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New insights into APOBEC editing functions suggest this family of enzymes might protect against invasive attack by foreign DNA.

Functional antibody genes assembled by V(D)J joining are subsequently diversified by somatic hypermutation, gene conversion and class-switch recombination. Recent evidence indicates that all three processes are caused by the deamination of cytosine to uracil at sites within the immunoglobulin (Ig) loci, with the pattern of diversification depending on the pathway used for resolving the initiating dU-dG lesion. Whereas DNA deamination targeted to the endogenous Ig locus triggers a program of somatic gene diversification that underpins adaptive immunity, deamination targeted to foreign DNA might have arisen initially as a form of innate immunity. Furthermore, the observation that members of the DNA deaminase family can target inappropriate genes suggests they might also contribute to mutations during genome evolution, as well as in cancer.

Adaptive reversion of a +1 frameshift mutation in Escherichia coli, which requires homologous recombination functions, is shown here to occur by -1 deletions in regions of small mononucleotide repeats. This pattern makes improbable recombinational mechanisms for adaptive mutation in which blocks of sequences are transferred into the mutating gene, and it supports mechanisms that use DNA polymerase errors. The pattern appears similar to that of mutations found in yeast cells and in hereditary colon cancer cells that are deficient in mismatch repair. These results suggest a recombinational mechanism for adaptive mutation that functions through polymerase errors that persist as a result of a deficiency in post-synthesis mismatch repair.

After gene rearrangement, immunoglobulin variable genes are diversified by somatic hypermutation or gene conversion, whereas the constant region is altered by class-switch recombination. All three processes depend on activation-induced cytidine deaminase (AID)1,2,3,4,5,6,7, a B-cell-specific protein that has been proposed (because of sequence homology1) to function by RNA editing. But indications that the three gene diversification processes might be initiated by a common type of DNA lesion8,9,10,11, together with the proposal that there is a first phase of hypermutation that targets dC/dG12, suggested to us that AID may function directly at dC/dG pairs. Here we show that expression of AID in Escherichia coli gives a mutator phenotype that yields nucleotide transitions at dC/dG in a context-dependent manner. Mutation triggered by AID is enhanced by a deficiency of uracil-DNA glycosylase, which indicates that AID functions by deaminating dC residues in DNA. We propose that diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of dC residues in the immunoglobulin locus with the outcome—that is, hypermutation phases 1 and 2, gene conversion or switch recombination—dependent on the way in which the initiating dU/dG lesion is resolved.

Somatic hypermutation introduces single base changes into the rearranged variable (V) regions of antigen activated B cells at a rate of approximately 1 mutation per kilobase per generation. This is nearly a million-fold higher than the typical mutation rate in a mammalian somatic cell. Rampant mutation at this level could have a devastating effect, but somatic hypermutation is accurately targeted and tightly regulated. Here, we provide an overview of immunoglobulin gene somatic hypermutation; discuss mechanisms of mutation in model organisms that may be relevant to the hypermutation mechanism; and review recent advances toward understanding the possible role(s) of DNA repair, replication, and recombination in this fascinating process.

Summary: We review some experiments designed to test recombination-based mechanisms for somatic hypermutation in twice, particularly mechanisms involving templated mutation or gene conversion. As recombination and repair functions are highly conserved among prokaryotes and eukaryotes, pathways of mutation in microorganisms may prove relevant to the mechanism of somatic hypermutation. Estherichia coli initates a recombination-based pathway of mutation in response to environmental stimuli, and this “adaptive” pathway of mutation has striking similarities with somatic hypermutation, as does a process of mutagenic repair that occurs at double-strand breaks in Saccharomyces cerevisiae. We present model for recombination-based hypermutation of the immunoglobulin loci which could result in cither templated cir non-templated mutation.

Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase (“adaptive”) mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Stationary-phase mutation in microbes can produce selected ('adaptive') mutants preferentially. In one system, this occurs via a distinct, recombination-dependent mechanism. Two points of controversy have surrounded these adaptive reversions of an Escherichia coli lac mutation. First, are the mutations directed preferentially to the selected gene in a Lamarckian manner? Second, is the adaptive mutation mechanism specific to the F plasmid replicon carrying lac? We report that lac adaptive mutations are associated with hypermutation in unselected genes, in all replicons in the cell. The associated mutations have a similar sequence spectrum to the adaptive reversions. Thus, the adaptive mutagenesis mechanism is not directed to the lac genes, in a Lamarckian manner, nor to the F' replicon carrying lac. Hypermutation was not found in non-revertants exposed to selection. Therefore, the genome-wide hypermutation underlying adaptive mutation occurs in a differentiated subpopulation. The existence of mutable subpopulations in non-growing cells is important in bacterial evolution and could be relevant to the somatic mutations that give rise to cancers in multicellular organisms.

