Figure 2
Schematic representation of intra- and intersubtype recombination systems. Single cycle tissue culture system (panel A) for recombination employed heterozygous VSV-pseudotyped env particles produced by transient co-transfection of two genomic and two helper plasmids in 293T cells. Following production from 293T cells, virus particles were used to transduce MT4 cells. PCR products cleaved with BamHI and SacII were then cloned into vectors for transfection into E. coli followed by screening of blue and white colonies. Calculations for the frequency of recombination are outlined in the Materials and Methods. Structure of the genomic plasmids and reverse transcription products are shown in panel A. The in vitro experimental system is outline in panel B and involves reverse transcription across a donor RNA template that shares a region of homology with an acceptor RNA template upstream of a genetic marker (lacZ') on the acceptor RNA or a truncated, non-functional portion of the malT gene from E.coli on the donor template. The donor RNA also contains at its 3' end an extension which is used to selectively prime reverse transcription after hybridization of a complementary oligonucleotide. Processive copying of the donor template will yield lac- genotypes, while template switching during reverse transcription of the retroviral sequence will produce lac+ genotypes. The resulting double-stranded DNAs are restricted with BamHI and PstI and, after ligation to a plasmid vector, used for bacterial transformation. On appropriate media, recombinant DNAs will yield blue colonies distinguishable from the white colonies given by the parental DNAs. The same LacZ screening system is employed for single cycle assay (A). Multiple cycle tissue culture system (panel C) was performed by infecting U87.CD4.CXCR4 cells with subtype A and D HIV-1 isolates in pairs (0.001 MOI). After the first round of replication, co-infected cells can produce both parental and heterodiploid viruses. Infection of new cells with heterodiploid virions can lead to intersubtype recombination. The schema for PCR amplification of intersubtype HIV-1 env fragments is outlined in panel C and the calculation for frequency is described in the Materials and Methods. Finally, panel D describes the reconstituted in vitro reverse transcription assay which involves initiating HIV-1 DNA synthesis from a radiolabeled DNA primer annealed to a defined donor RNA template (e.g. C3-V4) and in the same reaction mixtures with an acceptor RNA template slightly longer and with a region of sequence homology with the donor to promote strand transfer. RNA-dependent DNA synthesis is catalyzed by reverse transcriptase and a 20 nt 5' [32P]-labeled DNA primer on the RNA donor templates (225 nt), RNA acceptor templates (225 nt), and with or without NC. The templates have a 205 nt overlap region to promote intersubtype recombination. Products from these reactions were resolved on a 8% denaturing polyacrylamide gel.