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A Rev-CBP80-Eif4ai Complex Drives Gag Synthesis from the HIV-1

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A Rev-CBP80-Eif4ai Complex Drives Gag Synthesis from the HIV-1 bioRxiv preprint doi: https://doi.org/10.1101/313312; this version posted May 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA 2 3 Daniela Toro-Ascuy1#; Bárbara Rojas-Araya1#; Francisco García-de-Gracia1#; Cecilia 4 Rojas-Fuentes1; Camila Pereira-Montecinos1; Aracelly Gaete-Argel1; Fernando Valiente- 5 Echeverría1; Théophile Ohlmann2,3 and Ricardo Soto-Rifo1* 6 7 1 Laboratory of Molecular and Cellular Virology, Virology Program, Institute of 8 Biomedical Sciences, Universidad de Chile Faculty of Medicine, Independencia 834100, 9 Santiago, Chile 10 2 INSERM U1111, CIRI, Lyon, F-69364 France. 11 3 Ecole Normale Supérieure de Lyon, Lyon, F-69364 France. 12 13 14 * To whom correspondence should be addressed. 15 RSR: Tel: (56) 2 978 68 69; Fax: (56) 2 978 61 24; Email: [email protected] 16 17 # The authors wish it to be known that, in their opinion, the first three authors should be 18 regarded as joint First Authors 19 20 21 Running title: Role of Rev in HIV-1 gene expression 22 23 24 Keywords: HIV-1 unspliced mRNA, Rev, CBP80, eIF4AI, gene expression 25 26 27 28 29 30 31 bioRxiv preprint doi: https://doi.org/10.1101/313312; this version posted May 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 33 Abstract 34 Gag synthesis from the full-length unspliced mRNA is critical for the production of the 35 viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While 36 most spliced mRNAs follow the canonical gene expression pathway in which the 37 recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex 38 (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA 39 relies on the viral protein Rev to reach the cytoplasm and recruit the host translational 40 machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving 41 nuclear export and translation of the unspliced mRNA. These functions of Rev are 42 supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the 43 nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box 44 RNA helicase eIF4AI, which translocates to the nucleus and cooperates with Rev to 45 promote Gag synthesis. Interestingly, molecular docking analyses revealed the assembly of 46 a Rev-CBP80-eIF4AI complex that is organized around the Rev response element (RRE). 47 Together, our results provide further evidence towards the understanding of the molecular 48 mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 49 replication. 50 51 52 53 54 55 56 57 58 59 60 61 62 bioRxiv preprint doi: https://doi.org/10.1101/313312; this version posted May 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 63 Introduction 64 Human Immunodeficiency Virus type-1 (HIV-1) gene expression is a complex process that 65 leads to the synthesis of fifteen proteins from one single primary transcript (1,2). Once the 66 proviral DNA has been integrated into the host cell genome, the RNA polymerase II drives 67 the synthesis of a 9-kb, capped and polyadenylated pre-mRNA that undergoes alternative 68 splicing generating more than 100 different transcripts classified into three main 69 populations (3,4). The so-called 2-kb multiply spliced transcripts code for the key 70 regulatory proteins Tat and Rev and the accessory protein Nef and are the dominant viral 71 mRNA species at early stages of viral gene expression (1,2,5). Unlike cellular mRNAs or 72 the 2-kb transcripts, which are spliced to completion before they exit the nucleus, HIV-1 73 and other complex retroviruses produce an important fraction of viral transcripts that 74 remain incompletely spliced (2,6). These 4-kb transcripts are expressed during the 75 intermediate phase of gene expression and are used for the synthesis of the envelope 76 glycoprotein (Env) and the accessory proteins Vif, Vpr and Vpu (2,6). Finally, the full- 77 length 9-kb pre-mRNA in its unspliced form also reaches the cytoplasm to be used as an 78 mRNA template during the late stages of viral gene expression for the synthesis of the 79 major structural proteins Gag and Gag-Pol (1,2,6). 