Reuben S Harris

Publications (68)626.81 Total impact

• Article: Crystal Structure of the DNA Cytosine Deaminase APOBEC3F: The Catalytically Active and HIV-1 Vif-Binding Domain.

  Markus-Frederik Bohn, Shivender M D Shandilya, John S Albin, Takahide Kouno, Brett D Anderson, Rebecca M McDougle, Michael A Carpenter, Anurag Rathore, Leah Evans, Ahkillah N Davis, Jingying Zhang, Yongjian Lu, Mohan Somasundaran, Hiroshi Matsuo, Reuben S Harris, Celia A Schiffer
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  ABSTRACT: Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.
  Structure 05/2013; · 6.35 Impact Factor
• Article: Endogenous APOBEC3A is Cytoplasmic and Non-Genotoxic.

  Allison M Land, Emily K Law, Michael A Carpenter, Lela Lackey, William L Brown, Reuben S Harris
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  ABSTRACT: APOBEC3A (A3A) is a myeloid lineage specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14+-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14+ cells were stimulated with interferon. Immunofluorescent microscopy of interferon stimulated CD14+ and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell wide localization. A3A constructs engineered to be cytoplasmic are also non-toxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.
  Journal of Biological Chemistry 05/2013; · 4.77 Impact Factor
• Article: Impact of H216 on the DNA binding and catalytic activities of the HIV restriction factor APOBEC3G.

  Stefan Harjes, William C Solomon, Ming Li, Kuan-Ming Chen, Elena Harjes, Reuben S Harris, Hiroshi Matsuo
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  ABSTRACT: APOBEC3G has an important role in human defense against retroviral pathogens including HIV-1. Its single-stranded DNA cytosine deaminase activity, located in its C-terminal domain (A3Gctd), can mutate viral cDNA and restrict infectivity. We used time resolved NMR to determine kinetic parameters of A3Gctd's deamination reactions within a 5' -CCC hot spot sequence. A3Gctd exhibited 45-fold preference for 5' -CCC substrate over 5' -CCU substrate, which explains why A3G displays almost no processivity within a 5' -CCC motif. In addition, A3Gctd's shortest substrate sequence was found to be a pentanucleotide containing 5' -CCC flanked on both sides by a single nucleotide. A3Gctd as well as full-length A3G showed peak deamination velocities at pH 5.5. We found that H216 is responsible for this pH dependence, suggesting that protonation of H216 could play a key role in substrate binding. Protonation of H216 appeared important for HIV-1 restriction activity as well, since substitutions of H216 resulted in lower restriction in vivo.
  Journal of Virology 04/2013; · 5.40 Impact Factor
• Article: D316 is critical for the enzymatic activity and HIV-1 restriction potential of human and rhesus APOBEC3B.

  Rebecca M McDougle, Judd F Hultquist, Alex C Stabell, Sara L Sawyer, Reuben S Harris
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  ABSTRACT: APOBEC3B is one of seven human APOBEC3 DNA cytosine deaminases that function to inhibit the replication and persistence of retroelements and retroviruses. Human APOBEC3B restricts the replication of HIV-1 in HEK293 cells, while our laboratory clone of rhesus macaque APOBEC3B did not. We mapped the restriction determinant to a single amino acid difference that alters enzymatic activity. Human APOBEC3B D316 is catalytically active and capable of restricting HIV-1 while rhesus APOBEC3B N316 is not; swapping these residues alters the activity and restriction phenotypes respectively. Genotyping of primate center rhesus macaques revealed uniform homozygosity for aspartate at position 316. Considering the C-to-T nature of the underlying mutation, we suspect that our rhesus APOBEC3B cDNA was inactivated by its own gene product during subcloning in Escherichia coli. This region has been previously characterized for its role in substrate specificity, but these data indicate it also has a fundamental role in deaminase activity.
  Virology 03/2013; · 3.35 Impact Factor
• Article: Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination.

  Lela Lackey, Emily K Law, William L Brown, Reuben S Harris
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  ABSTRACT: Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.
  Cell cycle (Georgetown, Tex.) 02/2013; 12(5). · 5.36 Impact Factor
• Article: APOBEC3B is an enzymatic source of mutation in breast cancer.

  Michael B Burns, Lela Lackey, Michael A Carpenter, Anurag Rathore, Allison M Land, Brandon Leonard, Eric W Refsland, Delshanee Kotandeniya, Natalia Tretyakova, Jason B Nikas, Douglas Yee, Nuri A Temiz, Duncan E Donohue, Rebecca M McDougle, William L Brown, Emily K Law, Reuben S Harris
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  ABSTRACT: Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.
  Nature 02/2013; · 36.28 Impact Factor
• Article: Dispersed sites of HIV Vif-dependent polyubiquitination in the DNA deaminase APOBEC3F.

