 646-650Crossref PubMed Scopus (1077) Google Scholar). In the strand-transfer assay used by Hazuda et al. (5Hazuda 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: 646-650Crossref PubMed Scopus (1077) Google Scholar), an artificial, preassembled complex was formed between recombinant integrase protein and oligonucleotides designed to model the ends of the viral cDNA. Then, candidate inhibitors were added, followed by the target DNA (6Hazuda D.J. Hastings J.C. Wolfe A.L. Emini E.A. Nucleic Acids Res. 1994; 22: 1121-1122Crossref PubMed Scopus (121) Google Scholar). Although more reliable than the original strand transfer assay (7Craigie R. Mizuuchi K. Bushman F.D. Engelman A. Nucleic Acids Res. 1991; 19: 2729-2734Crossref PubMed Scopus (103) Google Scholar), this assay still scores as positive occasional compounds which fail to inhibit integration in vitro by authentic preintegration complexes (PICs)1 isolated from the cytoplasm of infected cells (5Hazuda 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: 646-650Crossref PubMed Scopus (1077) Google Scholar, 8Farnet C.M. Wang B. Lipford J.R. Bushman F.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9742-9747Crossref PubMed Scopus (124) Google Scholar) or virus-mediated integration in cultured cells (5Hazuda 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: 646-650Crossref PubMed Scopus (1077) Google Scholar). This suggests important roles for the other components present in PICs, which include viral proteins (reverse transcriptase, MA, NC, and Vpr) (9Bukrinsky M.I. Sharova N. McDonald T.L. Pushkarskaya T. Tarpley W.G. Stevenson M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6125-6129Crossref PubMed Scopus (387) Google Scholar, 10Heinzinger N.K. Bukinsky M.I. Haggerty S.A. Ragland A.M. Kewalramani V. Lee M.A. Gendelman H.E. Ratner L. Stevenson M. Emerman M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7311-7315Crossref PubMed Scopus (753) Google Scholar) together with cellular factors that are essential for the efficient integration of retroviral cDNA (11Lee M.S. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9823-9827Crossref PubMed Scopus (124) Google Scholar, 12Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Crossref PubMed Google Scholar, 13Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 14Kalpana G.V. Marmon S. Wang W. Crabtree G.R. Goff S.P. Science. 1994; 266: 2002-2006Crossref PubMed Scopus (458) Google Scholar, 15Chen H. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15270-15274Crossref PubMed Scopus (166) Google Scholar). These host cell factors may provide additional drug targets or otherwise influence the ability of a putative inhibitor to successfully inhibit integration. However, relatively little is known about PIC-mediated integration as an enzymatic process, and it has been difficult to construct a high-throughput assay for integration by PICs. Several groups have studied the influence of the target DNA upon PIC integration. PICs prefer to integrate into regions of distorted DNA (16Bor Y.C. Bushman F.D. Orgel L.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10334-10338Crossref PubMed Scopus (58) Google Scholar) such as nucleosomes (17Pryciak P.M. Varmus H.E. Cell. 1992; 69: 769-780Abstract Full Text PDF PubMed Scopus (232) Google Scholar, 18Pruss D. Bushman F.D. Wolffe A.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5913-5917Crossref PubMed Scopus (207) Google Scholar) and tend to avoid integration sites upstream of a pyrimidine nucleotide (19Bor Y.C. Miller M.D. Bushman F.D. Orgel L.E. Virology. 1996; 222: 283-288Crossref PubMed Scopus (38) Google Scholar). Not addressed in these studies, however, is the issue of target DNA size, which has inherent effects on substrate mobility. The present report describes an entirely fluid phase assay in which integration junctions are directly detected using PCR amplification. The only molecules that score as positive in this assay result from an actual joining of viral cDNA and target DNA. A uniquely designed DNA substrate is used to maintain amplicon size within the optimum limits for real-time fluorescence-monitored PCR detection. By comparing a single target DNA sequence of 105 bp with longer concatemers of the same sequence, the preference of