HIV-1 Proviral Genome Engineering with CRISPR-Cas9 for Mechanistic Studies

Abstract

HIV-1 latency remains a barrier to a functional cure because of the ability of virtually silent yet inducible proviruses within reservoir cells to transcriptionally reactivate upon cell stimulation. HIV-1 reactivation occurs through the sequential action of host transcription factors (TFs) during the "host phase" and the viral TF Tat during the "viral phase", which together facilitate the positive feedback loop required for exponential transcription, replication, and pathogenesis. The sequential action of these TFs poses a challenge to precisely delineate the contributions of the host and viral phases of the transcriptional program to guide future mechanistic and therapeutic studies. To address this limitation, we devised a genome engineering approach to mutate tat and create a genetically matched pair of Jurkat T cell clones harboring HIV-1 at the same integration site with and without Tat expression. By comparing the transcriptional profile of both clones, the transition point between the host and viral phases was defined, providing a system that enables the temporal mechanistic interrogation of HIV-1 transcription prior to and after Tat synthesis. Importantly, this CRISPR method is broadly applicable to knockout individual viral proteins or genomic regulatory elements to delineate their contributions to various aspects of the viral life cycle and ultimately may facilitate therapeutic approaches in our race towards achieving a functional cure.

Keywords: CRISPR; Cas9; HIV-1; Tat; genome engineering; transcription; viruses.

Figure 1

The HIV-1 transcriptional program consists of three phases: basal, host, and viral. Cellular TFs facilitate activation of the host phase to synthesize Tat and promote the viral phase and positive feedback loop. Without Tat, the viral phase is never turned on, thereby preventing the feedback loop and sustained HIV-1 transcription activation.

Figure 2

A flow cytometry-based method for the generation of Tat-null 10.6 clones. (a) Scheme for the generation of 10.6 Tat^KO cells. (b) Scheme for the generation of 10.6 Ctrl^KO cells.

Figure 3

The 10.6 Tat^KO cells can be generated using a CRISPR-based approach. (a) Flow cytometry histogram showing ATTO 550 levels upon electroporation with the gRNA RNP complex. (b) Flow cytometry histogram(s) of 10.6 Ctrl^KO and 10.6 Tat^KO ATTO 550+ cell populations treated with vehicle or TNF-α for 16 h. (c) Agarose gel showing the 345 bp PCR product of 10.6, 10.6 Ctrl^KO, 10.6 Tat^KO, and 10.6 Tat^KO deletion clones. (d) The HIV-1 proviral genome scheme and Sanger sequencing results for the 4 cell lines in panel (c). The cross (X) indicates a mutation that prevents env expression. (e) Western blot of Tat, Gag, and β-Actin in 3 cell lines (10.6, 10.6 Ctrl^KO, and 10.6 Tat^KO) treated with vehicle or PMA for 16 h.

Figure 4

Precise tat genomic mutations and large deletions lead to differences in HIV-1 induction upon cell stimulation. (a) Flow cytometry quantitation of GFP fluorescence intensity (geometric median) in 4 cell lines (10.6, 10.6 Ctrl^KO, 10.6 Tat^KO, and 10.6 Tat^KO deletion) −/+ 16 h in PMA. Unpaired Student’s t-test, n = 3, −/+ SEM, ns = non-significant, *** p < 0.001. (b) Flow cytometry histograms of the data in panel (a).

Figure 5

Tat sustains enhanced HIV-1 expression upon cell stimulation. (a) HIV-1 genome organization highlighting the genomic location of HIV-1 primers used in RT-qPCR analysis. The cross (X) indicates a mutation that prevents env expression. (b) RT-qPCR in 10.6 Ctrl^KO and 10.6 Tat^KO cells prior to cell stimulation. Unpaired Student’s t-test, n = 3, −/+ SEM, *** p < 0.001. (c,d) RT-qPCR for HIV-1 initiation (c) and HIV-1 elongation (d) in 10.6 Ctrl^KO and 10.6 Tat^KO cells across the PMA time course. All data presented in these plots are normalized to the respective vehicle control for that cell line. Statistics are compared between 10.6 Ctrl^KO and 10.6 Tat^KO for each time point using an unpaired Student’s t-test, n = 3, −/+ SEM, ns = non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (e,f) RT-qPCR for HIV-1 initiation (e) and HIV-1 elongation (f) in 10.6 Ctrl^KO and 10.6 Tat^KO clones across the TNF-α time course. All data presented in these plots are normalized to the respective vehicle control for that cell line. Statistics are compared between 10.6 Ctrl^KO and 10.6 Tat^KO for each time point using an unpaired Student’s t-test, n = 3, −/+ SEM, ns = non-significant, ** p < 0.01, *** p < 0.001.

Figure 6

Tat sustains enhanced Pol II levels at the HIV-1 proviral genome upon cell stimulation. Pol II ChIP-qPCR using the HIV-1 elongation primer in the 10.6 Ctrl^KO and 10.6 Tat^KO clones across the PMA time course. Data presented are normalized to the percentage input. Statistics are compared between both clones for each time point using unpaired Student’s t-test, n = 2, −/+ SEM, ns = non-significant, * p < 0.05.