  John S Albin, John S Anderson, Jeffrey R Johnson, Elena Harjes, Hiroshi Matsuo, Nevan J Krogan, Reuben S Harris
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  ABSTRACT: APOBEC3F and APOBEC3G are DNA cytosine deaminases that potently restrict Human Immunodeficiency Virus-type 1 replication when the virus is deprived of its accessory protein Vif. Vif counteracts these restriction factors by recruiting APOBEC3F and APOBEC3G to an E3 ubiquitin ligase complex that mediates their polyubiquitination and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream ubiquitin acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in APOBEC3G and proposed an antiparallel model of APOBEC3G interaction with the Vif-E3 ubiquitin ligase complex wherein Vif binding at one terminus of APOBEC3G orients the opposite terminus for polyubiquitination [Iwatani Y, et al. (2009) PNAS 106(46):19539-19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in APOBEC3F and used a complementary, unbiased proteomic approach to identify ubiquitin acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant ubiquitin acceptor sites in both APOBEC3F and APOBEC3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyubiquitination of APOBEC3F and APOBEC3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 ubiquitin ligase complex and diminish enthusiasm for the amenability of APOBEC3 ubiquitin acceptor sites to therapeutic intervention.
  Journal of Molecular Biology 01/2013; · 4.00 Impact Factor
• Article: The APOBEC3 Family of Retroelement Restriction Factors.

  Eric W Refsland, Reuben S Harris
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  ABSTRACT: The ability to regulate and even target mutagenesis is an extremely valuable cellular asset. Enzyme-catalyzed DNA cytosine deamination is a molecular strategy employed by vertebrates to promote antibody diversity and defend against foreign nucleic acids. Ten years ago, a family of cellular enzymes was first described with several proving capable of deaminating DNA and inhibiting HIV-1 replication. Ensuing studies on the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) restriction factors have uncovered a broad-spectrum innate defense network that suppresses the replication of numerous endogenous and exogenous DNA-based parasites. Although many viruses possess equally elaborate counter-defense mechanisms, the APOBEC3 enzymes offer a tantalizing possibility of leveraging innate immunity to fend off viral infection. Here, we focus on mechanisms of retroelement restriction by the APOBEC3 family of restriction enzymes, and we consider the therapeutic benefits, as well as the possible pathological consequences, of arming cells with active DNA deaminases.
  Current topics in microbiology and immunology 01/2013; 371:1-27. · 4.93 Impact Factor
• Source

  Available from: dx.plos.org

  Article: Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G

  David J. Stanley equal contributor, Koen Bartholomeeusen equal contributor, David C. Crosby, Dong Young Kim, Eunju Kwon, Linda Yen, Nathalie Caretta Cartozo, Ming Li, Stefanie Jäger, Jeremy Mason-Herr, Fumiaki Hayashi, Shigeyuki Yokoyama, Nevan J. Krogan, Reuben S. Harris, Boris Matija Peterlin, John D. Gross
  PLoS Pathogens 12/2012; 8(12). · 9.13 Impact Factor
• Article: Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G.

  David J Stanley, Koen Bartholomeeusen, David C Crosby, Dong Young Kim, Eunju Kwon, Linda Yen, Nathalie Caretta Cartozo, Ming Li, Stefanie Jäger, Jeremy Mason-Herr, Fumiaki Hayashi, Shigeyuki Yokoyama, Nevan J Krogan, Reuben S Harris, Boris Matija Peterlin, John D Gross
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  ABSTRACT: Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.
  PLoS Pathogens 12/2012; 8(12):e1003085. · 9.13 Impact Factor
• Article: Small-Molecule APOBEC3G DNA Cytosine Deaminase Inhibitors Based on a 4-Amino-1,2,4-triazole-3-thiol Scaffold.

  Margaret E Olson, Ming Li, Reuben S Harris, Daniel A Harki
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  ABSTRACT: APOBEC3G (A3G) is a single-stranded DNA cytosine deaminase that functions in innate immunity against retroviruses and retrotransposons. Although A3G can potently restrict Vif-deficient HIV-1 replication by catalyzing excessive levels of G→A hypermutation, sublethal levels of A3G-catalyzed mutation may contribute to the high level of HIV-1 fitness and its incurable prognosis. To chemically modulate A3G catalytic activity with the goal of decreasing the HIV-1 genomic mutation rate, we synthesized and biochemically evaluated a class of 4-amino-1,2,4-triazole-3-thiol small-molecule inhibitors identified by high-throughput screening. This class of compounds exhibits low-micromolar (3.9-8.2 μM) inhibitory potency and remarkable specificity for A3G versus the related cytosine deaminase, APOBEC3A. Chemical modification of inhibitors, A3G mutational screening, and thiol reactivity studies implicate C321, a residue proximal to the active site, as the critical A3G target for this class of molecules.
  ChemMedChem 11/2012; · 3.15 Impact Factor
• Article: THE RESTRICTION FACTORS OF HUMAN IMMUNODEFICIENCY VIRUS.

