Fig 1.
HIV-1 genome conservation analysis to select TALEN sites.
A. Schematic diagram of HIV-1 genome adapted from the Los Alamos National Laboratory HIV website [44]. Bolded boxes are regions with HT-TALEN DNA targets, one of which is shown in B. B. 5’ LTR DNA TALEN target sequence. The TALE binding targets are indicated by black lines. The endonuclease target site sequence is in lower case font and indicated by grey lines. C. TAR RNA with partial 5’ TALE binding site in upper-case font and endonuclease target site in lower-case font. D. HIV-1 DNA sequences (274,874 total) from the Los Alamos HIV Sequence Database were aligned with ClustalΩ to determine conservation which is presented in a position specific-scoring matrix [33,44]. The nucleotide frequency for the most conserved regions were chosen as TALEN target sequences found in the TAR coding region (B) of the LTRs (226 sequences) (A).
Fig 2.
HT-TALENs and NS-TALENs cleave an HIV-1 DNA fragment in vitro.
A. Schematic diagram representing HT-TALENs and NS-TALENs bound to their cognate DNA target sequence (thick lines). Relative locations of the Fok1 endonuclease, Flag epitope tag, and nuclear localization sequence (NLS) are indicated. Asterisks and grey boxes designate where a “NS” coding TALE repeat was used in the 5’ NS-TALEN construction. B. Western blot of in vitro transcription/translation reactions containing no expression plasmids, each TALEN alone, the HT-TALEN pair, or the NS-TALEN pair. C. Gel electrophoresis analysis of in vitro cleavage reactions containing no TALEN plasmids, the HT-TALEN pair, or the NS-TALEN pair. The HIV-1 target DNA fragment size is 747 bp, with expected on-target cleavage products of approximately 430 bp and 317 bp. Quantification of cleavage was performed using ImageJ software and is shown below the gel image. D. The HIV-1 target DNA fragment from (C) was mutated in the 5’ TALE binding site to create a set of triple mutant templates (Mut1-Mut4). The sequences of Mut1-Mut4 are depicted in bold, lowercase font and mutated positions are indicated by asterisks. Cleavage reactions containing either the HT-TALEN or NS-TALEN pairs incubated with the HIV-1 target templates were size fractionated by electrophoresis and quantified by densitometry with ImageJ [39].
Fig 3.
HT-TALEN and NS-TALEN targeting of an HIV-1 LTR in cell culture.
A. Schematic diagram of DNA GFP reporter to be targeted by HT-TALENs and NS-TALENs. The target DNA contains the 5’ LTR of HIV-1 fused upstream of the coding region of d1EGFP. B. Western blot analysis of HeLa-tat-III/LTR/d1EGFP cells transfected with either the HT-TALEN pair or NS-TALEN pair. The blot was probed with anti-Flag and anti-Actin as a loading control. C. Dose-response plot based on quantification of flow cytometry analysis of GFP reporter expression. Transiently transfected HeLa-tat-III/LTR/d1EGFP samples were analyzed for GFP and mCherry expression. Cells with mCherry contained the transfected plasmids. Cells containing the functional HIV-1 LTR fused d1EGFP reporter expressed GFP. Samples were done in triplicate. Those samples not expressing GFP, only mCherry were compared. Standard deviations from triplicate samples are smaller than the symbols and not shown. Statistically significant differences between slopes for TALEN treatment and control indicated is by a * (p<0.000001); NS-TALEN and HT-TALENs were not significantly different (p<0.08). D. Sequences of genomic clones containing mutated target regions. Upper-case bolded font indicates designated 5’ TALE and 3’ TALE binding sites. Inserted nucleotides are in lower-case italicized font. A deletion is represented by dashes. Lengths of the insertions (+) and deletions (-) are at the right of each sequence.
Fig 4.
TALEN targeting of integrated complete HIV-1 proviral DNA in cell culture.
A. Schematic diagram of the complete HIV-1 proviral DNA to be targeted by the HT-TALEN pair or the NS-TALEN pair. The target region is found in both the 5’ and 3’LTRs. The host genome is indicated in grey. B. Western blot analysis of HeLa/LAV cells transfected with a HT-TALEN plasmid pair or the NS-TALEN pair. The blot was probed with anti-Flag and anti-Actin as a loading control. C. Bar graph showing quantitation of flow cytometry analysis of cytotoxicity. Transiently transfected HeLa/LAV cells were analyzed by flow cytometry (n = 3) to identify Annexin V positive cells. Standard deviations are indicated by error bars with no statistical significance (NS) p>0.05 in cytotoxicity between the control and the TALEN pairs. D. Sequences of clones containing mutated target regions represented as in Fig 3. E. A schematic of the 5’ target region of wild type plai.2 HIV-1 proviral DNA and the mutated plai.2 HIV-1 proviral DNA. The mutated proviral DNA was designed based on the sequence from HeLa/LAV clone HL-16 (Fig 4D). The Gag coding region (containing capsid) is indicated. Western blot analysis of cell lysates harvested from pEAK Rapid cells transfected with mutant or wild type plai.2 proviral DNA. The blot was probed with anti-Capsid qingto detect Gag production and anti-Actin as a loading control.