Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo 英文参考文献.docVIP

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Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo 英文参考文献.doc

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Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo 英文参考文献

QuantificationofUnintegratedHIV-1DNAattheSingle CellLevelInVivo RodolpheSuspe`ne*¤a,AndreasMeyerhans¤b DepartmentofVirology,InstituteofMedicalMicrobiology,UniversityoftheSaarland,Homburg/Saar,Germany Abstract InthenucleusofHIV-1infectedcells,unintegratedHIV-1DNAmoleculesexistintheformofoneandtwoLTRcirclesand linear molecules with degraded extremities. In tissue culture they are invariably more numerous than the provirus, the relativeproportionofintegratedtounintegratedformsvarieswidelyfrom,1:1to1:10andevenover1:100.Invivo ,this ratio is unknown. To determine it, single nuclei from two infected patients with a known provirus copy number were microdissected,HIVDNAwasamplifiedbynestedPCR,clonedandindividualclonessequenced.Giventheextraordinary sequencecomplexity,wemadetheassumptionthatthetotalnumberofdistinctsequencesapproximatedtorealnumber ofamplifiableHIV-1DNAtemplatesinthenucleus.WefoundthatthenumberofunintegratedDNAmoleculesincreased linearlywiththeproviralcopynumbertherebeingonaverage86unintegratedmoleculesperprovirus. Citation: Suspe`ne R, Meyerhans A (2012) Quantification of Unintegrated HIV-1 DNA at the Single Cell Level In Vivo. PLoS ONE 7(5): e36246. doi:10.1371/ journal.pone.0036246 Editor:FatahKashanchi,GeorgeMasonUniversity,UnitedStatesofAmerica ReceivedJanuary30,2012;AcceptedMarch28,2012;PublishedMay4,2012 Copyright:?2012Suspe`ne,Meyerhans.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: RSwas supportedby an EMBO LongTerm Fellowship.AM was supported bygrants from the Deutsche Forschungsgemeinschaftand the Spanish MinistryofScienceandInnovation(SAF2010-21336).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationof themanuscript. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:rodolphe.suspene@pasteur.fr ¤aCurrentaddress:Ins