The sequences of adaptive reversions of a lac frameshift mutation in Escherichia coli resemble DNA polymerase errors, and the adaptive reversions decrease in strains with an antimutator DNA polymerase III (PolIII) allele. The latter finding could imply that DNA PolIII itself makes adaptive mutations. Alternatively, normal DNA PolIII errors could saturate post-synthesis mismatch repair during adaptive mutation. If so, the antimutator strain would produce fewer adaptive mutations because it possesses greater capacity for mismatch repair which could correct errors made by a polymerase other than DNA PolIII. Mismatch repair capacity is limited specifically during adaptive mutation, necessitating a test of this indirect model. This indirect model is ruled out here by the observation that the antimutator PolIII allele decreases adaptive mutation even in mismatch repair-defective cells. This supports a direct role for DNA PolIII in recombination-dependent adaptive mutation.

Two recent observations have connected the innate immune DNA cytosine deaminase APOBEC3B to the genetic evolution of breast cancer. First, APOBEC3B was shown to be up-regulated in the majority of breast cancers, and, in breast cancer cell lines, its activity was causally linked to a doubling of the number of C-to-T transitions over time and to a delay in cell cycle progression (1). Second, sequencing of the complete genome of 21 breast cancers independently suggested that APOBEC deaminase activity could be responsible for 2 of 5 mutational imprints identified, which involved clustered (also called kataegis) and dispersed C-to-T transition mutations in the context of 5’TC dinucleotide motifs (2). In the current study, we addressed a possible association of APOBEC3B expression with outcome in clinical breast cancer. For this we measured using real-time RT-PCR APOBEC3B mRNA levels in 1,491 primary invasive breast cancers and correlated these levels with disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS) using univariate and multivariable Cox regression analysis. In addition, we independently validated our findings in available gene expression datasets with appropriate follow-up. In univariate analyses including all patients, increasing levels of APOBEC3B mRNA analyzed as a continuous variable were significantly associated with shorter DFS, MFS and OS (Hazard Ratio [HR] = 1.29, 1.31 and 1.36, respectively, all P To substantiate and validate our findings, we analysed 4 independent available datasets containing in total 5,760 breast cancer cases in which APOBEC3B mRNA expression was measured by probes on microarrays and found that higher APOBEC3B mRNA expression (dichotomised by mean) was significantly associated with poor outcome in all 4 cohorts ([Metabric, 1,491 ER+ cases, HR = 1.82; P5 years], and [Affymetrix dataset-2, 643 ER+ cases, HR = 2.04; P = 0.001]). Altogether, our analyses show that APOBEC3B mRNA - and as a result likely DNA deamination – is a validated predictor of poor outcome in breast cancer, supporting the notion that APOBEC3B is a potentially interesting clinical target for therapeutic intervention to prevent breast cancer progression and metastasis, particularly in ER+ disease. 1. Burns, M.B. et al. Nature 494, 366-70 (2013); 2. Nik-Zainal, S. et al. Cell 149, 979-93 (2012). Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S6-05.