PICs for longer target DNA was clearly demonstrated. A model is presented which suggests that long DNA is preferred because its decreased rate of diffusion allows more time for the rate-limiting “target commitment” stage of PIC integration. Incorporating this information into the assay yielded a sensitive, robust, and adaptable platform for the discovery of drugs that inhibit integration by PICs. This assay was used to detect host cell factors that reconstituted the integration competence of salt-stripped PICs. Plasmid DNA containing head-to-tail concatemers encoding for drag-line silk protein was kindly provided by Dr. David Kaplan, Biotechnology Center, Tufts University (20Prince J.T. McGrath K.P. DiGirolamo C.M. Kaplan D.L. Biochemistry. 1995; 34: 10879-10885Crossref PubMed Scopus (241) Google Scholar, 21Winkler S. Wilson D. Kaplan D.L. Biochemistry. 2000; 39: 12739-12746Crossref PubMed Scopus (132) Google Scholar). Plasmid DNA was isolated from transformed Escherichia coliDH5a and purified using a Maxi Prep Kit (Qiagen Inc., Valencia, CA). The insert in the plasmid contained monomeric units of 105 bp repeated 32 times. Insert DNA was prepared by overnight digestion withBamHI (which creates the protruding 5′ ends favored by λ exonuclease, described below), purified by electrophoresis in agarose, and isolated using a QIAquick Gel Extraction Kit (Qiagen). To prepare single monomeric units of the 105-bp sequence, insert DNA was digested with StyI (which cleaves between each unit) and similarly gel purified. The real-time PCR (TaqMan) detection of integration junctions used the following oligonucleotides: forward primer LTRTaq5, 5′-GTGTGTGCCCGTCTGTTGTG-3′; reverse primer SILKREV1a, 5′-CAGCACCGCCCATTGC-3′; and probe LTRTaqP, 5′-FAM-CTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAG-TAMRA-3′ (purchased from TriLink Biotechnologies, Inc., San Diego, CA). In some experiments, integration reactions were also amplified by hemi-nested PCR using the following additional oligonucleotides: HIVout, 5′-CAATAAAGCTTGCCTTGAGTGC-3′; and HIVin2, 5′-AGTAGTGTGTGCCCGTCTGTTGTG-3′. PICs were prepared from the detergent lysates of cells acutely infected by HIV-1LAI (LAV-1 strain) (22Farnet C.M. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4164-4168Crossref PubMed Scopus (202) Google Scholar, 23Ellison V. Abrams H. Roe T. Lifson J. Brown P. J. Virol. 1990; 64: 2711-2715Crossref PubMed Google Scholar). Briefly, CEM cells were infected by HIV-1LAI under BSL-3 conditions and cultured for 7–10 days until cytopathic changes (ballooning and apoptosis) were evident in the majority of cells by phase microscopic examination (24Terai C. Kornbluth R.S. Pauza C.D. Richman D.D. Carson D.A. J. Clin. Invest. 1991; 87: 1710-1715Crossref PubMed Scopus (382) Google Scholar). Then, the cells were counted and co-cultured with a 10-fold excess of SupT1 cells (American Type Culture Collection, Manassas, VA) in RPMI 1640 with 10% fetal bovine serum at 5% CO2 in a flask placed on end so that the infected CEM cells and the uninfected SupT1 cells came into contact. After 5–7 h, when the cultures consisted of about 80% syncytia just large enough to be visible to the unaided eye, the cells were pelleted by centrifugation at 160 × gfor 5 min. The cells were washed once by gently resuspending them in 10 mm Tris-HCl (pH 7.4), 150 mm KCl, 5 mm MgCl2, and 20 μg/ml aprotinin (Buffer A of Ellison et al. (23Ellison V. Abrams H. Roe T. Lifson J. Brown P. J. Virol. 1990; 64: 2711-2715Crossref PubMed Google Scholar)) and pelleting them again by centrifugation. Then, based on the original cell counts of the culture, the pellet was resuspended in Buffer A plus 0.025% digitonin (Calbiochem-Novabiochem Corp., San Diego, CA) at a concentration of 2 × 107 cells/ml for 10 min at room temperature. The nuclei in the cell lysate were then pelleted at 1,000 ×g at 4 °C for 3 min in an Avanti 30 centrifuge with a F2402H rotor (Beckman Coulter Inc., Fullerton, CA). The post-nuclear supernatant was moved to a new microtube and the remaining cellular debris was removed by centrifugation at 8,000 × g at 4 °C for 10 min. Forty percent sucrose was added to the supernatant to a concentration of 8% sucrose final, and the PIC-containing cytoplasmic extract was aliquotted, snap frozen in liquid nitrogen, and stored at −80 °C. PIC activity was stable at this temperature for at least six months. Typically, these extracts contained ∼0.8 mg/ml total protein measured using the BCA reagent (Pierce Chemical Corp., Rockford, IL), and 0.5–2.0 × 108/ml copies of HIV-1 cDNA as quantified by real-time fluorescence-monitored PCR using the LTR-specific primers and TaqMan probe described by Rossio et al. (25Rossio J.L. Esser M.T. Suryanarayana K. Schneider D.K. Bess Jr., J.W. Vasquez G.M. Wiltrout T.A. Chertova E. Grimes M.K. Sattentau Q. Arthur L.O. Henderson L.E. Lifson J.D. J. Virol. 