Real-Time PCR Analysis of HIV-1 Replication Post-entry Events

Chapter5 Real-Time PCR Analysis of HIV-1ReplicationPost-entry EventsJean L.Mbisa∗,Krista A.Delviks-Frankenberry∗,James A.Thomas, Robert J.Gorelick,Vinay K.PathakAbstractThe reverse transcriptase enzyme plays an essential role in the HIV-1life cycle by converting a single-stranded viral RNA genome into a double-stranded viral DNA through a complex process known as reverse transcription.The resulting double-stranded DNA is integrated into the host chromosome to form a provirus.A small proportion of the viral DNAs form dead-end circular products,which never-theless can serve as useful surrogate markers for monitoring viral replication.Utilizing real-time PCR technology,it is possible to track and quantify different stages of the reverse transcription process,the proviruses,and the nonintegrated dead-end reverse transcription products.Key words:HIV-1,reverse transcription,real-time PCR,1-LTR circle,2-LTR circle,Alu-LTR real-time PCR.1.IntroductionReal-time PCR uses conventional PCR methods;however,theaddition of a quenched probe that becomesfluorescent uponeach PCR amplification step allows for direct quantificationof the number of DNA copies initially present in the sample(1–4).The probe that is added to the PCR reaction(com-monly referred to as the TaqMan probe),is an oligonucleotidethat contains a quencher dye at the3 end and afluorescentreporter dye at the5 end of the probe(4).Differentflu-orescent reporter dyes can be added to the5 end,such as∗These two authors contributed equally to this work.Vinayaka R.Prasad,Ganjam V.Kalpana(eds.),HIV Protocols:Second Edition,vol.485C 2009Humana Press,a part of Springer Science+Business MediaDOI10.1007/5556Mbisa et al.6-carboxy-fluorescein (FAM),hexachloro-6-carboxy-fluorescein,or tetrachloro-6-carboxy-fluorescein,each of which emit light atdifferent wavelengths.The quencher at the 3 end of the probe istypically 6-carboxy-tetramethyl-rhodamine (TAMRA).As shownin Fig.5.1,a typical reaction starts when the probe binds to thetarget template during the cool-down phase after the melting stepfollowed by binding of the forward and reverse primers (Step 1).At this stage,the quencher is in close proximity to the fluorescentreporter dye and therefore no fluorescence is emitted.The Taqpolymerase that catalyzes the PCR reaction not only has a 5 -3polymerase activity,but also a 5 -3 exonuclease activity (Step 2).Therefore,as the Taq polymerase synthesizes DNA,it cleaves anddisplaces the probe,releasing the quencher,and allowing fluo-rescence to be emitted (Step 3)before finishing each amplifi-cation cycle (Step 4).As a result,for each amplification cycle,the emitted fluorescence is recorded and the cycle number atwhich the fluorescence becomes detectable (cycle threshold;C t )is directly correlated to the number of templates in the reaction atthat cycle.This correlation only holds true during the exponen-tial phase of the PCR reaction.Utilizing a known copy numberof input templates as a standard,a standard curve can be gener-ated to determine the exact number of templates initially presentin any unknown sample.Therefore,this technology can moni-tor,in real time,the progress of any PCR reaction and determinethe exact DNA copy numbers per sample,with a high sensitivitythat allows detection of 10or fewer copies per sample (5).As an5'3'3'probe1.2.4.3.Fig.5.1.PCR amplification using TaqMan probe chemistry.After probe and primersbind to the template (Step 1),Taq polymerase initiates synthesis,displaces and cleavesthe probe (Step 2and 3),which allows fluorescence to be emitted.Polymerization iscompleted after one cycle of PCR amplification (Step 4).Analysis of HIV-1Post-entry Events57 alternative to the TaqMan system,one can use SYBR Green I dye which intercalates into double-stranded