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

Abstract

33Gag synthesis from the full-length unspliced mRNA is critical for the production of the 34 viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While 35 most spliced mRNAs follow the canonical gene expression pathway in which the 36 recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex 37 (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA 38 relies on the viral protein Rev to reach the cytoplasm and recruit the host translational 39 machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving 40 nuclear export and translation of the unspliced mRNA. These functions of Rev are 41 supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the 42 nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box 43 RNA helicase eIF4AI, which translocates to the nucleus and cooperates with Rev to 44 promote Gag synthesis. Interestingly, molecular docking analyses revealed the assembly of 45 a Rev-CBP80-eIF4AI complex that is organized around the Rev response element (RRE). 46 Together, our results provide further evidence towards the understanding of the molecular 47 mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 48 replication. 49 50 51 52 53 54 55 56 57 58 59 60 61 62Human Immunodeficiency Virus type-1 (HIV-1) gene expression is a complex process that 64 leads to the synthesis of fifteen proteins from one single primary transcript (1,2). Once the 65 proviral DNA has been integrated into the host cell genome, the RNA polymerase II drives 66 the synthesis of a 9-kb, capped and polyadenylated pre-mRNA that undergoes alternative 67 splicing generating more than 100 different transcripts classified into three main 68 populations (3,4). The so-called 2-kb multiply spliced transcripts code for the key 69 regulatory proteins Tat and Rev and the accessory protein Nef and are the dominant viral 70 mRNA species at early stages of viral gene expression (1,2,5). Unlike cellular mRNAs or 71 the 2-kb transcripts, which are spliced to completion before they exit the nucleus, HIV-1 72 and other complex retroviruses produce an important fraction of viral transcripts that 73 remain incompletely spliced (2,6). These 4-kb transcripts are expressed during the 74 intermediate phase of gene expression and are used for the synthesis of the envelope 75 glycoprotein (Env) and the accessory proteins Vif, Vpr and Vpu (2,6). Finally, the full-76 length 9-kb pre-mRNA in its unspliced form also reaches the cytoplasm to be used as an 77 mRNA template during the late stages of viral gene expression for the synthesis of the 78 major structural proteins Gag and Gag-Pol (1,2,6). 79Gene expression in eukaryotic cells occurs through the intricate connection of different 80 processes including transcription, splicing, nuclear export, translation and mRNA decay 81 and is regulated by the specific recruitment of nuclear proteins that together form the 82 messenger ribonuc...