Figure 1

PCR and NGS-based confirmation of recombination between the DHIV and pLET-LAI constructs as a source of replication-competent virus. To generate a vector capable of a single round of replication only, the vector employed in the original TCM model is produced with the env-deficient DHIV construct co-transfected with intact env. Recombination between overlapping sequences of these plasmids restores an intact env sequence in the viral genome and produces replication-competent virus. (cfr. Additional file 1). A-C. PCR based data A. Primer pairs used in the study aligned to the envelope gene of DHIV construct. Primer pair ENV aligns to the common sequence of env of DHIV and of the full length env, primer pair DEL aligns to the intact env sequence within the deleted part (dotted line) of DHIV. B-C. Electrophoretic separation of PCR products performed on plasmid DNA-NL4.3-IRES-HSA-E* and DHIV (B) and on DNA from cells infected with the indicated viruses (C). The positive signal in cells infected with DHIV + pLET-LAI indicates the presence of a full length env sequence in the viral DNA. D-F. Expression of HIV RNA in the latent TCM model derived from RNA-Seq analysis of 4 donor cells infected with DHIV + pLET-LAI. D. The plot representing the number of times individual nucleotides were mapped to the HIV genome. The delineated region indicates the env region deleted in the original DHIV. The high number of mapped reads indicates that the complete env sequence is expressed. E-F. The plot representing reads spanning the deletion in DHIV env region at the beginning (E) and end (F) of the deletion. Collectively, the electrophoretic analysis of integrated proviral DNA and the alignment of NGS reads in the region originally containing a deletion in env indicate that an intact sequence of env was restored in the construct.