DNA as it accumulates during the PCR reaction thereby producing afluorescent signal that can be quantified(6–8).The benefits of the SYBR Green dye intercalation technique are that it is less expensive and can be uni-versally applied to all PCR reactions.However,TaqMan probe chemistry is generally preferred over SYBR Green I dye chem-istry because it provides a greater level of specificity.The Taq-Man probe is specific for a given sequence whereas SYBR Green I dye binds all double-stranded DNA,including any PCR artifacts (primer-dimers or nonspecific amplification products),and there-fore requires afinal dissociation curve analysis to verify that the reactions are optimized so that thefinal amplified signal is specific to the target sequence(6–8).The HIV-1genome is initially plus-stranded RNA that must be converted into double-stranded DNA for the virus to com-plete its life cycle(9).The unique process by which the HIV-1reverse transcriptase carries out DNA synthesis enables one to utilize specific primer and probe sets to track and quantify early, intermediate,and late events in reverse transcription(Fig.5.2) (10–19).Early reverse transcription products can be monitored by using a primer/probe set that specifically amplifies minus-strand strong-stop DNA(Fig.5.2A);minus-strand DNA trans-fer can be monitored by using a primer/probe set thatflanks the U3-R-U5junction(Fig. 5.2B);intermediate stages of reverse transcription during minus-strand DNA synthesis and elongation can be monitored with a primer/probe set specific to regions of the viral genome that are between the two LTRs,e.g.,gag (Fig. 5.2C).Finally,plus-strand DNA transfer and late reverse transcription products can be monitored by a primer/probe set specific to the U5and5 untranslated regions of the viral DNA(Fig. 5.2D).Quantifying thefinal number of integrated proviruses into the host cell chromosome relies upon the pres-ence of natural Alu repeats found throughout all human chro-mosomal DNA(11,20,21).A U5specific forward primer and probe are used with a reverse primer that binds to the near-est Alu repeat,amplifying the integrated provirus(Fig.5.2G). Every reverse transcription reaction does not always result in an integration event as some viral DNAs remain linear or cir-cularize to form dead-end1-LTR-and2-LTR-circle products (Fig. 5.2E,F,respectively).The2-LTR circular products can be quantified using primer/probe sets specific to U5and U3,and the1-LTR circle products can also be quantified using a specific primer/probe set which binds gag and env during real-time PCR (11,22–25).The advent of real-time PCR has been instrumental to the field of retrovirology.Not only can real-time PCR be used for basic laboratory research,but it can also be used clinically to58Mbisa et al.R U5 PBS ΨPPT U3 Rviral RNA'integrationPCR integrated provirusesPCR minus strand transfer(A)(D)(G)(E)Fig.5.2.Schematic representation of the HIV-1reverse transcription and integration.Real-time PCR (shaded gray boxes containing arrows)can be used to analyze and quan-tify early,intermediate,and late reverse transcription products as well as integratedand unintegrated (1-and 2-LTR circles)HIV-1viral DNA (Stages A–G).Thin black lines,RNA;thick black lines,DNA;thick gray lines,chromosomal DNA;dashed lines,RNase Hdegradation;cloverleaf,tRNA.Analysis of HIV-1Post-entry Events59quantify HIV-1viral loads and mutation frequencies in patients(5,26–30).Real-time PCR can be used to quantify the efficiencyof reverse transcription and overall amount of viral DNA synthe-sis,quantify the levels of nonintegrated vs.integrated viral DNAfor any antiviral drug treatment,and determine the effects ofan HIV-1mutant or change in experimental conditions on viralDNA synthesis.Overall,real-time PCR technology has greatlyenhanced our basic knowledge of