Background: R-spondins are secreted agonists of Wnt signaling that function in development and promote tissue stem cell proliferation. Rspo2 was identified as a candidate oncogene in intestinal tumors via a Sleeping Beauty transposon insertional mutagenesis screen in mice (1). Further, oncogenic activation of RSPO2 and RSPO3 mediated by recurrent genomic rearrangements has been identified in human colorectal cancer (2). The purpose of the current study was to determine if R-spondins function as oncogenes in other cancer types characterized by active Wnt signaling. Methods: R-spondin mRNA expression was determined in primary human breast tumors and adjacent normal tissue by quantitative RT-PCR. Affymetrix microarrays were used to assay gene expression and classify by molecular subtype human hepatocellular carcinomas compared to pre-malignant lesions and normal livers. RSPO2 was depleted or overexpressed in breast cell lines by stable transduction with anti-RSPO2 shRNA or RSPO2 cDNA. Resulting changes in gene expression and proliferation were measured by qRT-PCR and MTS assay respectively. RSPO2 was somatically overexpressed in murine liver by hydrodynamic injection and Fah selection in an Fah-null model. Subsequent gene expression studies were conducted by qRT-PCR and immunohistochemistry. Results: RSPO2 was highly expressed in 12% of primary human breast tumors compared to adjacent normal tissue. Similarly, RSPO2 expression was elevated in the subset of primary human liver cancers with activated Wnt/beta-catenin signaling. In human breast cancer cells with elevated RSPO2 expression, RSPO2 knockdown decreased Wnt signaling and proliferation, while RSPO2 overexpression in a non-tumorigenic breast epithelial cell line potentiated Wnt signaling. Overexpression of RSPO2 in the mouse liver increased Wnt signaling and promoted an enlarged liver phenotype and tumor formation. Conclusions: These data strongly suggest that RSPO2 is a driver of human breast and liver cancer. Future work will further characterize signaling pathways implicated in RSPO-driven phenotypes, and develop targeted therapy to inhibit RSPO signaling. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A242. Citation Format: Caitlin B. Conboy, Bobbi R. Tschida, Hsiangyu Hu, Michael B. Burns, Nuri A. Temiz, Timothy Kuka, Vincent W. Keng, Sara Toffanin, Reuben S. Harris, Josep Llovet, Timothy K. Starr, David A. Largaespada. R-spondin 2 drives Wnt signaling and tumor formation in breast and liver cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A242.

A typical breast cancer accumulates hundreds to thousands of somatic mutations. Breast cancer mutation spectra are often dominated by C-to-T transition mutations, which occur in a mostly dispersed manner but can also occur in localized clusters called kataegis. Several lines of evidence indicate that the innate immune DNA cytosine deaminase APOBEC3B is the primary cause of these mutations. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is over-represented in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with more aggressive disease including shorter durations of survival. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that may help to explain how some tumors evolve rapidly and manifest gross heterogeneity. Citation Format: Reuben Harris. The DNA cytosine deaminase APOBEC3B drives mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A133.

Nature 494, 366–370 (2013); doi:10.1038/nature11881 We reported a comparison of the DNA cytosine deamination context of APOBEC3B in vitro with the observed C-to-T mutation context in breast cancer (see Fig. 4c of the original Letter). We incorrectly stated in the Fig. 4c legend that the data represent all cytosines.

Recent studies have revealed a role for the DNA cytosine deaminase APOBEC3B in breast cancer mutagenesis. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is evident in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with poorer prognostic outcomes including shorter survival durations. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that helps to explain how some tumors evolve rapidly and manifest gross heterogeneity. Citation Format: Reuben S. Harris. Molecular and clinical impact of APOBEC3B mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA23.

Knowledge of cancer genomic DNA sequences has created unprecedented opportunities for mutation studies. Computational analyses have begun to decipher mutational signatures that identify underlying causes. A recent analysis encompassing 30 cancer types reported 20 distinct mutation signatures, resulting from ultraviolet light, deficiencies in DNA replication and repair, and unexpectedly large contributions from both spontaneous and APOBEC-catalyzed DNA cytosine deamination. Mutational signatures have the potential to become diagnostic, prognostic, and therapeutic biomarkers as well as factors in therapy development.

Aspects of the molecular mechanism of “adaptive” mutation are emerging from one experimental system: reversion of an Escherichia coli lac frameshift mutation carried on a conjugative plasmid. Homologous recombination is required and the mutations resemble polymerase errors. Reports implicating a role for conjugal transfer proteins suggested that the mutation mechanism is ordinary replication error occurring during transfer synthesis, followed by conjugation-like recombination, to capture the replicated fragment into an intact replicon. Whereas conjugational recombination uses either of two systems of Holliday junction resolution, we find that the adaptive lac reversions are inhibited by one resolution system and promoted by the other. Moreover, temporary absence of both resolution systems promotes mutation. These results imply that recombination intermediates themselves promote the mutations.

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (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 non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data co...