1998; 72: 7992-8001Crossref PubMed Google Scholar). Integration reactions (15 μl total volume) were prepared on ice in 200-μl thin-walled PCR tubes (either in 8-tube strips or in 96-well plates) by adding 5 μl of a master mixture consisting of 1 μl of target DNA (3.3–100 ng/μl, with 11–33 ng/μl S32 concatemer being optimal for general use), 1.5 μl of 10 × integration buffer (200 mm HEPES, pH 7.4, 50 mm MgCl2, 10 mm dithiothreitol), and 2.5 μl of 30% PEG-8000 (Sigma). Then, 10 μl of PIC-containing cytoplasmic extract was added and the tube contents were mixed by pipetting up and down. The tubes were kept on ice and placed in a thermocycler (GeneAmp 9600, Applied Biosystems, Foster City, CA) and incubated at 4 °C for 10 min, 37 °C for 45 min, and then at 60 °C for 5 min. Unreacted target DNA was removed by adding 3–5 units of λ exonuclease in 15 μl of a 2 × concentration of its supplied buffer (New England Biolabs, Beverly, MA). The tubes (now containing 30 μl) were returned to the thermocycler and incubated at 37 °C for 45 min (which allows the λ exonuclease to degrade the target DNA) followed by 75 °C for 10 min (to heat-inactivate the λ exonuclease). The processing was completed by adding 20 μl of proteinase K (1 mg/ml in water) and incubating in the thermocycler at 60 °C for 30 min, followed by heat inactivation at 95 °C for 15 min. The processed integration reactions (now 50 μl in volume) could be stored at 4 °C for at least 1 week prior to TaqMan analysis. In some experiments, processed and unprocessed reactions were analyzed by polyacrylamide gel electrophoresis, staining with SYBR Gold (Molecular Probes, Eugene, OR), and visualization with UV light. In some experiments, the λ exonuclease and proteinase K-processed integration reaction mixtures were diluted 10-fold in water and then analyzed by real-time PCR. For integration reactions containing colored integration inhibitors, the inhibitor was removed by concentration/filtration of the processed integration reaction mixtures using Microcon PCR 96-well plates according to the manufacturer's protocol (Millipore, Bedford, MA). TaqMan reactions were set up in optical grade 96-well thermocycler plates by adding 20 μl of a master mixture containing 12.5 μl of TaqMan Universal PCR Master Mix 2X (Applied Biosystems), 2.5 μl each of 9 μm LTRTaq5 and SILKREV1a amplification primers (900 nm final concentration), and 2.5 μl of 2 μm LTRTaq5P probe (200 nm final concentration). Five μl of the processed integration reaction was added to each well, and the tube contents were mixed by pipetting up and down. Real-time fluorescence-monitored PCR reactions (TaqMan) were performed on an Applied Biosystems Model 7700 Sequence Detection System. The temperature profile for the reaction was: 50 °C for 2 min, 95 °C for 10 min, and then 95 °C for 15 s and 60 °C for 1 min for 45 cycles. Using the manufacturer's software, the cycle number at which fluorescence exceeded background (C t) was determined for each well. For each real-time PCR analysis, a standard curve was generated using dilutions in water of a cloned integration junction (below) calculated to provide 3, 10, 100, and 1000 integration junctions per well. The reactions were set up as 2- or 3-fold replicates and typically differed by less than 0.4 C t. To deduce the number of integration junctions in each sample, its C tvalue was compared with the cloned integration junction standard curve following linear regression analysis. Because each TaqMan reaction used only 5 μl of the 50 μl of processed integration reaction, the number of integration junctions was then multiplied by 10 to arrive at the number of integration events produced by the