HIV-1post-entry events.2.Materials2.1.Cell Culture, Virus Production, Infections1.293T cells and/or a cell line transduced with an HIV-1-basedvector.For example,a293T cell line has been made that sta-bly expresses the plasmid pHDV-eGFP(31)(named293T-HIV-GFP cell line for remainder of text)(see Note1).2.An integration standard cell line generated by transducingcells with an HIV-1-based vector capable of a single round of replication(11,19)(see Note2).3.Dulbecco’s Modified Eagle’s Medium(DMEM)(Gibco/BRL,Bethesda,MD)supplemented with10% fetal bovine serum(FBS;Hyclone,Ogden,UT),penicillin (50U/mL;Gibco/BRL)and streptomycin(50μg/mL;Gibco/BRL).4.Plasmids:an HIV-1-based vector,e.g.,pHDV-eGFP(31);pHCMV-G,a vector expressing the G glycoprotein of vestic-ular stomatis virus(VSV-G)(32).5.CalPhos Mammalian Transfection Kit(Clontech,MountainView,CA).6.Phosphate-buffered saline(PBS)without Ca2+or Mg2+(Cambrex,Walkersville,MD).lex GS0.45-μm pore sizefilter(Nalgene,Rochester,NY).8.HIV-1p24ELISA Kit(Perkin Elmer,Wellesley,MA).9.DNase I(Roche,Indianapolis,IN).10.Heat block set at65◦C.11.Hexadimethrine bromide(Polybrene),1mg/mL stock(Sigma,St.Louis,MO).2.2.DNA Isolation 1.QIAamp DNA Blood Mini Kit(Qiagen,Germantown,MD).2.2mL Collection tubes(Qiagen,Germantown,MD).3.Dpn I restriction enzyme(NEB,Beverly,MA).2.3.Real-Time PCR 1.A dedicated set of pipettes andfilter tips for real-time PCRuse only.2.AmpliTaq Gold R DNA Polymerase,5U/μL(PE-AppliedBiosystems,Foster City,CA).3.100mM dNTP Set,PCR grade,4×25μmol(Invitrogen,Carlsbad,CA).60Mbisa et al.Table5.1Primer/probe sets for analysis of different stages of reverse transcription,1-and 2-LTR circle formation,integrated proviruses,and cellular genesStage of reverse transcription a TargetsequenceForwardprimerReverseprimer ProbeStandardtemplateA RU5hRU5-F2hRU5-R hRU5-P pHDV-eGFPB U3U5FST-F1SS-R4P-HUS-SS1pHDV-eGFPC Gag GagF1GagR1P-HUS-103pHDV-eGFPD U5 MH531MH532LRT-P pHDV-eGFP E1-LTR LA1LA151-LTR-P pCR-1LTRF2-LTR FJ SS-F4LTR-R5P-HUS-SS1pJB1041F2-LTR TOT SS-F4LTR-R3P-HUS-SS1pJB1041G Alu-LTR MH535SB704P-HUS-SS1Special b control PBGD nPBGD-For nPBGD-Rev nPBGD-Pr pJB1057 control CCR5CCR5-For CCR5-Rev P-CCR5-01pRB2569(19)a Stages labeled in Fig.5.2.b Standard for the Alu-LTR assay is the Integration Standard cell line,prepared as described in Section3.8.4.10X TaqMan R Buffer II(PE-Applied Biosystems).5.10X TaqMan R Buffer A(containing FAM-10-ROX internalreference)(PE-Applied Biosystems).6.25mM MgCl2(PE-Applied Biosystems).7.Forward and reverse primers(100μM stocks).8.Dual-labeled probe(100μM stocks).9.Plasmid DNA to be used as standard template as listed inTable5.1(see Note3).10.DNA from uninfected293T cells.11.PCR grade nuclease-free water(Ambion,Austin,TX).12.MicroAmp R Optical96-well Reaction Plates(PE-AppliedBiosystems).13.MicroAmp R Optical Caps for96-well Reaction Plates(PE-Applied Biosystems).14.Microfuge tubes(DNase/RNase free)15.ABI7900HT(PE-Applied Biosystems)or equivalent Real-time PCR instrument.3.MethodsThe protocol outlined here describes the analysis of HIV-1post-entry events during infection of cultured cells utilizing the Taq-Man probe chemistry.In addition,this protocol can be easilyAnalysis of HIV-1Post-entry Events61 modified to analyze other types of samples,for example clinical samples.To analyze post-entry steps during HIV-1infection of cultured cells by real-time PCR,virus is produced by transfection of293T cells with an HIV-1-based vector and an envelope pro-tein expression construct,target cells are infected,and DNA is isolated from the infected cells.3.1.Virus Production from Transfected Cell Lines 1.Plate293T target cells in DMEM at density of2.5×106cellsper100-mm-diameter dish(see Note1).2.Twenty four hours later,replace medium with fresh DMEM.Transfect with10μg of pHDV-eGFP and2μg of pHCMV-G using CalPhos transfection