Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency

Abstract

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replicationcompetent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a wholegenome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4 + cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.

SARS-CoV-2 spike variants exhibit differential infectivity and neutralization resistance to convalescent or post-vaccination sera

“…Generation of HEK-hACE2 stable cell line hACE2 (received from S. Pohlmann lab, University Gö ttingen, Germany) was re-cloned into lentiviral expression vector. Lentiviral particles were produced as described previously (Krasnopolsky et al, 2020) Briefly, HEK293T cells were stably transduced with lentivirus expressing ACE2. Cells were analyzed for hACE2 expression by FACS, using biotinylated-labeled spike (ACROBiosystems).…”

“…Transfections were done in a 10cm format, as previously described and supernatant containing virus were harvested 72 h post transfection, filtered and stored at À80 C (Krasnopolsky et al, 2020). Neutralization assays were performed in a 96 well format, in the presence of pseudotyped viruses that were incubated with increasing dilutions of the tested sera (1:2000; 1:8000: 1;32000: 1:128000) or without sera as a control.…”

SARS CoV-2 escape variants exhibit differential infectivity and neutralization sensitivity to convalescent or post-vaccination sera

“…Briefly, LTR-PGK luciferase lentivector was transfected into cells together with other lentiviral expression plasmids coding for either Gag, Pol Tat Rev, and the corresponding wild type or mutate S envelopes. Transfections were done in a 10cm format, as previously described(Krasnopolsky et al, 2020).…”

Experimental Systems for Measuring HIV Latency and Reactivation

“…This maintains viral latency by keeping these cells in a transcriptionally silent state. Multiple cellular signaling pathways can control HIV transcription positively: positive transcription elongation factor b (P-TEFb), the nuclear factor kappa B (NFκB) pathway [ 16 ], the nuclear factor of activated T cells (NFAT) pathway [ 17 ], the mitogen-activated protein kinase (MAPK) pathway [ 18 ], the toll-like receptor (TLR) pathway [ 19 ], and the mammalian target of rapamycin (mTOR) pathway [ 20 ]); as well as negatively: via recruitment of the polycomb repressive complex (PRC) [ 21 , 22 , 23 ], the Cullin 3 E3 ligase pathway [ 24 ], and the zinc-finger protein 304 [ 25 ]. The proviral 5′-LTR is an inducible promoter containing a TATA-box and upstream enhancer binding regions [ 26 ].…”