Abstract
The mechanisms involved in the posttranscriptional control of the replicative cycle of the human immunodeficiency virus (HIV), specifically the molecular events which allow the interaction between the viral genomic RNA (gRNA) and the cellular machinery for the transport, translation, or intracellular packaging, have not been yet elucidated. In this chapter, we describe the in situ hybridization-proximity ligation assay (ISH-PLA) to characterize interactions between the genomic RNA (gRNA) of HIV-1 and viral proteins or host proteins involved in nuclear export and translation initiation. We also present data that validate the ISH-PLA as a simple and useful tool to study HIV-1 gRNA-protein interactions within cells.
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References
Coffin JM, Hughes SH, Varmus H (1997) Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, NY
Fields BN, Knipe DM, Howley PM (2013) Fields virology, 6th edn. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia
Brass AL, Dykxhoorn DM, Benita Y et al (2008) Identification of host proteins required for HIV infection through a functional genomic screen. Science 319:921–926
Bushman FD, Malani N, Fernandes J et al (2009) Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 5:e1000437
Konig R, Zhou Y, Elleder D et al (2008) Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 135:49–60
Ramanathan M, Porter DF, Khavari PA (2019) Methods to study RNA-protein interactions. Nat Methods 16:225–234
Soderberg O, Gullberg M, Jarvius M et al (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3:995–1000
Fredriksson S, Gullberg M, Jarvius J et al (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20:473–477
Toro-Ascuy D, Rojas-Araya B, Garcia-de-Gracia F et al (2018) A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA. Nuclei Acid Res 46:11539–11552
García-de-Gracia F, Toro-Ascuy D, Riquelme-Barrios S et al (2019) CBP80/20-dependent translation initiation factor (CTIF) inhibits HIV-1 Gag synthesis by targeting the function of the viral protein Rev. bioRxiv:710137
Pereira-Montecinos C, Toro-Ascuy D, Rojas-Fuentes C et al (2019) An epitranscriptomic switch at the 5′-UTR controls genome selection during HIV-1 genomic RNA packaging. bioRxiv:676031
Soto-Rifo R, Rubilar PS, Ohlmann T (2013) The DEAD-box helicase DDX3 substitutes for the cap-binding protein eIF4E to promote compartmentalized translation initiation of the HIV-1 genomic RNA. Nucleic Acids Res 41:6286–6299
Reed SE, Staley EM, Mayginnes JP et al (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98
Pillai RS, Artus CG, Filipowicz W (2004) Tethering of human Ago proteins to mRNA mimics the miRNA-mediated repression of protein synthesis. RNA 10:1518–1525
Rajanala K, Nandicoori VK (2012) Localization of nucleoporin Tpr to the nuclear pore complex is essential for Tpr mediated regulation of the export of unspliced RNA. PLoS One 7:e29921
Soto Rifo R, Ricci EP, Decimo D et al (2007) Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation. Nucleic Acids Res 35:e121
Acknowledgments
This work was supported by grants from CONICYT Chile through the FONDECYT 11180621 (D.T-A.) and FONDECYT 1180798 (F.V-E) and CONICYT grant no. 21181508 (AG-A).
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Toro-Ascuy, D., Gaete-Argel, A., Rojas-Celis, V., Valiente-Echeverria, F. (2021). In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to Study the Interaction of HIV-1 RNA and Remodeling Proteins. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 2209. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0935-4_19
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DOI: https://doi.org/10.1007/978-1-0716-0935-4_19
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