kit following manufacturer’sinstructions(see Note4).3.Incubate the plates in3%CO2incubator at37◦C for6h.4.Gently rinse the plates three times with10mL of PBS at roomtemperature supplemented with1%FBS(v/v)(see Note5).Add10mL of fresh DMEM media and incubate overnight in 5%CO2incubator at37◦C.5.Next morning,gently rinse the plates again three times with10mL PBS and add10mL of fresh DMEM media.Return to incubator.6.Gently rinse the plates again5–6h later once with10mL PBSand add10mL of fresh DMEM media.Incubate overnight.7.Harvest the virus containing supernatant the next morningand clarify the samples by centrifuging at400×g for4min at 4◦C.Pass the supernatant through Millex GS0.45μm pore sizefilter.8.Take an aliquot and perform p24ELISA analysis followingmanufacturer’s instructions.9.Store the remaining sample at−80◦C until ready to use forinfection of target cells.3.2.Virus Production from Stable Cell Lines 1.Plate293T-HIV-GFP cells at density of2.5×106cells per100-mm-diameter dish(see Note1).2.Twenty four hours later,replace medium with fresh DMEM.Transfect with2μg of pHCMV-G using CalPhos transfection kit following manufacturer’s instructions(see Note4).3.Incubate the plates in3%CO2incubator at37◦C for6h.4.Remove the media and add10mL of fresh DMEM media.5.Incubate the plates in5%CO2incubator at37◦C for36–48h.6.Harvest the virus containing supernatant and clarify the sam-ples by centrifuging at400×g for4min at4◦C.Pass the supernatant through Millex GS0.45-μm pore sizefilter.7.Take an aliquot and perform p24ELISA analysis followingmanufacturer’s instructions.8.Store the remaining sample at−80◦C until ready to use forinfection of target cells.62Mbisa et al.3.3.Infection 1.Plate293T target cells at a density of1×105cells per60-mm-diameter dish(see Note1).2.Twenty four hours later,infect target cells with p24-normalized virus as determined by HIV-1p24ELISA(seeNote6).Before infection,treat the virus with10U/mL ofDNase I plus4mM MgCl2at37◦C for1h to reduce trans-fected plasmid DNA carryover,which can contribute to thesignal during real-time PCR.3.For each virus,prepare in parallel a heat-inactivated controlsample by incubating the virus at65◦C for an hour to be usedfor determining carryover contamination from plasmid DNA.4.Make the inoculum,for both live and heat-inactivated virus,bydiluting so that p24values are comparable,adjustfinal volumeto2mL per plate with DMEM,and add5μg/mL of Poly-brene.5.Infect for3h with gentle rocking every30–60min.6.Remove inoculum,gently rinse twice with PBS plus1%FBSand add4mL of fresh DMEM.7.Harvest total cellular DNA from both live virus-and heat-inactivated virus-infected cells at desired time points by remov-ing the media and adding1mL of PBS.Scrape the plates orpipet vigorously to dislodge cells,and transfer cell suspensionto1.5mL centrifuge tubes and spin at1000×g for4minat4◦C.8.Discard supernatant and proceed to isolate DNA(see Section3.4)or store cell pellets at−80◦C until ready to isolate DNA.3.4.DNA Isolation 1.Resuspend cell pellets in200μL of PBS.2.Proceed to extract the total cellular DNA following the proto-col as stated in the Qiagen QIAamp DNA Blood Mini Kit.3.Elute the DNA in200μL of elution buffer EB provided in thekit or nuclease-free water.4.Digest DNA sample with Dpn I restriction enzyme,whichdigests only plasmid DNA and therefore helps further toreduce plasmid DNA detection during real-time e0.1U/μL in NEB Buffer2for2–4h at37◦C.3.5.Real-Time PCR Analysis of Postentry Reverse Transcription Steps 1.Analysis of the different stages of reverse transcription is nor-mally carried out by completing a time course from0to6or 0to12h,and subsequently comparing the different levels of reverse transcription products as time progresses.For example, collect and process samples at0,0.5,1,3,6and12h postin-fection.2.If