We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

Department of Biochemistry and Microbiology University of Chemistry and Technology 166 28 Prague Czech Republic

Department of Biotechnology University of Chemistry and Technology 166 28 Prague Czech Republic

Department of Informatics and Chemistry University of Chemistry and Technology 166 28 Prague Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences 142 20 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Research Centre and Gilead Sciences Czech Academy of Sciences 166 10 Prague Czech Republic

Zobrazit více v PubMed

Bourgeois C. F., Mortreux F., Auboeuf D., The multiple functions of RNA helicases as drivers and regulators of gene expression. Nat. Rev. Mol. Cell Biol. 17, 426–438 (2016). PubMed

Marecki J. C., Belachew B., Gao J., Raney K. D., RNA helicases required for viral propagation in humans. Enzymes 50, 335–367 (2021). PubMed PMC

Fairman-Williams M. E., Guenther U.-P., Jankowsky E., SF1 and SF2 helicases: Family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010). PubMed PMC

Singleton M. R., Dillingham M. S., Wigley D. B., Structure and mechanism of helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50 (2007). PubMed

Pyle A. M., Translocation and unwinding mechanisms of RNA and DNA helicases. Ann. Rev. Biophys. 37, 317–336 (2008). PubMed

Robert-Paganin J., et al. , Functional link between DEAH/RHA helicase Prp43 activation and ATP base binding. Nucleic Acids Res. 45, 1539–1552 (2017). PubMed PMC

Robert-Paganin J., Rety S., Leulliot N., Regulation of DEAH/RHA helicases by G-patch proteins. BioMed Res. Int. 2015, 931857 (2015). PubMed PMC

Tauchert M. J., Fourmann J.-B., Lührmann R., Ficner R., Structural insights into the mechanism of the DEAH-box RNA helicase Prp43. eLife 6, e21510 (2017). PubMed PMC

Hamann F., Enders M., Ficner R., Structural basis for RNA translocation by DEAH-box ATPases. Nucleic Acids Res. 47, 4349–4362 (2019). PubMed PMC

Bohnsack K. E., Ficner R., Bohnsack M. T., Jonas S., Regulation of DEAH-box RNA helicases by G-patch proteins. Biol. Chem. 402, 561–579 (2021). PubMed

De Bortoli F., Espinosa S., Zhao R., DEAH-box RNA helicases in pre-mRNA splicing. Trends Biochem. Sci. 46, 225–238 (2021). PubMed PMC

Linder P., Jankowsky E., From unwinding to clamping—The DEAD box RNA helicase family. Nat. Rev. Mol. Cell Biol. 12, 505–516 (2011). PubMed

Jarmoskaite I., Russell R., RNA helicase proteins as chaperones and remodelers. Annu. Rev. Biochem. 83, 697–725 (2014). PubMed PMC

Bohnsack K. E., Kanwal N., Bohnsack M. T., Prp43/DHX15 exemplify RNA helicase multifunctionality in the gene expression network. Nucleic Acids Res. 50, 9012–9022 (2022). PubMed PMC

Sloan K. E., Bohnsack M. T., Unravelling the mechanisms of RNA helicase regulation. Trends Biochem. Sci. 43, 237–250 (2018). PubMed

Fairman M. E., et al. , Protein displacement by DExH/D “RNA helicases” without duplex unwinding. Science 304, 730–734 (2004). PubMed

Jankowsky E., Gross C. H., Shuman S., Pyle A. M., Active disruption of an RNA-protein interaction by a DExH/D RNA helicase. Science 291, 121–125 (2001). PubMed

Aravind L., Koonin E. V., G-patch: A new conserved domain in eukaryotic RNA-processing proteins and type D retroviral polyproteins. Trends Biochem. Sci. 24, 342–344 (1999). PubMed

Bolinger C., Sharma A., Singh D., Yu L., Boris-Lawrie K., RNA helicase A modulates translation of HIV-1 and infectivity of progeny virions. Nucleic Acids Res. 38, 1686–1696 (2010). PubMed PMC

Studer M. K., Ivanovic L., Weber M. E., Marti S., Jonas S., Structural basis for DEAH-helicase activation by G-patch proteins. Proc. Natl. Acad. Sci. U.S.A. 117, 7159–7170 (2020). PubMed PMC

Hamann F., et al. , Structural analysis of the intrinsically disordered splicing factor Spp2 and its binding to the DEAH-box ATPase Prp2. Proc. Natl. Acad. Sci. U.S.A. 117, 2948–2956 (2020). PubMed PMC

Jeang K.-T., Yedavalli V., Role of RNA helicases in HIV-1 replication. Nucleic Acids Res. 34, 4198–4205 (2006). PubMed PMC