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., Author Summary The APOBEC3 family of editing enzymes catalyzes lethal hypermutation of retroviral genomes to block spread of virus in host. HIV Vif targets APOBEC3 family members for destruction by a cellular ubiquitin ligase containing CUL5. A major goal in the design of the next generation of antiretroviral therapies is to find an inhibitor of Vif so that the activity of the APOBEC3 family of antiretroviral enzymes can be restored. We define a three-enzyme cascade that is required to activate Vif by addition of the ubiquitin-like NEDD8 protein to CUL5. MLN4924, an anti-cancer compound currently in phase 1 clinical trials, inhibits the NEDD8 cascade, blocks the action of Vif, and thus has potent anti-HIV activity. Furthermore, our studies define downstream drug targets in the NEDD8 cascade more selective for inhibition of HIV Vif. We demonstrate pharmacological inhibition of HIV replication through a mechanism that restores the innate immunity provided by APOBEC3 enzymes by targeting a host pathway, providing additional candidates that could be further exploited for therapeutic development. Inhibition of this NEDD8 cascade alone, or in combination with existing antiretroviral drugs could prove to be a useful treatment for HIV.

The genetic requirements for adaptive mutation in Escherichia coli parallel those for homologous recombination in the RecBCD pathway. Recombination-deficient recA and recB null mutant strains are deficient in adaptive reversion. A hyper-recombinagenic recD strain is hypermutable, and its hypermutation depends on functional recA and recB genes. Genes of subsidiary recombination systems are not required. These results indicate that the molecular mechanism by which adaptive mutation occurs includes recombination. No such association is seen for spontaneous mutation in growing cells.

In this work, we present evidence that an endogenous DNA mutating protein, APOBEC3, is massively overexpressed in a high proportion of breast cancers and that this overexpression may be responsible for the genetic variability seen in these cancers. Multiple driver mutations are required for cancer development and recent genome-wide approaches have revealed that many cancers, including breast, harbor staggering numbers of mutations, both point mutations as well as gross chromosomal rearrangements. The molecular origins of most of these somatic aberrations remain unclear. In this work, we test the hypothesis that APOBEC3, an endogenous DNA mutator, is a causative factor driving these mutations. One clue derives from the fact that C/G-to-T/A transition mutations, both within and outside of methyl-C-p-G dinucleotide motifs, predominate in subsets of many tumor types, including breast cancer. We demonstrate that APOBEC3, a DNA cytosine deaminase, is a likely endogenous source of mutation in breast cancer. We show, using quantitative reverse transcriptase PCR (qRT-PCR), that APOBEC3 is overexpressed in a majority of commonly available breast cancer cell lines (34/44) when compared to MCF-10A and hTERT-HMEC control lines. This overexpression phenotype persists when examining primary breast tumors (24/37) using patient-matched normal tissues as controls. APOBEC3 is predominantly localized to the nuclear compartment when overexpressed, as seen using fluorescently tagged APOBEC3. Additionally, its catalytic DNA cytosine to uracil deamination activity is dramatically elevated in this compartment in multiple breast cancer cell lines, as quantified using a FRET-based DNA oligonucleotide cleavage assay. This presentation will discuss the potential impact of this potent endogenous mutagen on the breast cancer genome and the implications for tumor evolution and therapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-193. doi:10.1158/1538-7445.AM2011-LB-193