possible,use a UV-decontaminated hood when setting upall PCR reactions to avoid any cross contamination.Only use pipettes,RNase and DNase-free Eppendorf tubes and sterileAnalysis of HIV-1Post-entry Events63 PCR racks that are dedicated for real-time PCR use.All pre-pared reaction mixtures should be kept on ice.3.Select the primer/probe set to use from the list in Table5.1depending on the stage of reverse transcription to be analyzed,i.e.,RU5for initiation of reverse transcription or early RTproducts,U3U5products for minus-strand DNA transfer,gag for late minus-strand DNA synthesis,and U5 for plus-strand DNA transfer and late RT products(see Note7).PBGD or CCR5primer/probe sets target cellular genes and are run for each sample to normalize for DNA recovery and the number of cells loaded in each reaction.The sequences of the primers and probes are listed in Table5.2.4.Prepare a standard curve from the appropriate standard tem-plate listed in Table5.1to be used to compute the copy num-bers in each sample.Make a stock of109DNA copies/μL and dilute the working stock solution to108copies/μL.Prepare 11dilutions for the standard curve:thefirst dilution should be made at afinal concentration of107copies per10μL.Start-ing with thefirst dilution,make10additional threefold serial dilutions down to169copies per10μL.5.In a nuclease-free tube on ice,prepare a PCR master mix withall of the PCR reagents except the DNA as outlined in Table5.3(see Note8).The reaction setup shown in Table5.3is fora50-μL reaction,but prepare enough PCR master mix for all the samples and standards in the experiment as well as a“no template”control(NTC).Include a5%overage to compen-sate for pipetting error.Mix well and centrifuge briefly.It is recommended that all samples should be run in duplicate or triplicate to increase the accuracy of results.6.Dispense40μL of PCR master mix into each well of aMicroAmp R Optical96-well Reaction Plate and add10μL of DNA isolated from step3.4(100–500ng),or10μL of the serial dilution of standard DNA template or10μL of water for the NTC.You may also run a blank containing50μL of water.Securely cover all the wells(including unused ones)with the MicroAmp R Optical Caps for96-well Reaction Plates.Mix well and centrifuge briefly to ensure that the entire sample is at the bottom of the well.7.Run the PCR reaction using the thermocycling parametersoutlined in Table5.4for analysis of different stages of reverse transcription or for the cellular gene controls,using an ABI Prism7900HT sequence detection system(see Note9).8.After the reaction is complete,analyze the results accord-ing to the manufacturer’s instructions,especially paying great attention to the baseline and threshold settings (see /cms/groups/mcb-marketing/documents/generaldocuments/cms-042502.pdf and https:///support/apptech/64Mbisa et al.Table5.2Sequences of oligonucleotide primers and probesSequenceName ofoligonucleotide ahRU5-F2b5 -GCCTCAATAAAGCTTGCCTTGA-3hRU5-R b5 -TGACTAAAAGGGTCTGAGGGATCT-3hRU5-P b5 -FAM-AGAGTCACACAACAGACGGGCACACACTA-TAMRA-3 FST-F1b5 -GAGCCCTCAGATGCTGCATAT-3SS-R4b5 -CCACACTGACTAAAAGGGTCTGAG-3P-HUS-SS1b5 -FAM-TAGTGTGTGCCCGTCTGTTGTGTGAC-TAMRA-3 GagF1b5 -CTAGAACGATTCGCAGTTAATCCT-3GagR1b5 -CTATCCTTTGATGCACACAATAGAG-3P-HUS-103b5 -FAM-CATCAGAAGGCTGTAGACAAATACTGGGA-TAMRA-3 MH531b5 -TGTGTGCCCGTCTGTTGTGT-3MH532b5 -GAGTCCTGCGTCGAGAGATC-3LRT-P b5 -FAM-CAGTGGCGCCCGAACAGGGA-TAMRA-3SS-F4b5 -TGGTTAGACCAGATCTGAGCCT-3LTR-R3b5 -AGGTAGCCTTGTGTGTGGTAGATCC-3LTR-R5b5 -GTGAATTAGCCCTTCCAGTACTGC-3LA1b5 -GCGCTTCAGCAAGCCGAGTCCT-3LA15b5 -CACACCTCAGGTACCTTTAAGA-31-LTR-P b5 -FAM-AGTGGCGAGCCCTCAGATGCTGC-TAMRA-3MH535b5 -AACTAGGGAACCCACTGCTTAAG-3SB704c5 -TGCTGGGATTACAGGCGTGAG-3nPBGD-For c5 -AGGGATTCACTCAGGCTCTTTCT-3nPBGD-Rev c5 -GCATGTTCAAGCTCCTTGGTAA-3nPBGD-Pr c5 -FAM-TCCGGCAGATTGGAGAGAAAAGCCTG-TAMRA-3CCR5-For c5 -CCAGAAGAGCTGAGACATCCG-3CCR5-Rev c5 -GCCAAGCAGCTGAGAGGTTACT-3P-CCR5-01c5 -FAM-TCCCCTACAAGAAACTCTCCCCGG-TAMRA-3a See Table5.1for the target sequence detected by each primer/probe set.b Primer sequences were designed to amplify NL4-3viral DNA,or vectors containing these sequences.Because of sequence variation between HIV-1isolates,it may be necessary to modify or redesign the sequences.c Primer sequences are designed for human genes.The use of cells from other species will require modification or redesign of these sequences.Setup of real-time PCR reactionsAmount Component Final concentration 3μL10X Buffer A2μL10X PCR Buffer II1X8μL MgCl2(25mM)4mM0.4μL dNTPs(25mM)0.2mM each0.3μL a,b Forward primer(100μM)600nM a,b0.3μL a,b Reverse primer(100μM)600nM a,b0.04μL a,b Probe(100μM)80nM a,b0.25μL AmpliTaq Gold R (5U/μL) 1.25U10μL DNA(100to500ng)to50μL PCR Grade Nuclease-free Watera For the reaction to analyze1-LTR circle formation,the forward and reverse primers are used at400nM,the probe is used at200nM(25).b For Alu-LTR reactions,the forward primer is used at50nM,the reverse primer is used at900nM,the probe is used at100nM(see(19),Bushman website, /).#rt pcr).The blank and NTC samples should not give any signal.The viral standard curve will be used to calculate the copy number for each unknown sample.A graph plot showing a typical standard curve with values from live and heat-inactivated virus samples is presented in Fig.5.3.The cellular gene copy numbers can be used to normalize for the number of the cells loaded in each reaction.A good standard curve should have a y-intercept of approximately39–41cycles and a slope between−3.321and−3.5,which corresponds to an efficiency of100–93%,respectively.3.6.Real-Time PCR Analysis of2-LTR-Circle Formation 1.Analysis of2-LTR-circle formation is normally done usingDNA collected24h after infection.2.Select a primer/probe set from the list in Table5.1depend-ing on whether you want to analyze the total number of 2-LTR circles(2-LTR TOT)or2-LTR circles with com-plete junctions only(2LTR FJ).Also run a duplicate plate with PBGD or CCR5primer/probe set for each sam-ple to normalize for the number of cells loaded in each reaction.3.Prepare a standard curve from the appropriate standard tem-plate listed in Table5.1,to be used to compute the copy num-bers in each sample as described in point3of Section3.5.Thermocycling parameters for real-time PCR reactionsCycling parametersTarget of amplification Step Temperature Duration RU5,gag or U5 Denature a95◦C10min45PCR cyclesDenature95◦C15sAnneal and extend60◦C1min PBGD or CCR5Denature95◦C10min45PCR cyclesDenature95◦C15sAnneal55◦C30sExtend60◦C30sAlu-LTR Denature95◦C10min45PCR cyclesDenature95◦C15sAnneal60◦C1minExtend72◦C1min2-LTR circles Denature95◦C10min45PCR cyclesDenature95◦C15sAnneal and extend66◦C1min1-LTR circles Denature95◦C10min45PCR cyclesDenature95◦C30sAnneal60◦C30sExtend72◦C1minFinal extension72◦C5mina AmpliTaq Gold(ABI)is acetylated to prevent DNA synthesis;10min at95◦C acti-vates the polymerase.Platinum Taq(Invitrogen)is bound to an antibody to prevent DNA synthesis;2min at95◦C is enough to activate the polymerase.4.Prepare the PCR master mix as described in point4ofSection 3.5.5.Dispense the PCR master mix and DNA into each well of aMicroAmp R Optical96-well Reaction Plate as described in point5of Section3.5.0510********3502468T h r e s h o l d C y c l e (C t )Copy Number (log)Fig.5.3.Typical real-time PCR standard curve with values from live and heat-inactivated virus samples.6.Run the PCR reaction using the thermocycling parameters outlined in Table 5.4for analysis of 2-LTR circles or for the cellular gene controls,using an ABI Prism 7900HT sequence detection system (see Note 9).7.Analyze the data as described in point 8of Section 3.5.The number of 2-LTR circles formed is normally