Genome-editing activity of the TALENs and CRISPR/Cas9 systems targeting HIV LTR.

<p>(A) LTR-driven GFP expression after TF with CRISPR/Cas9 or TALENs expressing plasmid DNA. Jurkat cells were infected with an LTIG lentiviral vector. Five days after infection, the GFP positive cells were sorted (Jurkat/LTIG) and co-transfected with T5 gRNA-expressing and hCas9-expressing DNA or LTR TALEN-L and -R expressing DNA. The level of GFP expression was analyzed by flow cytometry 5 days after TF. (B) Sequence analysis of TALENs targeting sites. The DNA sequences of the TAR and adjacent regions of LTR are indicated. Nineteen sequences were obtained from Jurkat/LTIG cells, which were transfected twice with TAR TALEN-LR. The WT reference sequence is shown at the top. The target sequences of TAR TALENs and T5 gRNA are indicated in orange and green, respectively. The putative cleavage site of T5 CRISPR is indicated with an orange arrowhead. (C) Genome-editing activity of TAR TALENs and T5 CRISPR in c19 cells, latently transduced with an LTIG lentiviral vector. The level of GFP expression after 48 hours of TNF-α stimulation is shown. The error bars in B and D show standard deviations (n = 3).</p

HIV LTR editing with mRNAs of TAR TALENs.

<p>(A) Kinetics of GFP transduction with mRNA and plasmid DNA in Jurkat cells. Jurkat cells were transfected with 1 μg of mRNA GFP or 1 μg of plasmid GFP under the control of a CMV promoter. The time course analysis of GFP expression was performed by flow cytometry. (B and C) A Jurkat cell line latently transduced with an LTIG vector was transfected with mTALENs. The level of GFP expression 48 hours after TNF-α stimulation is shown. Representative histograms are shown in B. The positive percentage of GFP is shown in B (n = 3). (D and E) ACH-2 cells were transfected with TAR mTALENs. The expression of p24 antigen 48 hours after TNF-α stimulation is shown. The percentage of p24 antigen expression in cells is shown in C (n = 3). The amount of p24 antigen in the culture supernatant is shown in E (n = 3). The error bars in A, C, D, and E show standard deviations (n = 3).</p

Excision of HIV provirus from host cell genome with TAR mTALENs.

<p>(A and B) HIV proviral excision in c19. (A) HIV provirus in c19 treated with TAR mTALENs was amplified using a primer set designed for the host cell genome sequence flanking the proviral integration site. The schematic of PCR products indicating genomic sequences, full-length provirus, and one LTR footprint resulting from proviral excision are shown on the left side. (B) The relative amount of <i>EGFP</i> DNA in TAR mTALENs—treated c19 is shown. (C and D) Excision of HIV-based lentiviral vector DNA with TAR mTALENs. Jurkat and MT-4 cells were transduced with a lentivirus vector containing a CMV promoter—derived GFP-expressing cassette, Jurkat/CS-CG and MT-4/CS-CG cells, respectively, and, these cells were treated with TAR mTALENs. A schematic of the lentiviral vector DNA used in this assay is shown at the top of C. The percentage of GFP positive cells after TF of mTALENs is shown in C (n = 3). The relative amount of <i>EGFP</i> DNA in treated TAR mTALENs is shown in D (n = 3). The error bars in B, C, and D show standard deviations (n = 3).</p

SCARB2 is absent from the cell surface, irrespective of EV-A71 susceptibility.