Singh G., Heng X., Boris-Lawrie K., “Chapter 7—Cellular RNA helicases support early and late events in retroviral replication” in Retrovirus-Cell Interactions, Parent L. J., Ed. (Academic Press, 2018), pp. 253–271, 10.1016/B978-0-12-811185-7.00007-8. DOI

Heaton S. M., Gorry P. R., Borg N. A., DExD/H-box helicases in HIV-1 replication and their inhibition. Trends Microbiol. 31, 393–404 (2023). PubMed

Chen C.-Y., Liu X., Boris-Lawrie K., Sharma A., Jeang K.-T., Cellular RNA helicases and HIV-1: Insights from genome-wide, proteomic, and molecular studies. Virus Res. 171, 357–365 (2013). PubMed PMC

Kohoutova Z., et al. , The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer monkey virus. Virology 384, 59–68 (2009). PubMed PMC

Zabransky A., et al. , Three active forms of aspartic proteinase from Mason-Pfizer monkey virus. Virology 245, 250–256 (1998). PubMed

Rumlova-Klikova M., Hunter E., Nermut M. V., Pichova I., Ruml T., Analysis of Mason-Pfizer monkey virus gag domains required for capsid assembly in bacteria: Role of the N-terminal proline residue of CA in directing particle shape. J. Virol. 74, 8452–8459 (2000). PubMed PMC

Barabas O., et al. , dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency. J. Biol. Chem. 278, 38803–38812 (2003). PubMed

Bauerova-Zabranska H., et al. , The RNA binding G-patch domain in retroviral protease is important for infectivity and D-type morphogenesis of Mason-Pfizer monkey virus. J. Biol. Chem. 280, 42106–42112 (2005). PubMed

Křížová I., et al. , The G-patch domain of Mason-Pfizer monkey virus is a part of reverse transcriptase. J. Virol. 86, 1988–1998 (2012). PubMed PMC

Roy B. B., et al. , Association of RNA helicase a with human immunodeficiency virus type 1 particles. J. Biol. Chem. 281, 12625–12635 (2006). PubMed

Schur F. K., et al. , Structure of the immature HIV-1 capsid in intact virus particles at 8.8 A resolution. Nature 517, 505–508 (2015). PubMed

Memet I., Doebele C., Sloan K. E., Bohnsack M. T., The G-patch protein NF-kappaB-repressing factor mediates the recruitment of the exonuclease XRN2 and activation of the RNA helicase DHX15 in human ribosome biogenesis. Nucleic Acids Res. 45, 5359–5374 (2017). PubMed PMC

Niu Z., Jin W., Zhang L., Li X., Tumor suppressor RBM5 directly interacts with the DExD/H-box protein DHX15 and stimulates its helicase activity. FEBS Lett. 586, 977–983 (2012). PubMed

Yoshimoto R., Kataoka N., Okawa K., Ohno M., Isolation and characterization of post-splicing lariat-intron complexes. Nucleic Acids Res. 37, 891–902 (2009). PubMed PMC

Tanaka N., Aronova A., Schwer B., Ntr1 activates the Prp43 helicase to trigger release of lariat-intron from the spliceosome. Genes. Dev. 21, 2312–2325 (2007). PubMed PMC

Lebaron S., et al. , The ATPase and helicase activities of Prp43p are stimulated by the G-patch protein Pfa1p during yeast ribosome biogenesis. EMBO J. 28, 3808–3819 (2009). PubMed PMC

Chen Y. L., et al. , The telomerase inhibitor Gno1p/PINX1 activates the helicase Prp43p during ribosome biogenesis. Nucleic Acids Res. 42, 7330–7345 (2014). PubMed PMC

Uversky V. N., Intrinsically disordered proteins and their environment: Effects of strong denaturants, temperature, pH, counter ions, membranes, binding partners, osmolytes, and macromolecular crowding. Protein J. 28, 305–325 (2009). PubMed

Castoralova M., et al. , A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein. eLife 13, e93489 (2024). PubMed PMC

Heininger A. U., et al. , Protein cofactor competition regulates the action of a multifunctional RNA helicase in different pathways. RNA Biol. 13, 320–330 (2016). PubMed PMC

Fouraux M. A., et al. , The human La (SS-B) autoantigen interacts with DDX15/hPrp43, a putative DEAH-box RNA helicase. RNA 8, 1428–1443 (2002). PubMed PMC

Dostalkova A., et al. , Mutations in the basic region of the Mason-Pfizer monkey virus nucleocapsid protein affect reverse transcription, genomic RNA packaging, and the virus assembly site. J. Virol. 92, e00106-18 (2018). PubMed PMC

Rao S., Mahmoudi T., DEAD-ly affairs: The roles of DEAD-box proteins on HIV-1 viral RNA metabolism. Front. Cell Dev. Biol. 10, 917599 (2022). PubMed PMC

