A Rev–CBP80–eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA

Abstract

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with the viral protein to promote Gag synthesis. Finally, we show that the Rev/RRE axis is important for the assembly of a CBP80-eIF4AI complex onto the unspliced mRNA. Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.

An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging

“…Since results presented above indicate that m 6 A deposition by METTL3/14 affects the cytoplasmic fate of the full-length 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 full-length RNA and METTL3 by in situ hybridization coupled to the proximity ligation assay (ISH-PLA) 24 . Confocal microscopy analyses revealed a predominant interaction within the nucleus, which suggests that the full-length RNA must be methylated in the nucleus and reach the cytoplasm in a methylated form (Fig.…”

“…HEK293T and HeLa cells were maintained in DMEM (Life technologies) supplemented with 10% FBS (Hyclone) and antibiotics (Hyclone) at 37°C and 5% CO 2 atmosphere. Cells growing in 6-well plates (2.5×10 5 cells/well) were transfected using linear PEI ~25000 Da (Polyscience) as described previously 24 Cells were transfected using a ratio μg DNA/μl PEI of 1/15 and the DNA/PEI mix was incubated for 20 min at room temperature before adding to the cells. For experiments involving the FTO inhibitor, the culture medium was replaced by medium containing dimethyl sulfoxide (DMSO) as a control or 80 μM of an ethyl ester form of meclofenamic acid diluted in DMSO (MA2) prior DNA transfection.…”

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

“…The pNL4.3-ΔEnv, pNL4.3R, pNL4.3R-ΔRev, pROD10R and pNCAC proviral vectors were previously described (18,29,30). The pcDNA3-Flag-CTIF, pcDNA3-Flag-CTIF(1-305) and pcDNA3-Flag-CTIF(306-598) were previously described (25).…”

“…The pcDNA3-Flag-CTIF, pcDNA3-Flag-CTIF(1-305) and pcDNA3-Flag-CTIF(306-598) were previously described (25). The pcDNA-d2EGFP, pCDNA β-globin 5’-UTR, pCIneo-Renilla and pEGFP-Rev were previously described (18,29).…”

“…The Jurkat clone E6-1 was maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS (Pan-Biotech) and antibiotics (Sigma-Aldrich) at 37 °C and a 5% CO2 atmosphere. Cells were transfected using linear PEI ∼25,000 Da (Polysciences) as described previously (18,29).…”