RNA-dependent oligomerization of APOBEC3G is required for restriction of HIV-1

Department of Infectious Diseases, King's College London, London, United Kingdom.
PLoS Pathogens (Impact Factor: 7.56). 04/2009; 5(3):e1000330. DOI: 10.1371/journal.ppat.1000330
Source: PubMed


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.

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    • "These data demonstrate that neither movement alone can enable high levels of A3G-induced mutagenesis. Interestingly, the F126A/W127A mutant is monomeric, suggesting that the oligomeric state of A3G plays a role in efficient restriction of HIV not only by facilitating virion encapsidation but also by facilitating the DNA scanning process (Huthoff et al., 2009; Chelico et al., 2010; Feng and Chelico, 2011). The processivity determinants of A3G reside on predicted loop 7 and helix 6 of the non-catalytic NTD (Feng and Chelico, 2011; Ara et al., 2014, Figure 3A). "
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    ABSTRACT: The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-)DNA, APOBEC3 enzymes deaminate cytosines to form uracils in single-stranded (-)DNA regions. Upon replication of the (-)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite to the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but also by several degradation-independent mechanisms, such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective.
    Full-text · Article · Aug 2014 · Frontiers in Microbiology
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    • "This RNA dependence for protein multimerization has been observed in other cases as well. For example, the human cytidine deaminase APOBEC3G (A3G) was shown to oligomerize only in the presence of RNA (Huthoff et al. 2009), and, similarly, the p53 protein can interact with p68/p72 and the Drosha complex in an RNA-dependent manner (Suzuki et al. 2009). Moreover, the physical interaction of CTCF with the DEAD-box RNA-binding protein p68 (DDX5) was also shown to be dependent on its associated noncoding RNA, SRA (Yao et al. 2010). "
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    ABSTRACT: The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.
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    • "To fully understand the mechanism by which N-terminal 30 amino acids of hA3G regulate oligomerization, we analyzed the effect of the deletion of the N-terminal 30 amino acids on the predicted 3-D structure of the hA3G dimer that was reported to be the major form of hA3G oligomer [34,59]. Thermodynamically stable N-terminal structures of wild-type hA3G and its N-terminal 30-amino-acid deletion mutant were constructed by homology modeling using the hA2 crystal structure as a template. "
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