80 Gene expression in eukaryotic cells occurs through the intricate connection of different 81 processes including transcription, splicing, nuclear export, translation and mRNA decay 82 and is regulated by the specific recruitment of nuclear proteins that together form the 83 messenger ribonucleoprotein (mRNP) complex (7-9). As such, the early binding of the 84 nuclear cap-binding complex (CBC) to the 5´-end cap structure and the splicing-dependent 85 recruitment of nuclear proteins such as the exon-junction complex (EJC) onto the mRNA 86 has been shown to increase the rates of nuclear export and translation of spliced transcripts 87 (10-20). Eukaryotic cells have also evolved quality control mechanisms ensuring that only 88 properly processed mRNAs reach the cytoplasm and are decoded by the translational 89 machinery. These mechanisms include the EJC-dependent degradation of transcripts 90 containing premature stop codons through nonsense-mediated decay (NMD) or the NXF1- 91 dependent nuclear retention of unspliced transcripts mediated by the nucleoporin Tpr (21- 92 25). Consistent with these cellular quality control mechanisms, it has been widely reported 93 that viral intron-containing transcripts including the 4-kb and the 9-kb mRNA produced bioRxiv preprint doi: https://doi.org/10.1101/313312; this version posted May 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 during HIV-1 replication are retained and degraded in the host cell nucleus unless the viral 95 protein Rev is present (26-30). As such, Rev has been proposed to promote HIV-1 gene 96 expression from its target transcripts by i) avoiding mRNA degradation (26,31); ii) 97 promoting nuclear export (31-33) or by iii) promoting translation (34,35). In this study, we 98 developed Rev mutant proviruses and confirmed that Rev is required for both nuclear 99 export and translation of the HIV-1 unspliced mRNA. Interestingly, we show that the 100 nuclear cap-binding complex subunit CBP80 and the translation initiation factor eIF4AI 101 associate with Rev and the unspliced mRNA to promote Gag synthesis. Molecular docking 102 analyses suggest the assembly of a Rev-CBP80-eIF4AI complex that is reorganized when 103 Rev binds to the RRE. Together, our work provides further insights into the molecular 104 mechanism by which Rev drives Gag synthesis from the HIV-1 unspliced mRNA. 105 106 Materials and methods 107 DNA constructs: The pNL4.3 and pNL4.3R proviruses were previously described (36,37). 108 These vectors were digested with NheI and subjected to a 20 min polymerization reaction at 109 72 ºC using the Phusion® High-Fidelity DNA polymerase (New England Biolabs) in order 110 to create a frameshift that generates a premature stop codon within the env gene. The 111 resulting vectors were ligated with the T4 DNA ligase and transformed into E.coli DH5α. 112 To create the pNL4.3-ΔRev and pNL4.3R-ΔRev vectors, the above vectors were digested 113 with BamHI and subjected to the same polymerization/ligation reaction to create a 114 frameshift within the rev gene previously shown to abolish expression of a functional 115 protein (28). The pCMV-NL4.3R and pCMV-NL4.3R-ΔRev vectors were obtained by 116 replacing the FspAI/BssHII fragment of the corresponding vector by the CMV IE promoter 117 amplified from the pCIneo vector (Promega) as we previously reported (38). The pCDNA- 118 Flag-Rev vector was previously described (22). pCDNA-d2EGFP vector was generated by 119 inserting the d2EGFP ORF into pCDNA3.1 (Life Technologies). pCDNA HIV-1 5`-UTR 120 and pCDNA β-globin 5`-UTR were previously described (39). The dl HIV-1 IRES vector 121 was previously described (40). The pCIneo-HA-eIF4GI, -eIF4A and -eIF4E were 122 previously described (41). The pCMV-myc-eIF4E and CBP80 were previously described 123 (42). 124 bioRxiv preprint doi: https://doi.org/10.1101/313312; this version posted May 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 125 Cell culture and DNA transfection: HeLa cells and human microglia (C20 cells)(43) were 126 maintained in DMEM (Life Technologies) supplemented with 10% FBS (Hyclone) and 127 antibiotics (Hyclone) at 37 ºC and a 5% CO2 atmosphere. H9 T-lymphocytes (44) and 128 THP-1ATCC monocytes (45) were maintained in RPMI 1640 (Life Technologies) 129 supplemented with 10% FBS (Hyclone) and antibiotics (Hyclone) at 37 ºC and a 5% CO2 130 atmosphere. Cells were transfected using linear PEI ~25.000 Da (Polysciences) prepared as 131 described previously (46). Cells were transfected using a ratio µg DNA/µl PEI of 1/15. 132 133 Analysis of Renilla and firefly activities: Renilla activity was determined using the Renilla 134 Reporter Assay System (Promega) and Renilla/firefly activities were determined using the 135 Dual Luciferase Reporter Assay System (Promega) in a GloMax® 96 microplate 136 luminometer (Promega).
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