Publications (112)833.41 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: The human APOBEC3 family of DNA-cytosine deaminases comprises seven members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It is proposed that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other likely ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.
- [Show abstract] [Hide abstract] ABSTRACT: Stress-induced reactivation of latent herpesviruses requires disabling of repression, but the mechanism for converting silenced chromatin into an active state is unknown. In this issue of Cell Host & Microbe, Cliffe et al. (2015) suggest a methyl/phospho switch on histone H3 overcomes repression to facilitate reactivation of latent herpes simplex virus type 1 (HSV-1).
- [Show abstract] [Hide abstract] ABSTRACT: Viruses are obligate intracellular parasites that necessarily rely on hijacking cellular resources to produce viral progeny. The success of viral infection requires manipulation of host chromatin in order to activate genes useful for production of viral proteins as well as suppress antiviral responses. Host chromatin manipulation on a global level is likely reliant on modulation of post-translational modifications (PTMs) on histone proteins. Mass spectrometry (MS) is a powerful tool to quantify and identify novel histone PTMs, beyond the limitations of site-specific antibodies. Here, we employ MS to investigate global changes in histone PTM relative abundance in human cells during infection with adenovirus. Our method reveals several changes in histone PTM patterns during infection. We propose that this method can be used to uncover global changes in histone PTM patterns that are universally modulated by viruses to take over the cell. Copyright © 2015. Published by Elsevier Inc.
- [Show abstract] [Hide abstract] ABSTRACT: Telomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1 and loss of the telomere repeat DNA signal. The HSV-1-encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. Small hairpin RNA (shRNA) depletion of TPP1 increases viral replication, indicating that TPP1 inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins, and ICP8-null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated prereplication foci and to promote viral genomic replication. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
- [Show abstract] [Hide abstract] ABSTRACT: Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition. DOI: http://dx.doi.org/10.7554/eLife.02008.001
- [Show abstract] [Hide abstract] ABSTRACT: Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs.
- [Show abstract] [Hide abstract] ABSTRACT: Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.
- [Show abstract] [Hide abstract] ABSTRACT: Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus type 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1, and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in non-dividing macrophages.
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Analysis of putative miR155 oncomiR target genes. (A) Conservation of the seed sequences of miR155 in the 3′UTR of predicted target genes, DET1, JARID2 and TP53INP1, identified by computational analysis, in human, cow, mouse and chicken sequences. (B) Luciferase reporters containing the 3′UTR of DET1, JARID2 and TP53INP1 were compared with a control Luciferease reporter (pMIR-REP-dCMV), demonstrating that Buparvaquone (+Bup) induced Luciferase activity in parasitized THEI cells (average ± sd, n = 3). *p<0.05, **p<0.01 (C) Buparvaquone treatment had no effect on the mRNA levels of DET1 or TP53INP1 in the three different cell lines, as assessed by qPCR analysis for the bovine genes. Transcript levels in untreated cells are shown relative to the control and normalized against β-actin and β2M mRNA (average ± sd, n = 3). (TIF)
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: The effect of Buparvaquone treatment on the growth and survival of infected cell lines. (A) The parasite-infected THEI cells were grown in the presence or absence of Buparvaquone (+Bup) and cell numbers were monitored by counting live cells followed by trypan blue exclusion (left panel). Representation of flow cytometry analysis indicating the induction of apoptosis (sub-G1 population) and growth arrest (G1 population) of THEI cells following treatment with Buparvaquone (open histograms - right panel) (average ± sd, n = 3). *p<0.05, **p<0.01 (B) BL3 and TBL3 were treated with Buparvaquone for 64 h and cycling cells were measured by immunofluorescence using an anti-Ki67 antibody. Quantification of Ki67-positive cells was monitored by fluorescence microscopy (average ± sd, n = 3). *p<0.05, **p<0.01 (TIF)
Dataset: Table S2[Show abstract] [Hide abstract] ABSTRACT: Oligonucleotide primer sequences used to analyze the expression of genes. List of oligonucleotide sequences (sense and antisense) used for PCR analysis. (PPT)
Dataset: Table S1[Show abstract] [Hide abstract] ABSTRACT: Summary of additional microRNAs downregulated more than two-fold (Log2) upon Buparvaquone treatment in TBL3 or Thei cells. The table shows the known functions and known target genes and references. (PPT)
Dataset: Figure S3[Show abstract] [Hide abstract] ABSTRACT: miR155 does not affect c-Jun transcription. (A) The overexpression of miR155 or siDET1 or the miR-155 Sponge had no effect on c-Jun mRNA levels in BL3 cells (left) or TBL3 cells (right), as assessed by qPCR analysis. Transcript levels are shown relative to the control plasmids or scrambled siControls (gray bars) and normalized against β-actin and β2M mRNA (average ± sd, n = 3). (B) The miR155 Sponge had no significant effect on c-Jun mRNA levels in TBL3 cells treated or not with MG132, as assessed by qPCR analysis. Transcript levels are shown relative to the control plasmid and normalized against β-actin and B2M mRNA (average ± sd, n = 3). (C) miR155 inhibition in TBL3 cells increased c-Jun ubiquitination. Transfected TBL3 cells were treated with MG132 for 3 h, followed by immunoprecipitation of endogenous c-Jun protein and immunoblot analysis with antibodies against Ubiquitin or c-Jun (average ± sd, n = 3). (TIF)
- [Show abstract] [Hide abstract] ABSTRACT: The intracellular parasite Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet 'oncogene addiction' implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation.
