Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations

The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal β-strand, β2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the β2 strand for those structures that have a distorted starting conformation of β2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the β2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function

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

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Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA

The HIV-1 untranslated leader RNA can adopt two mutually exclusive conformations that represent alternative secondary structures. This leader RNA can fold either an extended duplex through long-distance base pairing or a branched conformation in which the RNA locally folds into hairpin structures. Both leader RNA conformations have the TAR hairpin in common, which forms the extreme 5′ end of all HIV-1 transcripts. We report that truncation of the TAR hairpin shifts the equilibrium between the two RNA conformations away from the thermodynamically favored long-distance interaction. However, the equilibrium is partially restored in response to the cations Na(+) and Mg(2+). The transcripts with mutant TAR structures allowed us to investigate conditions affecting the competition between the alternative conformations of the HIV-1 leader RNA. We also demonstrate that the change in conformation of the leader RNA due to TAR truncations severely affects formation of the HIV-1 RNA dimer

Identification of Amino Acid Residues in APOBEC3G Required for Regulation by Human Immunodeficiency Virus Type 1 Vif and Virion Encapsidation

The human immunodeficiency virus type-1 (HIV-1) accessory protein Vif serves to neutralize the human antiviral proteins apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G [A3G]) and A3F. As such, the therapeutic blockade of Vif function represents a logical objective for rational drug design. To facilitate such endeavors, we have employed molecular genetics to define features of A3G that are required for its interaction with Vif. Using alanine-scanning mutations and multiple different substitutions at key residues, we confirm the central role played by the aspartic acid at position 128 and identify proline 129 and aspartic acid 130 as important contributory residues. The overall negative charge of this 3-amino-acid motif appears critical for recognition by Vif, as single lysine substitutions are particularly deleterious and a double alanine substitution at positions 128 and 130 is far more inhibitory than single-residue mutations at either position. Our analyses also reveal that the immediately adjacent 4 amino acids, residues 124 to 127, are important for the packaging of A3G into HIV-1 particles. Most important are tyrosine 124 and tryptophan 127, and mutations at these positions can ablate virion incorporation, as well as the capacity to inhibit virus infection. Thus, while pharmacologic agents that target the acidic motif at residues 128 to 130 have the potential to rescue A3G expression by occluding recognition by Vif, care will have to be taken not to perturb the contributions of the neighboring 124-to-127 region to packaging if such agents are to have therapeutic benefit by promoting A3G incorporation into progeny virions

Restriction of retroviral replication by APOBEC3G/F and TRIM5 alpha

Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5α. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5α binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans

HIV-1 pathogenicity and virion production are dependent on the metabolic phenotype of activated CD4+ T cells

BACKGROUND: HIV-1, like all viruses, is entirely dependent on the host cell for providing the metabolic resources for completion of the viral replication cycle and the production of virions. It is well established that HIV-1 replicates efficiently in activated CD4+ T cells, whereas resting CD4+ T cells are refractory to infection with HIV-1. A hallmark of T cell activation is the upregulation of glycolysis to meet the biosynthetic and bioenergetic needs of cell proliferation and the execution of effector functions by the secretion of cytokines. To date, it has remained unknown if HIV-1 requires the high glycolytic activity of activated T cells to support its replication. RESULTS: We report that in primary CD4+ T cells, the flux through the glycolytic pathway is increased upon infection with HIV-1. This increase in glycolytic activity does not occur in T cell lines when infected with HIV-1. By providing cells with galactose instead of glucose, the former being a poor substrate for glycolysis, we monitored the effect of preventing glycolysis in CD4+ T cells on virus replication cycle and cell fate. We observed that HIV-1 infected primary CD4+ T cells cultured in galactose have a survival advantage over those cultured in glucose and this coincides with reduced caspase 3 activation and apoptosis in cultures with galactose. T cell lines do not recapitulate this difference in cell death. Finally, we demonstrate that virion production is dependent on glycolysis as cultures containing galactose yield reduced amounts of HIV-1 virions compared with cultures containing glucose. CONCLUSIONS: The replication of HIV-1 in primary CD4+ T cells causes an increase in glycolytic flux of the cell. Glycolysis is particularly required for virion production and additionally increases the sensitivity of the infected cell to virus-induced cell death. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0098-4) contains supplementary material, which is available to authorized users