Antiviral Protein APOBEC3G Localizes to Ribonucleoprotein Complexes Found in P Bodies and Stress Granules

Abstract

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.

TRIM5α Cytoplasmic Bodies Are Highly Dynamic Structures

“…TRIM5␣ cytoplasmic bodies were not observed to associate with cellular markers of actin, early endosomes, the trans-Golgi network, or lysosomes by imaging (data not shown). TRIM5␣ cytoplasmic bodies did not coincide with processing bodies (P-bodies) (Figure 2, C and D), another class of cytoplasmic body that is involved in mRNA storage and metabolism (Anderson and Kedersha, 2006) when the fluorescent P-body markers mRFP-Dcp1A ( Figure 2C) and RFP-rck/p54 ( Figure 2D) (Gallois-Montbrun et al, 2007) were expressed in HeLa cells stably expressing YFP-rhTRIM5␣. They also do not associate with vimentin as would be expected for aggresomes (data not shown) (Garcia-Mata et al, 2002).…”

“…Data are presented as Z-series compilations of 6 -10 images in a stack (Gallois-Montbrun et al, 2007).…”

“…These constructs were introduced into cells using a vesicular stomatitis virus-G protein (VSV-G) pseudotyped Moloney MLV vector. The pmRFP-Dcp1a vector that expresses the mRNA-decapping enzyme involved in 5Ј-to-3Ј mRNA degradation has been described previously (Kedersha et al, 2005;Gallois-Montbrun et al, 2007). …”

“…Colonies were subsequently screened for protein expression and viral restriction. These HeLa YFPrhTRIM5␣ cells were transiently transfected using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) as described previously (Gallois-Montbrun et al, 2007). Stable cell lines expressing both CFP-tubulin and GFP-huTRIM5␣ were generated by transduction of 8 ϫ 10 4 TE671 cells in a 12-well plate by using Moloney MLV-based vectors with above-mentioned fragments at an MOI of ϳ10.…”

APOBEC3G: an intracellular centurion

“…One possibility was suggested immediately by subcellular localization studies, which indicated that A3G is strongly retained in the cytoplasm (Mangeat et al 2003;Muckenfuss et al 2006;OhAinle et al 2006;Wichroski et al 2006;Gallois-Montbrun et al 2007). While such sequestration would limit promiscuous editing of genomic DNA during many phases of the cell cycle, nuclear access could occur during mitosis when nuclear membranes break down.…”