- [Show abstract] [Hide abstract] ABSTRACT: In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination.
- [Show abstract] [Hide abstract] ABSTRACT: Viral hijacking of cellular processes relies on the ability to mimic the structure or function of cellular proteins. Many viruses encode ubiquitin ligases to facilitate infection, although the mechanisms by which they select their substrates are often unknown. The Herpes Simplex Virus type-1-encoded E3 ubiquitin ligase, ICP0, promotes infection through degradation of cellular proteins, including the DNA damage response E3 ligases RNF8 and RNF168. Here we describe a mechanism by which this viral E3 hijacks a cellular phosphorylation-based targeting strategy to degrade RNF8. By mimicking a cellular phosphosite, ICP0 binds RNF8 via the RNF8 forkhead associated (FHA) domain. Phosphorylation of ICP0 T67 by CK1 recruits RNF8 for degradation and thereby promotes viral transcription, replication, and progeny production. We demonstrate that this mechanism may constitute a broader viral strategy to target other cellular factors, highlighting the importance of this region of the ICP0 protein in countering intrinsic antiviral defenses.
- [Show abstract] [Hide abstract] ABSTRACT: Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.
- [Show abstract] [Hide abstract] ABSTRACT: The human APOBEC3 family of cytidine deaminases constitutes a cellular intrinsic defense mechanism that is effective against a range of viruses and retro-elements. While it is well established that these enzymes are powerful mutators of viral DNA, the possibility that their activity could threaten the integrity of the host genome has only recently begun to be investigated. Here, we discuss the implications of new evidence suggesting that APOBEC3 proteins can mediate the deamination of cellular DNA. The maintenance of genomic integrity in the face of this potential off-target activity must require high fidelity DNA repair and strict regulation of APOBEC3 gene expression and enzyme activity. Conversely, the ability of specific members of the APOBEC3 family to activate DNA damage signaling pathways might also reflect another way that these proteins contribute to the host immune response.
Dataset: Figure S6[Show abstract] [Hide abstract] ABSTRACT: RNF8 represses viral genomes and is part of the intrinsic anti-viral defense. (A) RNF8-/- MEFs transduced with control retrovirus or retrovirus expressing RNF8 were infected with WT or ICP0 null virus at an MOI of 0.01. ICP27 transcripts were detected at 2 or 5 hrs post infection and normalized to a cellular control. In both cell lines, ICP0 null virus was less transcriptionally competent than wild-type, in keeping with the known phenotype of this mutant virus. Experiments were performed in duplicate and averaged. Results are representative of three independent experiments and error is one standard deviation of the duplicate samples. (B) RIDDLE cells complemented either with empty vector or RNF168 were treated with lentivirus expressing either control shRNA or shRNA targeting RNF8. Levels of RNF8 and uH2A were assessed by western blot. (C) Cells depleted for RNF168 and/or RNF8 were infected with wild-type or ICP0-null virus. Relative probabilities of plaque formation were calculated by comparing the numbers of plaques on the different cell lines at each separate dilution of virus. (TIF)
University of California, San DiegoSan Diego, California, United States