original 10 μl of PIC-containing cytoplasmic extract. If the processed reaction was diluted prior to TaqMan analysis, then an additional correction was made for the dilution factor. No correction was made, however, for the presumably equal number of integrations into the other, unmeasured strand of the target DNA. Statistical calculations were performed using the InStat software program (GraphPad Software Inc., San Diego, CA). Hemi-nested PCR was used to isolate PCR products in order to confirm that the method identifies integration junctions. For the first round of PCR, integration reactions were diluted 1:1,000 in water and 5 μl was amplified in a 25-μl reaction using HIVout and SILKREV1a (above) and HotStarTaq (Qiagen) according to the manufacturer's instructions. The reactions were heated to 95 °C for 15 min; then 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s for 25 cycles; and finally 72 °C for 10 min. The first round reaction products were then diluted 1:100 with water and re-amplified in an identical manner using HIVin2 and SILKREV1a. The final products were electrophoresed in agarose, isolated using a using a QIAquick Gel Extraction Kit (Qiagen), and cloned into the pCR4-TOPO vector using a TOPO TA Cloning Kit (Invitrogen Corp., Carlsbad, CA). Dye-terminator sequencing of eight clones was performed by the Molecular Biology Core of the UCSD Center for AIDS Research using the kit-supplied primers. One of the sequenced clones was used to create an integration junction standard curve in the TaqMan reaction above. Salt-stripped PICs were prepared by a modification of the methods of Lee and Craigie (26Lee M.S. Craigie R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1528-1533Crossref PubMed Scopus (217) Google Scholar) and Chen and Engelman (15Chen H. Engelman A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15270-15274Crossref PubMed Scopus (166) Google Scholar). 500 μl of PIC-containing cytoplasmic extract was diluted in an equal volume of Buffer A (above) containing 0.025% digitonin but formulated without KCl to reduce the salt concentration to 75 mm. The diluted PICs were incubated at 4 °C for 30 min and then pelleted at 8,000 × g for 20 min in a refrigerated microcentrifuge. The barely visible pellet was then resuspended in 170 μl of Buffer A with 1.2 m KCl and incubated for 30 min on ice. Then, the hypertonic PIC solution was loaded onto a Sepharose CL-4B column (2.2 ml bed volume) that had been pre-equilibrated in the same buffer (but not pretreated with bovine serum albumin). The column was placed in a tube and centrifuged at 800 × g for 3 min at 4 °C. About 1,500 μl was retrieved from each column and then concentrated to about 40 μl by centrifugation at 800 × g in a Microcon-100 ultrafiltration unit (Millipore), taking care not to allow the sample to concentrate to dryness. The amount of HIV LTR cDNA was then quantified by TaqMan (above) so that samples could be adjusted to equal numbers of PICs before placing them in the integration assay. Cytoplasmic extracts were prepared from uninfected SupT1 cells using exactly the same protocol used to prepare PIC-containing extracts. 5 μl of this extract was added to 2 μl of salt-stripped PICs and incubated on ice for 15 min. Then, 5 μl of Buffer A without salt was added to bring the KCl concentration down to about 262 mm, and the mixture was incubated on ice for another 15 min to allow the cytoplasmic proteins to assemble onto the salt-stripped PICs. Then, 10 μl of this mixture was analyzed in the integration assay above. To investigate the effect of target DNA length on the efficiency of integration, we chose a concatemeric DNA substrate containing 32 head-to-tail monomeric units encoding dragline silk, a natural protein composed of repeating units (27Winkler S. Szela S. Avtges P. Valluzzi R. Kirschner D.A. Kaplan D. Int. J. Biol. Macromol. 