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Human APOBEC3G inhibits the replication of Vif-deficient HIV-1 by hypermutating nascent viral cDNA (reviewed in [1]–[3]). Zheng and colleagues recently reported that the HIV-1 restriction activity of APOBEC3G requires a cellular co-factor [4]. Their conclusion depended on three critical observations: i) CEM-T4 cells support the replication of Vif-deficient HIV-1, ii) CEM-T4 cells express restrictive levels of APOBEC3G, and iii) CEM-T4 cells engineered to express more APOBEC3G still permitted Vif-deficient HIV-1 replication [4]. These observations suggested that APOBEC3G alone is insufficient for restriction, and together with subsequent cell fusion experiments, that a recessive cellular co-factor is required. However, the fact that APOBEC3G is capable of restricting a broad number of retroelements, including yeast Ty elements, strongly suggests that other human cellular proteins are not absolutely required for restriction (e.g., [5],[6] and reviewed in [1]–[3]). An alternative explanation that could account for the observed permissive phenotype of the CEM-T4 line is that it is mixed, composed of a population of cells expressing low and/or variable levels of APOBEC3G. If this were the case, then the permissive phenotype could simply be due to virus replication in the subset of cells expressing low levels of APOBEC3G. To address this hypothesis, we generated subclones of the CEM-T4 line by serial dilution and determined the level of APOBEC3G expression by immunoblotting (Figure 1A). First, we observed that CEM-T4 cells expressed levels of APOBEC3G that were considerably lower than those in the non-permissive line CEM, regardless of whether they were obtained from the AIDS Research and Reference Reagent Program (CEM-T4-A) or directly from the Zheng laboratory (CEM-T4-Z). Second, we found that representative CEM-T4 subclones, regardless of source, expressed both low and variable APOBEC3G levels. It is further notable that none of the subclones expressed fully non-permissive, CEM-like APOBEC3G levels. This heterogeneity is reflected by the kinetics of Vif-deficient virus replication, with some subclones being fully permissive and others being semi-permissive (Figure 1B and 1C). Nevertheless, these data demonstrated that APOBEC3G levels are both low and variable within individual cells of the CEM-T4 line, thus providing a reasonable molecular explanation for the permissive phenotype of this cell line. Figure 1 CEM-T4 cells express low APOBEC3G levels and subclones show additional heterogeneity. Curiously, Zheng and coworkers [4] also showed that CEM-T4 cells retrovirally transduced with APOBEC3G were still permissive for Vif-deficient HIV-1 replication. This result may be due to the distinct possibility that APOBEC3G itself inactivated some of the transducing viral cDNAs. Such an attempt at complementation would probably result in a CEM-T4 line that is mixed for APOBEC3G expression. To clarify this important point, we used electroporation to generate panels of HA-tagged and untagged APOBEC3G-expressing CEM-T4 clones and used them for virus replication experiments (Figures 2 and ​and3,3, respectively). Figure 2 Expression of exogenous APOBEC3G-HA is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1. Figure 3 Increasing levels of untagged APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. Contrary to the findings of Zheng and colleagues [4], our APOBEC3G-expressing CEM-T4 clones were fully restrictive for Vif-deficient HIV-1 replication. The observed non-permissive phenotype was not due to gross overexpression of APOBEC3G because many of the restrictive CEM-T4 clones stably expressed APOBEC3G to levels slightly lower to those of CEM, the original parent of CEM-T4 (e.g., clone G4 in Figure 3). It is also notable that some vector control clones such as V9, like the subclones in Figure 1, expressed low and non-restrictive levels of APOBEC3G. These data suggest that cells must pass an APOBEC3G expression “threshold” before they become fully non-permissive for Vif-deficient HIV-1. Nevertheless, together these virus replication data clearly demonstrated that expression of exogenous APOBEC3G is sufficient to render CEM-T4 cells non-permissive for Vif-deficient HIV-1 replication. We conclude that this cell line does not require a specific endogenous APOBEC3G “co-factor” to restrict Vif-deficient HIV-1. In this report, we question the obligate APOBEC3G co-factor hypothesis put forth by Zheng and colleagues [4]. Our data clearly provide an alternative explanation that—rather than lacking a co-factor for APOBEC3G—the CEM-T4 line is mixed with individual cells expressing heterogeneous and less-than-restrictive levels of APOBEC3G. Importantly, when transfections were used to generate stable APOBEC3G-expressing CEM-T4 clones, the resulting lines were non-permissive for Vif-deficient HIV-1 replication. Based on these results, we conclude that CEM-T4 cells do not lack an APOBEC3G co-factor and, more generally, that APOBEC3G may not strictly require another cellular protein for HIV-1 restriction. However, we would like to emphasize that neither study rules out the attractive possibility that APOBEC3G, like most other proteins, is likely to be regulated by a variety of cellular processes.

