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.

Analysis of the human immunodeficiency virus-1 RNA packageome

“…Together with reports that 7SL is packaged by HIV-1 without SRP54 (OnafuwaNuga et al 2006), and that overexpression of SRP19 prevents 7SL encapsidation Bach et al 2008;Apolonia et al 2015), our finding that HIV-1 selectively packages pre-snRNAs, nascent rDNA transcripts, and pretRNAs indicates that HIV-1, similar to MLV (Eckwahl et al 2015), preferentially encapsidates newly made ncRNAs.…”

“…Together with reports that 7SL RNA is recruited by both MLV and HIV-1 before assembling into mature SRP (Onafuwa-Nuga et al 2006;Wang et al 2007;Bach et al 2008;Apolonia et al 2015;Eckwahl et al 2015) and that many nascent ncRNAs are enriched in MLV virions (Garcia et al 2009;Eckwahl et al 2015), our data suggest that the encapsidation of newly made RNAs may be a general property of retroviruses. One theme that emerges from these studies is that forms of RNAs that are rare and difficult to detect in cells are strikingly enriched in virions.…”

“…Because several of these ncRNAs were detected previously in HIV-1 virions produced by transfecting HEK293T cells with proviral clones (Houzet et al 2007;Khan et al 2007;Tian et al 2007;Bach et al 2008;Apolonia et al 2015), we determined whether encapsidation of the RNAs increased under these conditions. After transfecting HEK293T cells with pNL4-3 and purifying the resulting virus, Northern blotting revealed that vtRNA1-1 and snaR-A were both more selectively packaged in these virions than in CEM-SS cell-derived virions (Fig.…”

“…4) underscores the selectivity of their virion packaging. Moreover, if 7SL RNA is indeed recruited into virions before assembling with most core SRP proteins (Onafuwa-Nuga et al 2006;Wang et al 2007;Bach et al 2008;Apolonia et al 2015;Eckwahl et al 2015), retroviruses encapsidate a SRP biogenesis intermediate that has not otherwise been detected in cells.…”

“…Similarly, although 7SL, Y RNAs, and vault RNAs are all highly enriched in MLV virions, their protein partners (SRP19, Ro60, and TEP1, respectively) were not detected (Garcia et al 2009;Eckwahl et al 2015). Packaging of 7SL may occur before SRP19 binding, since overexpressing SRP19, the subunit that stabilizes a 7SL structure required for SRP54 association, significantly reduces 7SL packaging by HIV-1 Bach et al 2008;Apolonia et al 2015). Moreover, although Y RNAs are unstable in cells lacking Ro60, Y RNA packaging by MLV remains efficient in these cells (Garcia et al 2009), suggesting MLV recruits these RNAs prior to Ro60 binding.…”