A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin

Abstract

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74

“…As previously reported (40,41,51), CPSF6 depletion by itself did not largely affect HIV-1 infection, with an ϳ20% increase in WT virus infectivity being observed. However, we found that CPSF6 depletion counteracted the triphasic dose-response curve of the drug against the WT virus ( Fig.…”

“…Dulbecco modified Eagle medium (DMEM; Cellgro) supplemented with 10% fetal bovine serum (FBS; Cellgro) and 1ϫ penicillin-streptomycin (Pen-Str; Cellgro) was used to culture adherent cell lines. CPSF6 knockout HEK293T cells that carry an empty expression vector or express the 551-amino-acid isoform of CPSF6 (CPSF6[551]) were described previously (41). RPMI medium (Cellgro) supplemented with 10% FBS (Sigma), 1ϫ Pen-Str and 2 mM 2-glutamine (Cellgro) was used to culture an immortalized T cell line (MT4).…”

Structural basis for spumavirus GAG tethering to chromatin

“…Table S2. HIV-1 and MLV integration sites in HEK293T cells alongside the matched random control simulated data were from published work (11). HT1080, HEK293T, MRC5, or HepG2 cell line-specific gene-expression activity was reported previously (GEO accession codes GSE58968, GSE11892, GSE63577, and GSE87958) (54)(55)(56)(57).…”

“…Recent studies uncovered a hierarchical mechanism that depends on HIV-1 capsid, the major viral structural protein (a product of the viral gag gene), and integrase, which interact with cellular proteins CPSF6 and LEDGF/p75, respectively (6)(7)(8)(9)(10)(11). Ablation of the mRNA maturation factor CPSF6 results in the bulk of virus abandoning generich domains, whereas ablation of the transcriptional coactivator LEDGF leads to a reduction of integration into transcription units while largely preserving the virus's ability to locate gene-rich domains (11). Thus, the capsid-CPSF6 and integrase-LEDGF axes appear to direct HIV-1 integration on the global and local scale, respectively.…”

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein

“…SupT1 and CEM-SS cells were propagated in similarly supplemented RPMI 1640 medium. Viruses used for infectivity measures and transmission electron microscopy were generated by plasmid DNA transfection of HEK293T cells as previously described (58).…”