1999; 24: 265-270Crossref PubMed Scopus (98) Google Scholar). To amplify integration junctions, PCR primers were designed to hybridize to the 3′ LTR of HIV-1 (forward primer) and the silk target DNA (reverse primer). The concatemeric arrangement of the target DNA places a binding site for the reverse primer within a short distance (≤183 bp) from the LTR forward primer and effectively limits amplicon size to a length that is optimal for TaqMan detection. Although both the 5′ and 3′ LTRs integrate into the target DNA during concerted integration, only the integration of the 3′ LTR was analyzed in these studies. Also, although HIV can integrate into the target DNA in either orientation, the assay only detects integration junctions in one of the two target DNA strands (shown as the top strand in Fig. 1). As a first test of the PCR system depicted in Fig. 1, amplicons containing putative integration junctions were isolated by hemi-nested PCR and cloned into a plasmid. Sequencing analysis revealed that the integrations had occurred into various sites of the target DNA, with a possible “hot spot” immediately 5′ to the reverse primer (data not shown). In several cases, the cloned amplicons contained an additional monomeric unit of target DNA on their 3′ end resulting from the hybridization of the reverse primer to a binding site in the next unit adjacent to the integration site. To validate the real-time PCR detection step, control reactions were set up lacking either PICs or target DNA. As expected for a 45-cycle real-time PCR, these negative control integration reactions yielded C t values equal to 45, indicating that no integration junctions were detected. To quantify positive reactions, dilutions of a cloned integration junction were prepared to create the standard curves used to relate the C t values obtained from an unknown sample to the number of integration events that occurred. When reaction mixtures were diluted prior to detection, the final number of integration events reported was calculated by multiplying by the dilution factor. No correction was performed to account for the presumably equal number of integrations that occurred into the opposite, unmeasured strand of the target DNA. Fig. 2 presents data representative of five experiments. When PICs and target DNA were both present in an integration reaction and the λ exonuclease processing step was omitted, a signal corresponding to 500–1,000 integration events was detected. If the reaction mixtures were diluted prior to PCR, however, the number of integration events detected (corrected for the dilution factor) increased, indicating that the reaction mixtures contained an inhibitor for the PCR detection step. This disturbing nonlinearity likely explains why there has been no previous report of a quantitative PIC assay based upon the PCR quantification of integration junctions. After an exhaustive analysis, it was determined that the principal cause of this inhibition of detection is the carryover into the PCR detection step of unreacted target DNA, which contains a very large number of binding sites for the reverse primer. For example, the addition of target DNA, but not irrelevant plasmid DNA, to a cloned integration junction dramatically inhibited real-time PCR detection. As a first approach to solve this problem, the concentration of the reverse primer was increased 10-fold (to 9 μm final concentration) which completely reversed the inhibitory effects of target DNA on the detection of 10 and 100 copies of a cloned integration junction. However, for the integration junctions generated by PICs in the reaction mixtures, PCR amplification critically depends upon the reverse primer creating the complementary strand to which the forward primer binds (whereas this strand pre-exists when a cloned integration junction is used). In this case, a simple increase in the reverse primer concentration was not sufficient to eliminate the nonlinearity of the assay (data not shown). A close examination of the amplification plots revealed an additional problem caused by the carry-over of target DNA into the TaqMan reaction. In these cases, the slope of the amplification plot was markedly less than that of the cloned integration junction that was used as a standard. In effect, the target DNA prevented the amount of product from doubling with each cycle of PCR. The atypical shape of the amplification plots from the samples containing target DNA reduced the sensitivity of the TaqMan analysis by raising the apparentC t, and also precluded the possibility of converting the assay to an end point detection format. Fortunately, two strategies were found which succeeded in removing the