Characterization of APOBEC3G binding to 7SL RNA

Abstract

Human APOBEC3 proteins are editing enzymes that can interfere with the replication of exogenous retroviruses such as human immunodeficiency virus (HIV), hepadnaviruses such as hepatitis B virus (HBV), and with the retrotransposition of endogenous retroelements such as longinterspersed nuclear elements (LINE) and Alu. Here, we show that APOBEC3G, but not other APOBEC3 family members, binds 7SL RNA, the common ancestor of Alu RNAs that is specifically recruited into HIV virions. Our data further indicate that APOBEC3G recognizes 7SL RNA and Alu RNA by its common structure, the Alu domain, suggesting a mechanism for APOBEC3G-mediated inhibition of Alu retrotransposition. However, we also demonstrate that APOBEC3F and APOBEC3G are normally recruited into and inhibit the infectivity of ΔVif HIV1 virions when 7SLRNA is prevented from accessing particles by RNA interference against SRP14 or by over expression of SRP19, both components of the signal recognition particle. We thus conclude that 7SL RNA is not an essential mediator of the virion packaging of these antiviral cytidine deaminases.

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.…”

“…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.…”

HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants

“…However, the P129D mutation confers resistance to Vif2, perhaps by inducing a conformational change that alters the structure of another distal domain that interacts with Vif2. To further analyze the region surrounding D128 in A3G, we determined the sensitivity of the A3G-W127A mutant, which has been shown to be deficient in virion incorporation (59)(60)(61). Because this mutant lacks antiviral activity, we tested the ability of Vif1 and Vif2 to induce the degradation of A3G-W127A by Western blotting (Fig.…”