Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRFO1_AE) generated by the polymerase chain reaction

We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend t hese findings by describing a novel vector system to recover infectious mol ecular clones from long PCR amplicons. Directional ligation of 9.2-kb provi ral amplicons into a recovery vector reconstitutes missing LTR sequences, p roviding candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional i nfectious molecular clones generated, from primary isolates of subtype B (H IV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235 )) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first repor t of infectious molecular clones from circulating recombinant form 01_AE (s ubtype E). (C) 2000 Academic Press.