CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev

Abstract

Translation initiation of the human immunodeficiency virus type-1 (HIV-1) unspliced mRNA has been shown to occur through cap-dependent and IRES-driven mechanisms. Previous studies suggested that the nuclear cap-binding complex (CBC) rather than eIF4E drives cap-dependent translation of the unspliced mRNA and we have recently reported that the CBC subunit CBP80 supports the function of the viral protein Rev during nuclear export and translation of this viral transcript. Ribosome recruitment during CBC-dependent translation of cellular mRNAs relies on the activity CBP80/20 translation initiation factor (CTIF), which bridges CBP80 and the 40S ribosomal subunit through interactions with eIF3g. Here, we report that CTIF restricts HIV-1 replication by interfering with Gag synthesis from the unspliced mRNA. Our results indicate that CTIF associates with Rev through its N-terminal domain and is recruited onto the unspliced mRNA ribonucleoprotein complex in order to block translation. We also demonstrate that CTIF induces the cytoplasmic accumulation of Rev impeding the association of the viral protein with CBP80. We finally show that CTIF restricts HIV-2 but not MLV Gag synthesis indicating an inhibitory mechanism conserved in Rev-expressing human lentiviruses..

Epitranscriptomic regulation of HIV-1 full-length RNA packaging

“…Since results presented above suggest that m 6 A deposition by METTL3/14 affects the cytoplasmic fate of the FL-RNA, we wanted to investigate where within the cell the m 6 A writer complex modifies the viral RNA. For this, we analyzed the interaction between the FL-RNA and METTL3 by in situ hybridization coupled to the proximity ligation assay (ISH-PLA), which we have successfully used to quantify FL-RNA-protein interactions in intact cells ( 27 , 37 ). Confocal microscopy analyses revealed a predominant interaction within the nucleus, which suggests that the FL-RNA must be methylated in the nucleus and reach the cytoplasm in a methylated form (Figure 1E and Supplementary Figure S1E ).…”

“…The ISH-PLA protocol was developed by mixing the RNA-FISH and PLA protocols described above essentially as we have previously reported ( 37 ). Briefly, HeLa cells transfected with pNL4.3 and pCDNA-Flag-METTL3 or pCDNA-3XFlag-FTO growing on coverslips were fixed, permeabilized for 10 min at RT with 0.2% Triton X-100 and hybridized overnight at 37ºC in 200 μl of hybridization mix (10% dextran sulphate, 2 mM vanadyl–ribonucleoside complex, 0.02% RNase-free bovine serum albumin, 50% formamide, 300 μg of tRNA and 120 ng of 11-digoxigenin-UTP probes) in a humid chamber.…”

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency

“…One other protein potentially involved in HIV-1 latency is host restriction factor SLFN11, that, as mentioned before, prevents the synthesis of viral proteins and has been found to correlate with HIV-1 persistance in ART-suppressed PLWH ( Abdel-Mohsen et al., 2015 ). Besides, as discussed above, viral protein Rev and its co-factors, for example DDX3 or CBP80/20, have a very prominent role in HIV RNA translation ( Figure 2C ) ( Arrigo and Chen, 1991 ; Perales et al., 2005 ; Soto-Rifo et al., 2012 ; Frohlich et al., 2016 ; Garcia-de-Gracia et al., 2021 ). Thus, regulation of Rev production and DDX3-mediated translation of viral RNAs are important mechanisms involved in latency at this level.…”

Anomalous HIV-1 RNA, How Cap-Methylation Segregates Viral Transcripts by Form and Function

“…Notably, CTIF-CBC mRNPs released to the cytoplasm were shown to form eIF4E-independent polysomes, demonstrating the translatability of CBC-bounded mRNAs. The CTIF-DDX19B interaction was shown to play an inhibitory role in nonsense mediated decay [ 98 , 99 , 100 ]. CTIF is an inhibitor of HIV Gag synthesis [ 99 ], which may also be attributable to perinuclear trapping [ 98 ].…”

“…The CTIF-DDX19B interaction was shown to play an inhibitory role in nonsense mediated decay [ 98 , 99 , 100 ]. CTIF is an inhibitor of HIV Gag synthesis [ 99 ], which may also be attributable to perinuclear trapping [ 98 ].…”