expressed as a percentage of late RT products at 6h after infection in a paral-lel infection.On average,during wild-type virus infection,1–5%of late RT products at 6h ultimately form 2-LTR circles,depending on the infection target cell line (11,19).3.7.Real-Time PCR Analysis of 1-LTR-Circle Formation1.Analysis of 1-LTR-circle formation is normally done using DNA collected 24h after infection.2.Prepare a standard curve from the appropriate standard tem-plate listed in Table 5.1,to be used to compute the copy num-bers in each sample as stated in point 3of Section3.5.3.Prepare the PCR master mix as described in point 4of Section 3.5using the 1-LTR primer/probe set (Table 5.1).Note the difference in the 1-LTR primer and probe concentrations.Also run a duplicate plate with PBGD or CCR5primer/probe set to normalize for the number of cells loaded for each reaction.4.Dispense the PCR master mix and DNA into each well of aMicroAmp ROptical 96-well Reaction Plate as described in point 5of Section 3.5.5.Run the PCR reaction using the thermocycling parameters outlined in Table 5.4for analysis of 1-LTR circle-formation or for the cellular gene controls,using an ABI Prism 7900HT sequence detection system (see Note 9).6.Analyze the data as described in point 8of Section 3.5.The number of 1-LTR circles formed is normally expressed as a per-centage of late RT product at 6h after infection determinedfrom a parallel infection.On average,the proportion of1-LTR:2-LTR circles formed during wild-type virus infection is 9:1(11).3.8.Alu-LTR Real-Time PCR Analysis of Viral Integration 1.Analysis of integrated proviruses is normally done using DNAcollected24–48h after infection.2.Prepare integration standard DNA from a293T cell line trans-duced with an HIV-1-based vector as previously described (11,19)(see Note2).3.Determine the equivalent provirus copy number by corre-lating the Alu-LTR signal with late RT product using U5 primer/probe set as previously described(11,20,21)(see Note10).It is important to make this determination in the presenceof the amount of cell-extract that will be present when measur-ing the unknowns(see Note11).Make single use aliquots of 105–106provirus/μL and keep at−80◦C until needed.Keep the cellular DNA as concentrated as is practical.4.Measure the quantities of either PBGD or CCR5to deter-mine the number of cells present in each reaction,and dilute if necessary to insure that100–500ng of DNA is present when quantifying provirus.Otherwise,the signal obtained cannot be equated with quantities on the standard curve.5.Prepare dilutions for the standard curve:thefirst dilutionshould be made at afinal concentration of approximately106 copies per10μL.Starting with this dilution,prepare threefold serial dilutions down to approximately1,000copies per10μL.Each dilution should also contain100–500ng of DNA from uninfected293T cells per10μL,as a control for cellular DNA in the sample(see Note11).6.Prepare the PCR master mix as described in point4of Sec-tion3.5using the Alu-LTR primer/probe set(Table5.1).Note the difference in the Alu-LTR primer and probe concen-trations.7.Dispense the PCR master mix and DNA into each well of aMicroAmp R Optical96-well Reaction Plate as described in point5of Section3.5.8.Run the PCR reaction using the thermocycling parametersoutlined in Table5.4for analysis of integrated proviruses or for the cellular gene controls,using an ABI Prism7900HT sequence detection system(see Note9).9.Analyze the data as described in point8of Section3.5.Thenumber of integrated proviruses is normally expressed as a per-centage of late RT product at6h after infection determined from a parallel infection.On average,during wild-type virus infection,5–20%of late RT products at6h eventually inte-grate into the host genome,depending on the infection target cell line(11).。