Freeze-substitution was performed on strains of Escherichia coli, Pasteurella multocida, Campylobacter fetus, Vibrio cholerae, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas salmonicida, Proteus mirabilis, Haemophilus pleuropneumoniae, Caulobacter crescentus, and Leptothrix discophora with a substitution medium composed of 2% osmium tetroxide and 2% uranyl acetate in anhydrous acetone. A thick periplasmic gel ranging from 10.6 to 14.3 nm in width was displayed in E. coli K-12, K30, and His 1 (a K-12 derivative containing the K30 capsule genes), P. multocida, C. fetus, P. putida, A. salmonicida, H. pleuropneumoniae, and P. mirabilis. The other bacteria possessed translucent periplasms in which a thinner peptidoglycan layer was seen. Capsular polysaccharide, evident as electron-dense fibers radiating outward perpendicular to the cell surface, was observed on E. coli K30 and His 1 and P. mirabilis cells. A more random arrangement of fibers forming a netlike structure was apparent surrounding cells of H. pleuropneumoniae. For the first time a capsule, distinct from the sheath, was observed on L. discophora. In all instances, capsular polysaccharide was visualized in the absence of stabilizing agents such as homologous antisera or ruthenium red. Other distinct envelope structures were observed external to the outer membrane including the sheath of L. discophora and the S layers of A. salmonicida A450 and C. crescentus CB15A. We believe that the freeze-substitution technique presents a more accurate image of the structural organization of these cells and that it has revealed complex ultrastructural relationships between cell envelope constituents previously difficult to visualize by more conventional means of preparation.

APOBEC3G is capable of providing a cellular defense against retroviruses by deaminating deoxycytidine to deoxyuridine in the first strand of nascent retroviral cDNA. However, this defense can be counteracted by the HIV virion-infectivity factor (Vif), which targets APOBEC3G for degradation by the proteasome. The delicate balance between the anti-viral effect of APOBEC3G and the pro-viral effect of Vif will be discussed.

CEM15/APOBEC3G is a cellular protein required for resistance to infection by virion infectivity factor (Vif)-deficient human immunodeficiency virus (HIV). Here, using a murine leukemia virus (MLV)-based system, we provide evidence that CEM15/APOBEC3G is a DNA deaminase that is incorporated into virions during viral production and subsequently triggers massive deamination of deoxycytidine to deoxyuridine within the retroviral minus (first)-strand cDNA, thus providing a probable trigger for viral destruction. Furthermore, HIV Vif can protect MLV from this CEM15/APOBEC3G-dependent restriction. These findings imply that targeted DNA deamination is a major strategy of innate immunity to retroviruses and likely also contributes to the sequence variation observed in many viruses (including HIV).

The important contributions of many investigators could not be specifically cited due to space considerations, but many of these can be found within the bibliography. We thank Tim Behrens, Bill Brown, Eric Hendrickson, Stefan Jonsson and Michael Neuberger for thoughtful comments on this manuscript. R.S.H. is supported in part by a Searle Scholarship, a University of Minnesota McKnight Land Grant Assistant Professorship, and a grant from the NIH NIAID (AI064046).

APOBEC3G is a human cellular enzyme that is incorporated into retroviral particles and acts to restrict retroviral replication in infected cells by deaminating dC to dU in the first (minus)-strand cDNA replication intermediate [1–5]. HIV, however, encodes a protein (virion infectivity factor, Vif [6, 7]), which overcomes APOBEC3G-mediated restriction but by an unknown mechanism. Here, we show that Vif triggers APOBEC3G degradation by a proteasome-dependent pathway and that an 80 amino acid region of APOBEC3G surrounding its first zinc coordination motif is sufficient to confer the ability to partake in an interaction involving Vif. Inhibitors of this interaction might therefore prove therapeutically useful in blocking Vif-mediated APOBEC3G destruction.

SummaryHIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV-1 Vif protein, which subverts cellular CBF-β and a polyubiquitin ligase complex to neutralize these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host-pathogen conflict has been elusive. We combine virus adaptation and computational studies to interrogate the APOBEC3F-Vif interface and build a robust structural model. A recurring compensatory amino acid substitution from adaptation experiments provided an initial docking constraint, and microsecond molecular dynamics simulations optimized interface contacts. Virus infectivity experiments validated a long-lasting electrostatic interaction between APOBEC3F E289 and HIV-1 Vif R15. Taken together with mutagenesis results, we propose a “wobble model” to explain how HIV-1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, explain how pathogens may evolve to escape innate host defenses.

APOBEC3 proteins are DNA cytosine deaminases that restrict the replication of human immunodeficiency virus deficient in the counterdefense protein Vif. Here, we address the capacity of APOBEC3F to restrict via deaminase-dependent and -independent mechanisms by monitoring spreading infections in diverse T cell lines. Our data indicate that only a deaminase-proficient protein is capable of long-term restriction of Vif-deficient HIV in T cells, analogous to prior reports for APOBEC3G. This indicates that the principal mechanism of APOBEC3F restriction is deaminase-dependent.

