A Virus-Packageable CRISPR System Identifies Host Dependency Factors Co-Opted by Multiple HIV-1 Strains

Abstract

With a small genome of ~9.2 kb that encodes 14 major proteins, HIV must hijack host cellular machinery to successfully establish infection. These host proteins necessary for HIV replication are called “dependency factors.” Whole-genome, and then targeted screens were done to try to comprehensively identify all dependency factors acting throughout the HIV replication cycle.

A CRISPR Screen of HIV Dependency Factors Reveals That CCNT1 Is Non-Essential in T Cells but Required for HIV-1 Reactivation from Latency

“…The HIV-Dep library containing 525 genes (4191 sgRNAs) was previously described [ 17 ]. For the transduction of J-Lat cells, HEK293T cells were seeded in 20 × 6 well cell culture plates, transfected with the HIV-DEP plasmid (667 ng), psPax2 (GagPol, Addgene, 12260; 500 ng), and MD2.G (VSVG, Addgene, 12259; 333 ng) per well in 200 μL of serum-free DMEM (Thermo Fisher Scientific, Grand Island, NY, USA, 11965092) along with 4.5 μL of TransIT-LT1 reagent (Mirus Bio LLC, Madison, WI, USA, MIR2305).…”

“…vRNA and gDNA were both amplified by PCR using R1_forward primer and R1_Reverse primer using Herculase II Fusion DNA Polymerase (Agilent, Santa Clara, CA, USA, 600677). PCR products were cleaned up using the QIAquick PCR clean-up kit (Qiagen, Germantown, MD, USA, 28104), and a second round of PCR was performed using R2_reverse primer and R2_IndexX primer (described in [ 12 , 17 , 19 ]). The 230 bp band was verified to be present and the amplified PCR products were cleaned up using double-sided SPRI via AMPure Beads (Beckman Coulter, Indianapolis, IN, USA, A63880).…”

“…Here, we performed a CRISPR-Cas9 screen using the Latency HIV-CRISPR technique [ 12 ] for factors necessary for HIV-1 to be released from latency in the presence of a combination of LRAs. We used a custom CRISPR guide library, called the HIV dependency factor gene library (HIV-Dep), that had been previously used to identify novel host dependency factors across multiple HIV strains [ 17 ]. We identified and validated factors important in latency reactivation including ELL1, TBL1XR1, UBE2M, HDAC3, AMBRA1, and ALYREF.…”

HIV-1 transcriptional modulation: novel host factors and prospective therapeutic strategies

“…Furthermore, inhibition of the TRIM24-C terminal bromodomain using the small molecule IACS-9571 [46,47], in combination with the PKC agonist PEP005 [48], promotes HIV-1 reactivation in primary CD4 + lymphocytes, supporting their potential use as latency reversing agents (LRAs) [49 ▪ ]. A CRISPR/Cas genome-wide screen in Jurkat T cells identified novel host factors and pathways contributing to HIV expression [50], including UBE2 M, FBXW7, SLC39A7, or ING3 [50,51 ▪ ], but their specific roles in the HIV-1 life cycle remains to be elucidated. In summary, numerous approaches are being used to gain insight into host protein involved in HIV transcriptional regulation, and additional work is needed towards depth in their mechanism of action in cells and tissues.…”

Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

“…Three days later, the cultures are examined for enrichment of specific sgRNAs in HIV-1 virions compared to their frequency in genomic DNA by RT-PCR/PCR and deep sequencing 26 . The initial screen identified IFN-induced antiviral factors 26 and has subsequently been modified to identify cellular that promote HIV-1 replication 27 , restrict HIV-1 by targeting the viral capsid 28 , or affect viral latency 29 . In addition, it has been used to identify ISGs restricting HIV-1 in primary CD4 + T cells 30 .…”