title: Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.
abstract: The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences.
authors.affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.
authors.forename: Luis
authors.initials: L
authors.lastname: Apolonia
authors.affiliation: Department of Medical and Molecular Genetics, King's College London, London, United Kingdom.
authors.forename: Reiner
authors.initials: R
authors.lastname: Schulz
authors.affiliation: Faculty of Computer and Information Science, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Tomaž
authors.initials: T
authors.lastname: Curk
authors.affiliation: Department of Statistical Science, University College London, London, United Kingdom.
authors.forename: Paula
authors.initials: P
authors.lastname: Rocha
authors.affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.
authors.forename: Chad M
authors.initials: CM
authors.lastname: Swanson
authors.affiliation: Department of Infectious Diseases, King's College London, London, United Kingdom.
authors.forename: Torsten
authors.initials: T
authors.lastname: Schaller
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom; Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, United Kingdom.
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Malim
mesh_terms: D000071478:APOBEC Deaminases; D000071480:APOBEC-3G Deaminase; D001324:Autoantigens / Q000378:metabolism; D002478:Cells, Cultured; D003564:Cytidine Deaminase / Q000378:metabolism; D043525:Cytosine Deaminase / Q000378:metabolism*; D057809:HEK293 Cells; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D020733:RNA, Small Cytoplasmic / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*; D000099207:SS-A Antigen
chemical_list: D000071478:APOBEC Deaminases; D000071480:APOBEC-3G Deaminase; D001324:Autoantigens; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase; D020733:RNA, Small Cytoplasmic; D058727:RNA, Small Untranslated; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle; C115990:7SL RNA; C116006:RO60 protein, human; D000099207:SS-A Antigen; C492906:APOBEC3F protein, human; C502889:APOBEC3 proteins, human; C464924:APOBEC3G protein, human