Human Retroviral Host Restriction Factors APOBEC3G and APOBEC3F Localize to mRNA Processing Bodies

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

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Programmed Cellular Necrosis Mediated by the Pore-Forming α-Toxin from Clostridium septicum

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The α-toxin of Clostridium septicum is a β-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that α-toxin formed Ca2+-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca2+ levels. This Ca2+ influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca2+ influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of α-toxin-treated cells. Collectively, these data show that α-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced

RNA-Dependent Oligomerization of APOBEC3G Is Required for Restriction of HIV-1

The human cytidine deaminase APOBEC3G (A3G) is a potent inhibitor of retroviruses and transposable elements and is able to deaminate cytidines to uridines in single-stranded DNA replication intermediates. A3G contains two canonical cytidine deaminase domains (CDAs), of which only the C-terminal one is known to mediate cytidine deamination. By exploiting the crystal structure of the related tetrameric APOBEC2 (A2) protein, we identified residues within A3G that have the potential to mediate oligomerization of the protein. Using yeast two-hybrid assays, co-immunoprecipitation, and chemical crosslinking, we show that tyrosine-124 and tryptophan-127 within the enzymatically inactive N-terminal CDA domain mediate A3G oligomerization, and this coincides with packaging into HIV-1 virions. In addition to the importance of specific residues in A3G, oligomerization is also shown to be RNA-dependent. Homology modelling of A3G onto the A2 template structure indicates an accumulation of positive charge in a pocket formed by a putative dimer interface. Substitution of arginine residues at positions 24, 30, and 136 within this pocket resulted in reduced virus inhibition, virion packaging, and oligomerization. Consistent with RNA serving a central role in all these activities, the oligomerization-deficient A3G proteins associated less efficiently with several cellular RNA molecules. Accordingly, we propose that occupation of the positively charged pocket by RNA promotes A3G oligomerization, packaging into virions and antiviral function

Analysis of HIV-1 viral infectivity factor-mediated proteasome-dependent depletion of APOBEC3G: correlating function and subcellular localization

To study how HIV-1 viral infectivity factor (Vif) mediates proteasome-dependent depletion of host factor APOBEC3G, functional and nonfunctional Vif-APOBEC3G interactions were correlated with subcellular localization. APOBEC3G localized throughout the cytoplasm and co-localized with gamma-tubulin, 20 S proteasome subunit, and ubiquitin at punctate cytoplasmic bodies that can be used to monitor the Vif-APOBEC3G interaction in the cell. Through immunostaining and live imaging, we showed that a substantial fraction of Vif localized to the nucleus, and this localization was impaired by deletion of amino acids 12-23. When co-expressed, Vif exhibited more pronounced localization to the cytoplasm and reduced the total cellular levels of APOBEC3G but rarely co-localized with APOBEC3G at cytoplasmic bodies. On the contrary, Vif(C114S), which is inactive but continues to interact with APOBEC3G, stably associated with APOBEC3G in the cytoplasm, resulting in complete co-localization at cytoplasmic bodies and a dose-dependent exclusion of Vif(C114S) from the nucleus. Following proteasome inhibition, cytoplasmic APOBEC3G levels increased, and both proteins co-accumulated nonspecifically into a vimentin-encaged aggresome. Furthermore in the presence or absence of APOBEC3G, Vif localization was significantly altered by proteasome inhibition, suggesting that aberrant localization may also contribute to the loss of Vif function. Finally mutations at Vif Ile(9) disrupted the ability of Vif or Vif(C114S) to coimmunoprecipitate and to co-localize with APOBEC3G, suggesting that the N terminus of Vif mediates interactions with APOBEC3G. Taken together, these results demonstrate that cytoplasmic Vif-APOBEC3G interactions are required but are not sufficient for Vif to modulate APOBEC3G and can be monitored by co-localization in vivo

APOBEC3G Cytoplasmic Bodies Are P-Bodies

<div><p>(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.</p><p>(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.</p></div

APOBEC3G Localizes to Cytoplasmic Bodies

<p>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.</p

APOBEC3G Interacts with P-Body Proteins through RNA-Dependent Interactions

<p>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.</p

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

<div><p>(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.</p><p>(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.</p><p>(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.</p></div