The Anti-HIV-1 Editing Enzyme APOBEC3G Binds HIV-1 RNA and Messenger RNAs That Shuttle between Polysomes and Stress Granules

Abstract

Deoxycytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family) have RNA-binding motifs, invade assembling human immunodeficiency virus (HIV-1), and hypermutate reverse transcripts. Antagonistically, HIV-1 viral infectivity factor degrades these enzymes. A3G is enzymatically inhibited by binding RNA within an unidentified large cytosolic ribonucleoprotein, implying that RNA degradation during reverse transcription may activate intravirion A3G at the necessary moment. We purified a biologically active tandem affinity-tagged A3G from human HEK293T cells. Mass spectrometry and coimmunoprecipitation from HEK293T and T lymphocyte extracts identified many RNAbinding proteins specifically associated with A3G and A3F, including poly(A)-binding proteins (PABPs), YB-1, Ro-La, RNA helicases, ribosomal proteins, and Staufen1. Most strikingly, nearly all A3G-associated proteins were known to bind exclusively or intermittently to translating and/or dormant mRNAs. Accordingly, A3G in HEK293T and T lymphocyte extracts was almost completely in A3G-mRNA-PABP complexes that shifted reversibly between polysomes and dormant pools in response to translational inhibitors. For example arsenite, which inhibits 5-cap-dependent translational initiation, shifted mRNA-A3G-PABP from polysomes into stress granules in a manner that was blocked and reversed by the elongation inhibitor cycloheximide. Immunofluorescence microscopy showed A3G-mRNA-PABP stress granules only partially overlapping with Staufen1. A3G coimmunoprecipitated HIV-1 RNA and many mRNAs. Ribonuclease released nearly all A3G-associated proteins, including A3G homo-oligomers and A3G-A3F hetero-oligomers, but the viral infectivity factor remained bound. Many proteins and RNAs associated with A3G are excluded from A3G-containing virions, implying that A3G competitively partitions into virions based on affinity for HIV-1 RNA.The viral infectivity factor (Vif) 4 encoded by human immunodeficiency virus type-1 (HIV-1) neutralizes a potent anti-HIV-1 defense system that occurs specifically in T lymphocytes and to a lesser degree in macrophages, thus explaining the efficient replication in T cells of wild-type HIV-1 but not HIV-1(⌬vif) that lacks a functional vif gene (1-4). This antiviral defense system involves the deoxycytidine deaminases APOBEC3G (A3G) and its paralog APOBEC3F (A3F) (members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 family), each of which contains two RNA-binding motifs and incorporates into assembling HIV-1 capsids where they cause lethal dC-to-dU hypermutations in the single-stranded viral DNA that transiently forms during reverse transcription (1, 2, 4 -10). This single-stranded DNA, which has a minus sense, is synthesized using viral RNA as a template and is released from the DNA-RNA hybrid when the RNA is degraded by a ribonuclease H inherent in reverse transcriptase. Vif binds specifically to A3G and A3F and recruits a multisubunit ubiquitin ligase complex containing...

Identification of Molecular Determinants from Moloney Leukemia Virus 10 Homolog (MOV10) Protein for Virion Packaging and Anti-HIV-1 Activity

“…However, it should be noted that Vif potently inhibits the encapsidation of A3G by HIV-1 (20 -24), which should completely disrupt this competition. On the other hand, it has been reported that A3G binds to MOV10 (25)(26)(27). Thus, even when the neutralization of A3G by Vif is incomplete, remnant A3G proteins may enhance MOV10 packaging as long as the domain on A3G that binds to MOV10 does not overlap with the domain that binds to NC.…”

APOBEC proteins and intrinsic resistance to HIV-1 infection

“…The identity of the RNA(s) that promote A3G packaging in HIV-1 infected cells remains somewhat unresolved, though HIV-1 genomic RNA and 7SL RNA (a small pol III transcript that is found in the signal recognition particle, SRP) have each been assigned stimulatory roles in the process (Luo et al 2004;Khan et al 2005Khan et al , 2007Wang et al 2007). The fact that A3G is an avid RNA-binding protein ( Navarro et al 2005;Iwatani et al 2006) raises an important question concerning packaging: specifically, since A3G is associated with an array of cellular ribonucleoprotein (RNP) complexes in an RNA-dependent manner (Chiu et al 2005(Chiu et al , 2006Chelico et al 2006;Kozak et al 2006;Wichroski et al 2006;Gallois-Montbrun et al 2007), how can its RNA-binding site(s) be liberated to allow engagement with the RNA that facilitates packaging? Accordingly, elegant pulse-chase analyses revealed that it is newly synthesized A3G that is incorporated into virions, presumably averting its recruitment into cytosolic RNPs (Soros et al 2007).…”

APOBEC3G: an intracellular centurion

“…An association of A3G with exogenously expressed components of stress granules (SGs) and processing bodies (PBs) has been reported (Kozak et al 2006;Wichroski et al 2006;Gallois-Montbrun et al 2007). The fact that Staufen RNA granules, SGs and PBs represent a continuum of granular-like structures in which cargos may be transferred back and forth could contribute to these results.…”

“…Numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism and fate determination are found in these HMM A3G complexes, and their participation in the complex occurs in an RNAdependent manner (Chiu et al 2006;Kozak et al 2006;Gallois-Montbrun et al 2007). Careful analysis suggests that these components fall into at least three previously defined multi-subunit RNP complexes in human cells: (i) Staufen-containing, polysome-associated RNA granules, (ii) Ro RNPs, and (iii) select components of prespliceosomes plus reservoirs for transcriptional regulators (Chiu et al 2006).…”

“…The HMM A3G RNP complexes contain at least 95 different proteins, as determined by tandem affinity purification and mass spectrometry (Chiu et al 2006;Kozak et al 2006;Gallois-Montbrun et al 2007). Numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism and fate determination are found in these HMM A3G complexes, and their participation in the complex occurs in an RNAdependent manner (Chiu et al 2006;Kozak et al 2006;Gallois-Montbrun et al 2007).…”