nonlinearity of the assay by reducing or eliminating the carryover of target DNA into the PCR detection step. The first strategy is simply to dilute the integration reaction mixtures prior to PCR detection. However, the drawback of diluting the reaction mixtures is that this also dilutes the integration junctions that the real-time PCR aims to detect. For example, diluting the reaction mixtures beyond 100-fold prior to TaqMan detection generally resulted in C tvalues outside of the dynamic range of the assay. A second strategy is to selectively remove the target DNA in order to prevent it from inhibiting the PCR detection step. This approach was not associated with the loss of sensitivity that occurred when the dilution method is employed. λ Exonuclease, which degrades double-stranded DNA containing phosphorylated, protruding 5′ ends, was employed for target DNA removal prior to the PCR detection step. An examination of the λ exonuclease-treated reactions by polyacrylamide gel electrophoresis and SYBR Gold nucleic acid staining showed that all visible DNA in the reactions had been removed (data not shown). However, integration junctions evidently survive this λ exonuclease step because PIC proteins bound to the DNA block the 5′ → 3′ digestive action of this exonuclease as it approaches the integration site (12Miller M.D. Farnet C.M. Bushman F.D. J. Virol. 1997; 71: 5382-5390Crossref PubMed Google Scholar). These PIC proteins may be part of the complex (the “intasome”) that has been detected on the ends of integrated Moloney murine leukemia virus LTR DNA using a sensitive DNA footprinting method (28Wei S.Q. Mizuuchi K. Craigie R. EMBO J. 1997; 16: 7511-7520Crossref PubMed Scopus (87) Google Scholar). Supporting the concept that λ exonuclease effects the selective removal of target DNA, pretreatment of an integration reaction with λ exonuclease prior to detection consistently resulted in a 4–10-fold increase in detection sensitivity (Fig. 2). Also, the amplification plots of the λ exonuclease-treated samples showed a normal slope in parallel to the cloned integration junction standards, which could be important for converting the assay into a simplified end point detection format. To study the effects of target DNA length on the efficiency of PIC integration in vitro, target DNAs consisting of either 1 (S1) or 32 (S32) monomeric units of the same silk coding sequence were used in the integration assay system (Fig. 3). Two major conclusions were drawn from these experiments: 1) for a given number of monomeric units of target DNA present in the integration reaction, the concatemerization of those units into a longer molecule resulted in a 2- to >10-fold increase in the integration efficiency. 2) Overall, integration efficiency rises with an increase in the amount of target DNA molecules (in terms of monomeric units) (Fig. 3). This indicates that the amount of target DNA in the integration reaction must be kept high in order to maximize the number of integration events, even though the carryover of target DNA inhibits the subsequent real-time PCR detection step. As a counterbalancing factor, the linearity of the assay for PICs is best at ≤9.27 × 1010 monomers/15 μl reaction (see below and data not shown), a concentration where only the 32-mer can be used. To validate that the integration assay system using long target DNA concatemers can actually measure integration-competent PICs, PIC extracts were serially diluted and then assayed. Based on the data obtained in the experiments described above, the number of integration events was quantified in undiluted integration reactions using 32-mer concatemeric target DNA and λ exonuclease treatment in order to maximize detection efficiency. As shown (Fig. 4), the number of integration events decreased proportionally to PIC dilution. For the PIC extract tested, dilutions beyond 9-fold producedC t values that were outside of the linear range of the assay. In other experiments, the target DNA concentration was reduced resulting in less inhibition of the real-time PCR detection step. In these cases, integration events were detectable in PIC extracts diluted 27-fold, even though the absolute number of integration events at eac
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