Human retroviral host restriction factors APOBEC3G and APOBEC3F localize to mRNA processing bodies

Abstract

APOBEC3G is an antiviral host factor capable of inhibiting the replication of both exogenous and endogenous retroviruses as well as hepatitis B, a DNA virus that replicates through an RNA intermediate. To gain insight into the mechanism whereby APOBEC3G restricts retroviral replication, we investigated the subcellular localization of the protein. Herein, we report that APOBEC3G localizes to mRNA processing (P) bodies, cytoplasmic compartments involved in the degradation and storage of nontranslating mRNAs. Biochemical analysis revealed that APOBEC3G localizes to a ribonucleoprotein complex with other P-body proteins which have established roles in cap-dependent translation (eIF4E and eIF4E-T), translation suppression (RCK/p54), RNA interference-mediated post-transcriptional gene silencing (AGO2), and decapping of mRNA (DCP2). Similar analysis with other APOBEC3 family members revealed a potential link between the localization of APOBEC3G and APOBEC3F to a common ribonucleoprotein complex and P-bodies with potent anti-HIV-1 activity. In addition, we present evidence suggesting that an important role for HIV-1 Vif, which subverts both APOBEC3G and APOBEC3F antiviral function by inducing their degradation, could be to selectively remove these proteins from and/or restrict their localization to P-bodies. Taken together, the results of this study reveal a novel link between innate immunity against retroviruses and P-bodies suggesting that APOBEC3G and APOBEC3F could function in the context of P-bodies to restrict HIV-1 replication.

Figure 1. APOBEC3G Localizes to Cytoplasmic Bodies

Subcellular localization images of living 293T cells transiently expressing APO3G-YFP (A), HeLa cells transiently expressing APO3G-YFP (B), HeLa-APO3G cells stably expressing APO3G-Myc (C), endogenous APOBEC3G in primary peripheral blood CD4⁺ T cells (D), and endogenous APOBEC3G in H9 T lymphocytes (E). APO3G-YFP was detected by direct YFP fluorescence while APO3G-Myc and endogenous APOBEC3G were detected by indirect immunostaining, using antibodies against the c-Myc epitope and APOBEC3G, respectively. The cells were stained with Hoechst 33258 to visualize nuclei and the imaged were merged digitally. Cell type is noted to the left of each image and arrows point to cytoplasmic bodies.

Figure 2. APOBEC3G Cytoplasmic Bodies Are P-Bodies

(A) Subcellular localization of APOBEC3G and LSM1 in T cells. Endogenous APOBEC3G and LSM1 were localized in peripheral blood CD4⁺ T cells (a) and H9 T lymphocytes (b) through indirect immunostaining using antibodies against APOBEC3G and LSM1, respectively. The cells were counterstained with Hoechst 33258 to visualize nuclei and the images were digitally merged to highlight regions of colocalization, which appear white in the merged images. A corresponding differential interference contrast (DIC) image is presented to the left and arrows point to cytoplasmic bodies. (B) APOBEC3G colocalizes with P-body proteins at P-bodies. Subcellular localization of APO3G-Myc and YFP-LSM1 (a), YFP-AGO2 (b), YFP-eIF4E (c), YFP-eIF4E-T (d), YFP-RCK/p54 (e), or YFP-DCP2 (f) in HeLa-APO3G cells. APO3G-Myc was detected through indirect immunostaining with an antibody against the c-Myc epitope and YFP was detected by direct fluorescence. The cells were counterstained with Hoechst 33258 to visualize nuclei and the images were digitally merged to highlight regions of colocalization, which appear white in the merged images. Arrows point to P-bodies.

Figure 3. APOBEC3G Interacts with P-Body Proteins through RNA-Dependent Interactions

Total cell extracts from 293T cells coexpressing APO3G-HA and YFP-LSM1, YFP-AGO2, YFP-eIF4E, YFP-eIF4E-T, YFP-RCK/p54, or YFP-DCP2 (A–F, respectively) were treated with RNase A (+) or vehicle alone (−) and APO3G-HA was immunoprecipitated (IP) with α-HA agarose (α-HA IP). Total cell extracts (TCE) and α-HA IPs were analyzed by immunoblot, and APO3G-HA was detected with an antibody against the HA epitope, while YFP-tagged P-body proteins were detected with an antibody against GFP.

Figure 4. Potent Anti–HIV-1 Activity of APOBEC3 Proteins Correlates to P-Body Localization

(A) Subcellular localization of endogenous RCK/p54 and APO3G-HA (a), APO3F-HA (b), or APO3B-HA (c) in 293T cells. Endogenous RCK/p54 was detected through indirect immunostaining with an antibody against RCK/p54 and APOBEC3 proteins with an antibody against the HA epitope. Arrows point to P-bodies. (B) Subcellular localization of endogenous RCK/p54, APO3G-CFP, and APO3F-HA in 293T cells. RCK/p54 was detected through indirect immunostaining with an antibody against RCK/p54, APO3F-HA with an antibody against the HA epitope, and APO3G-CFP was detected by direct CFP fluorescence. The cells were also stained with Hoechst 33258 to visualize nuclei and the images were digitally merged to highlight regions of colocalization, which appear white in the merged image. Arrows point to P-bodies. (C) RNA-dependent interaction of APOBEC3G and APOBEC3F. Total cell extracts from 293T cells coexpressing APO3G-CFP and APO3G-HA (top panel), APO3G-CFP and APO3F-HA (center panel), or APO3G-CFP and APO3B-HA (bottom panel) were treated with RNase A (+) or vehicle alone (−) and HA-tagged proteins were immunoprecipitated (IP) with α-HA agarose (α-HA IP). Total cell extracts (TCE) and α-HA IPs were analyzed by immunoblot and APO3G-CFP was detected using an antibody against GFP while APO3G-HA, APO3F-HA and APO3B-HA were detected using an antibody against the HA epitope.

Figure 5. Localization of Vif to P-Bodies Requires APOBEC3G

(A) Myc-AGO2 is not sensitive to Vif-mediated degradation in the absence or presence of APO3G-CFP. Total cell extracts from 293T cells expressing CFP or APO3G-CFP with Myc-AGO2 and either NL-A1Δvif or NL-A1 were analyzed by immunoblot with antibodies against CFP (α-GFP), c-Myc (α-Myc) and Vif (α-Vif). Numbers to the left indicate molecular mass in kDa. (B) Subcellular localization images of 293T cells coexpressing a combination of Myc-AGO2 and Vif^C114S (a), APO3G-CFP, Myc-AGO2, and Vif^C114S (b), or APO3G-CFP, Myc-AGO2, and Vif following a 4-h incubation with the proteasome inhibition ALLN (d) or the equivalent DMSO vehicle control–treated cells (c). APO3G-CFP was detected by direct CFP fluorescence while Myc-AGO2 and Vif protein were detected by indirect immunostaining with antibodies against c-Myc and Vif, respectively. Arrows mark regions of colocalization or lack of colocalization between proteins.