  Reuben S Harris, Judd F Hultquist, David T Evans
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  ABSTRACT: Cellular proteins called "restriction factors" can serve as powerful blockades to HIV replication, but the virus possesses elaborate strategies to circumvent these barriers. First, we discuss general hallmarks of a restriction factor. Then, we review how the viral Vif protein protects the viral genome from lethal levels of cDNA deamination by promoting APOBEC3 protein degradation, how the viral Vpu, Env, and Nef proteins facilitate internalization and degradation of the virus-tethering protein Tetherin/BST-2, and how the viral Vpx protein prevents the premature termination of reverse transcription by degrading the dNTPase SAMHD1. These HIV restriction and counter-restriction mechanisms suggest strategies for new therapeutic interventions.
  Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
• Article: Methylcytosine and Normal Cytosine Deamination by the Foreign DNA Restriction Enzyme APOBEC3A.

  Michael A Carpenter, Ming Li, Anurag Rathore, Lela Lackey, Emily K Law, Allison M Land, Brandon Leonard, Shivender M D Shandilya, Markus-Frederik Bohn, Celia A Schiffer, William L Brown, Reuben S Harris
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  ABSTRACT: Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.
  Journal of Biological Chemistry 08/2012; 287(41):34801-8. · 4.77 Impact Factor
• Article: Nanoscale structure and dynamics of ABOBEC3G complexes with single-stranded DNA.

  Luda S Shlyakhtenko, Alexander Y Lushnikov, Atsushi Miyagi, Ming Li, Reuben S Harris, Yuri L Lyubchenko
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  ABSTRACT: The DNA cytosine deaminase APOBEC3G (A3G) is capable of blocking retrovirus replication by editing viral cDNA and impairing reverse transcription. However, the biophysical details of this host-pathogen interaction are unclear. We applied atomic force microscopy (AFM) and hybrid DNA substrates to investigate properties of A3G bound to single-stranded DNA (ssDNA). Hybrid DNA substrates included ssDNA with 5' or 3' ends attached to DNA duplexes (tail-DNA) and gap-DNA substrates, in which ssDNA is flanked by two double-stranded fragments. We found that A3G binds with similar efficiency to the 5' and 3' substrates, suggesting that ssDNA polarity is not an important factor. Additionally, we observed that A3G binds the single-stranded region of the gap-DNA substrates with the same efficiency as tail-DNA. These results demonstrate that single-stranded DNA ends are not needed for A3G binding. The protein stoichiometry does not depend on the ssDNA substrate type, but the ssDNA length modulates the stoichiometry of A3G in the complex. We applied single-molecule high-speed AFM to directly visualize the dynamics of A3G in the complexes. We were able to visualize A3G sliding and protein association-dissociation events. During sliding, A3G translocated over a 69-nucleotide ssDNA segment in <1 s. Association-dissociation events were more complex, as dimeric A3G could dissociate from the template as a whole or undergo a two-step process with monomers capable of sequential dissociation. We conclude that A3G monomers, dimers, and higher-order oligomers can bind ssDNA substrates in a manner independent of strand polarity and availability of free ssDNA ends.
  Biochemistry 07/2012; 51(32):6432-40. · 3.42 Impact Factor
• Source

  Available from: PubMed Central

  Article: Endogenous origins of HIV-1 G-to-A hypermutation and restriction in the nonpermissive T cell line CEM2n.