Roy B. B., et al. , Association of RNA helicase A with human immunodeficiency virus type 1 particles*. J. Biol. Chem. 281, 12625–12635 (2006). PubMed

Sharma A., Boris-Lawrie K., Determination of host RNA helicases activity in viral replication. Methods Enzymol. 511, 405–435 (2012). PubMed PMC

Müller T. G., Zila V., Müller B., Kräusslich H.-G., Nuclear capsid uncoating and reverse transcription of HIV-1. Ann. Rev. Virol. 9, 261–284 (2022). PubMed

Veverka V., et al. , Three-dimensional structure of a monomeric form of a retroviral protease. J. Mol. Biol. 333, 771–780 (2003). PubMed

Hanson H. M., Willkomm N. A., Yang H., Mansky L. M., Human retrovirus genomic RNA packaging. Viruses 14, 1094 (2022). PubMed PMC

Pocock G. M., Becker J. T., Swanson C. M., Ahlquist P., Sherer N. M., HIV-1 and M-PMV RNA nuclear export elements program viral genomes for distinct cytoplasmic trafficking behaviors. PLoS Pathog. 12, e1005565 (2016). PubMed PMC

Maldonado Rebecca J. K., et al. , Visualizing association of the retroviral gag protein with unspliced viral RNA in the nucleus. mBio 11, e00524-20 (2020), 10.1128/mbio.00524-00520. PubMed DOI PMC

Scheifele L. Z., Garbitt R. A., Rhoads J. D., Parent L. J., Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein. Proc. Natl. Acad. Sci. U.S.A. 99, 3944–3949 (2002). PubMed PMC

Garbitt-Hirst R., Kenney Scott P., Parent Leslie J., Genetic evidence for a connection between Rous Sarcoma Virus Gag nuclear trafficking and genomic RNA packaging. J. Virol. 83, 6790–6797 (2009). PubMed PMC

Sfakianos J. N., LaCasse R. A., Hunter E., The M-PMV cytoplasmic targeting-retention signal directs nascent Gag polypeptides to a pericentriolar region of the cell. Traffic 4, 660–670 (2003). PubMed

Pasquinelli A. E., et al. , The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J. 16, 7500–7510 (1997). PubMed PMC

Li Y., et al. , An intron with a constitutive transport element is retained in a Tap messenger RNA. Nature 443, 234–237 (2006). PubMed

Edwards C. R., et al. , A dynamic intron retention program in the mammalian megakaryocyte and erythrocyte lineages. Blood 127, e24–e34 (2016). PubMed PMC

Dvinge H., Bradley R. K., Widespread intron retention diversifies most cancer transcriptomes. Genome Med. 7, 45 (2015). PubMed PMC

Müller B., et al. , Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J. Virol. 78, 10803–10813 (2004). PubMed PMC

Obr M., et al. , Stabilization of the beta-hairpin in Mason-Pfizer monkey virus capsid protein—A critical step for infectivity. Retrovirology 11, 94 (2014). PubMed PMC

Rumlova M., et al. , Breast cancer-associated protein—A novel binding partner of Mason-Pfizer monkey virus protease. J. Gen. Virol. 95, 1383–1389 (2014). PubMed

Hadravova R., et al. , In vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus XMRV. J. Virol. 86, 1297–1306 (2012). PubMed PMC

Hadravova R., Rumlova M., Ruml T., FAITH—Fast assembly inhibitor test for HIV. Virology 486, 78–87 (2015). PubMed

Vranken W. F., et al. , The CCPN data model for NMR spectroscopy: Development of a software pipeline. Proteins 59, 687–696 (2005). PubMed

Rumlova M., Krizova I., Zelenka J., Weber J., Ruml T., Does BCA3 play a role in the HIV-1 replication cycle? Viruses 10, 212 (2018). PubMed PMC

Diehl W. E., Stansell E., Kaiser S. M., Emerman M., Hunter E., Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells. J. Virol. 82, 11140–11151 (2008). PubMed PMC

Wang G. Z., Goff S. P., Postentry restriction of Mason-Pfizer monkey virus in mouse cells. J. Virol. 89, 2813–2819 (2015). PubMed PMC

Kutluay S. B., Bieniasz P. D., Analysis of HIV-1 Gag-RNA Interactions in cells and virions by CLIP-seq. Methods Mol. Biol. 1354, 119–131 (2016). PubMed PMC

Danecek P., et al. , Twelve years of SAMtools and BCFtools. GigaScience 10, giab008 (2021). PubMed PMC

Junková P., Rumlová M., Data from “Proteomic analysis of Mason-Pfizer monkey virus virions, project accession: PXD046672”. PRIDE. http://www.ebi.ac.uk/pride/archive/projects/PXD046672. Deposited 5 November 2023.