Background The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. Results HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. Conclusion These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1042-9) contains supplementary material, which is available to authorized users.

The RNA binding protein DEAD-END (DND1) is one of the few proteins known to regulate microRNA (miRNA) activity at the level of miRNA-mRNA interaction. DND1 blocks miRNA interaction with the 3′-untranslated region (3′-UTR) of specific mRNAs and restores protein expression. Previously, we showed that the DNA cytosine deaminase, APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide like 3), interacts with DND1. APOBEC3 has been primarily studied for its role in restricting and inactivating retroviruses and retroelements. In this report, we examine the significance of DND1-APOBEC3 interaction. We found that while human DND1 inhibits miRNA-mediated inhibition of P27, human APOBEC3G is able to counteract this repression and restore miRNA activity. APOBEC3G, by itself, does not affect the 3′-UTR of P27. We found that APOBEC3G also blocks DND1 function to restore miR-372 and miR-206 inhibition through the 3′-UTRs of LATS2 and CX43, respectively. In corollary experiments, we tested whether DND1 affects the viral restriction function or mutator activity of APOBEC3. We found that DND1 does not affect APOBEC3 inhibition of infectivity of exogenous retrovirus HIV (ΔVif) or retrotransposition of MusD. In addition, examination of Ter/Ter;Apobec3−/− mice, lead us to conclude that DND1 does not regulate the mutator activity of APOBEC3 in germ cells. In summary, our results show that APOBEC3 is able to modulate DND1 function to regulate miRNA mediated translational regulation in cells but DND1 does not affect known APOBEC3 function.

Background: HIV-1 Vif is essential for virus replication in natural target cells such as T cells and macrophages. Vif recruits a ubiquitin ligase to degrade restrictive APOBEC3 proteins. APOBEC3G is one of the most potent retroviral restriction factors targeted by Vif and, as such, the Vif-APOBEC3G interaction has emerged as a promising HIV-1 therapeutic target. Methods: 20,000 small molecules were used in live-cell screens for those that preserve EGFP-APOBEC3G fluorescence and luciferase-APOBEC3G luminescence in the presence of HIV-1 Vif. Results: 2 compounds with similar core structures preserved APOBEC3G levels in the presence of Vif. 10 μ Mo f compound restored APOBEC3G to levels sufficient for incorporation into vif-proficient virus particles and restriction of virus infectivity. Vif-dependent APOBEC3G polyubiquitination and general proteasomal activity were unaffected at the same concentration. Conclusions: The small molecules described here preserve APOBEC3G levels and activity in the presence of Vif. These molecules are starting points for further development as antiretrovirals.

Cell-free expression systems are increasingly being employed as platforms for biophysical, biochemical, and systems biology experiments. We demonstrate that combining fluorescence fluctuation spectroscopy (FFS) with cell-free expression provides quick and quantitative measurements of chromophore maturation, protein-protein interactions and protein-DNA interactions. We perform the first study of chromophore maturation as a function of temperature, and demonstrate pronounced temperature dependence of the maturation kinetics for EGFP, EYFP, and mCherry. The Eyring equation successfully reproduces the temperature-dependence of the maturation rate for each of the proteins. Our results for EGFP, EYFP and mCherry provide an explanation for the differences in the reported maturation times studied by de novo protein synthesis. A droplet sample protocol was developed to ensure sufficient oxygenation for chromophore maturation studies, while preventing evaporation of the sample. We further demonstrate the feasibility of protein titrations with the droplet protocol and characterize oligomerization of the nuclear transport factor 2 (NTF2) over a wide concentration range by brightness analysis. We employ the droplet setup to study APOBEC3G, a DNA cytosine deaminase enzyme with innate immune activity against retroviruses, notably HIV-1. Brightness analysis reports the homo-oligomerization of APOBEC3G, which is both concentration and temperature dependent. Using two-color FFS, we simultaneously measure APOBEC3G oligomerization and the interaction between APOBEC3G and single-stranded DNA (ssDNA) in order to characterize the molecular interactions of this important enzyme. This work was supported by grants from the National Institutes of Health (GM64589, GM091743).