  Eric W Refsland, Judd F Hultquist, Reuben S Harris
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  ABSTRACT: The DNA deaminase APOBEC3G converts cytosines to uracils in retroviral cDNA, which are immortalized as genomic strand G-to-A hypermutations by reverse transcription. A single round of APOBEC3G-dependent mutagenesis can be catastrophic, but evidence suggests that sublethal levels contribute to viral genetic diversity and the associated problems of drug resistance and immune escape. APOBEC3G exhibits an intrinsic preference for the second cytosine in a 5'CC dinucleotide motif leading to 5'GG-to-AG mutations. However, an additional hypermutation signature is commonly observed in proviral sequences from HIV-1 infected patients, 5'GA-to-AA, and it has been attributed controversially to one or more of the six other APOBEC3 deaminases. An unambiguous resolution of this problem has been difficult to achieve, in part due to dominant effects of protein over-expression. Here, we employ gene targeting to dissect the endogenous APOBEC3 contribution to Vif-deficient HIV-1 restriction and hypermutation in a nonpermissive T cell line CEM2n. We report that APOBEC3G-null cells, as predicted from previous studies, lose the capacity to inflict 5'GG-to-AG mutations. In contrast, APOBEC3F-null cells produced viruses with near-normal mutational patterns. Systematic knockdown of other APOBEC3 genes in an APOBEC3F-null background revealed a significant contribution from APOBEC3D in promoting 5'GA-to-AA hypermutations. Furthermore, Vif-deficient HIV-1 restriction was strong in parental CEM2n and APOBEC3D-knockdown cells, partially alleviated in APOBEC3G- or APOBEC3F-null cells, further alleviated in APOBEC3F-null/APOBEC3D-knockdown cells, and alleviated to the greatest extent in APOBEC3F-null/APOBEC3G-knockdown cells revealing clear redundancy in the HIV-1 restriction mechanism. We conclude that endogenous levels of APOBEC3D, APOBEC3F, and APOBEC3G combine to restrict Vif-deficient HIV-1 and cause the hallmark dinucleotide hypermutation patterns in CEM2n. Primary T lymphocytes express a similar set of APOBEC3 genes suggesting that the same repertoire may be important in vivo.
  PLoS Pathogens 07/2012; 8(7):e1002800. · 9.13 Impact Factor
• Article: APOBEC3B and AID have similar nuclear import mechanisms.

  Lela Lackey, Zachary L Demorest, Allison M Land, Judd F Hultquist, William L Brown, Reuben S Harris
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  ABSTRACT: Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.
  Journal of Molecular Biology 03/2012; 419(5):301-14. · 4.00 Impact Factor
• Article: Vif proteins of human and simian immunodeficiency viruses require cellular CBFβ to degrade APOBEC3 restriction factors.

  Judd F Hultquist, Mawuena Binka, Rebecca S LaRue, Viviana Simon, Reuben S Harris
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  ABSTRACT: HIV-1 requires the cellular transcription factor CBFβ to stabilize its accessory protein Vif and promote APOBEC3G degradation. Here, we demonstrate that both isoforms of CBFβ allow for increased steady-state levels of Vif, enhanced APOBEC3G degradation, and increased viral infectivity. This conserved functional interaction enhances the steady-state levels of Vif proteins from multiple HIV-1 subtypes and is required for the degradation of all human and rhesus Vif-sensitive APOBEC3 proteins by their respective lentiviral Vif proteins.
  Journal of Virology 12/2011; 86(5):2874-7. · 5.40 Impact Factor
• Article: Vif hijacks CBF-β to degrade APOBEC3G and promote HIV-1 infection.

  Stefanie Jäger, Dong Young Kim, Judd F Hultquist, Keisuke Shindo, Rebecca S LaRue, Eunju Kwon, Ming Li, Brett D Anderson, Linda Yen, David Stanley, Cathal Mahon, Joshua Kane, Kathy Franks-Skiba, Peter Cimermancic, Alma Burlingame, Andrej Sali, Charles S Craik, Reuben S Harris, John D Gross, Nevan J Krogan
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  ABSTRACT: Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host's antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β-Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.
  Nature 12/2011; 481(7381):371-5. · 36.28 Impact Factor
• Article: Human and rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity to restrict Vif-deficient HIV-1.

  Judd F Hultquist, Joy A Lengyel, Eric W Refsland, Rebecca S LaRue, Lela Lackey, William L Brown, Reuben S Harris
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  ABSTRACT: Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.
  Journal of Virology 08/2011; 85(21):11220-34. · 5.40 Impact Factor
• Article: Phosphorylation directly regulates the intrinsic DNA cytidine deaminase activity of activation-induced deaminase and APOBEC3G protein.

  Zachary L Demorest, Ming Li, Reuben S Harris
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  ABSTRACT: The beneficial effects of DNA cytidine deamination by activation-induced deaminase (AID; antibody gene diversification) and APOBEC3G (retrovirus restriction) are tempered by probable contributions to carcinogenesis. Multiple regulatory mechanisms serve to minimize this detrimental outcome. Here, we show that phosphorylation of a conserved threonine attenuates the intrinsic activity of activation-induced deaminase (Thr-27) and APOBEC3G (Thr-218). Phospho-null alanine mutants maintain intrinsic DNA deaminase activity, whereas phospho-mimetic glutamate mutants are inactive. The phospho-mimetic variants fail to mediate isotype switching in activated mouse splenic B lymphocytes or suppress HIV-1 replication in human T cells. Our data combine to suggest a model in which this critical threonine acts as a phospho-switch that fine-tunes the adaptive and innate immune responses and helps protect mammalian genomic DNA from procarcinogenic lesions.
  Journal of Biological Chemistry 06/2011; 286(30):26568-75. · 4.77 Impact Factor