RD, HeLa, HEp-2, 293T, and Hep G2 were obtained from the ATCC specifically for this study and used after limited passage. (A) Western blotting analysis by anti-SCARB2 pAb (left) and mAb (right, clone 12H5L1). Recombinant SCARB2-Fc (1 ng for pAb, 5 ng for mAb) was loaded as a positive control. RD-SCARB2-KO clones were loaded as negative controls. The figure is representative of three independent experiments. (B) Flow cytometric analysis by anti-SCARB2 pAb (top panels) and mAb (bottom panels), followed by Alexa Fluor 488-tagged secondary Ab. The solid line and the shaded area represent staining with anti-SCARB2 Ab and control Ab, respectively. Note that the solid line and the border of the shaded area are almost completely overlapped, indicating the absence of SCARB2 on the cell surface. Representative results with the following passage numbers after receiving from the ATCC; for pAb: RD, 3; HeLa, 3; HEp-2, 3; 293T, 3; Hep G2, 5; for mAb: RD, 13; HeLa, 5; HEp-2, 4; 293T, 4; Hep G2, 8. As a positive control of SCARB2 staining, cells expressing surface SCARB2 were always stained and analyzed in parallel. The figure is representative of three independent experiments. (C) EGFP expression in cells infected with EV-A71-EGFP. Cells were infected with EV-A71-EGFP at 10 CCID50 per cell and cultured for 18 h. Then EGFP expression was measured by flow cytometry. The EGFP-negative cells are not infected. The majority of EGFP-dim cells were infected early in the incubation period, and are dying and losing EGFP expression; some may have just been infected and are starting to express EGFP. The EGFP-bright cells were infected late in the incubation period are actively producing EGFP. The number indicates the percentage of EGFP-positive cells (mean and s.e. for three independent experiments).</p

Fixation prior to anti-SCARB2 pAb treatment causes non-specific intracellular staining in RD and RD-SCARB2-KO cells.

RD and RD-SCARB2-KO (clone No. 3) cells were used. Staining with anti-SCARB2 pAb, fixation with 4% PFA, and permeabilization were performed in the combination and order as indicated above the top panels. Finally the cells were stained with Alexa Fluor-tagged secondary Ab and observed under a regular fluorescence microscope. The figure is representative of three independent experiments. Scale bars, 200 μm. (TIF)</p

Cell authentication.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71–susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71–susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.</div

PSGL-1 expression confers EV-A71 susceptibility on Jurkat-PSGL-1-KO cells.

To eliminate the possibility of off-target effects of CRISPR/Cas9, PSGL-1 was stably re-expressed in Jurkat-PSGL-1-KO clones (No. 1 and No. 2). PSGL-1 expression was confirmed by a flow cytometry. The solid line and the shaded area represent staining with anti-PSGL-1 mAb and control mouse IgG1, respectively, followed by Alexa Fluor 488-tagged secondary Ab. The cells infected with EV-A71-EGFP for 12 h were observed under a fluorescence microscope for evaluation of the EGFP expression. The figure is representative of three independent experiments. Scale bars, 100 μm. (TIF)</p

RD-A cells do not express SCARB2 on the cell surface.

Flow cytometric analysis of RD-A cells used in [9]. RD-A cells was stained with anti-SCARB2 pAb, followed by Alexa Fluor 488-tagged secondary Ab. The solid line and the shaded area represent staining with anti-SCARB2 pAb and control Ab, respectively. Note that the solid line and the border of the shaded area are almost completely overlapped, indicating the absence of SCARB2 on the cell surface. As a positive control of SCARB2 staining, cells expressing surface SCARB2 were stained and analyzed in parallel whenever possible. The figure (RD-A cells at passage number 235) is representative of at least fifteen independent experiments. (TIF)</p

SCARB2 is necessary for viral replication, irrespective of EV-A71’s PSGL-1 binding phenotype.

EV-A71 with VP1-145G or VP1-145Q are the PSGL-1-binding (PB) phenotype. EV-A71 with VP1-145E is the PSGL-1-nonbinding (non-PB) phenotype. RD and RD-SCARB2-KO (clone No.3) cells were infected with EV-A71 (MOI around 10) at 4°C for 30 min. Then the cells were washed three times. Viral titers were determined immediately after washing (0 h) and following two days of incubation (2 d). Results are indicated as the mean and s.e. for triplicate samples. (TIF)</p

Plasmids and oligos for genome editing by CRISPR/Cas9.

Plasmids and oligos for genome editing by CRISPR/Cas9.</p