Linking retroelements to autoimmunity

In this issue, Stetson et al. (2008) report a mechanism by which host cells avert an autoimmune response to self-nucleic acids. They show that the nuclease Trex1 prevents the accumulation of DNA derived from endogenous retroelements that, if left unchecked, trigger elevated production of type I interferons leading to autoimmunity.     

TREX1 DNA exonuclease deficiency, accumulation of single stranded DNA and complex human genetic disorders

Aicardi-Goutieres syndrome (AGS) is an unusual condition that clinically mimics a congenital viral infection. Several genes have recently been implicated in the aetiology of this disorder. One of these genes encodes the DNA exonuclease TREX1. Recent work from Yang, Lindahl and Barnes has provided insight into the cellular consequence of TREX1-deficiency. They found that TREX1-deficiency resulted in the intracellular accumulation of single stranded DNA resulting in chronic activation of the DNA damage response network, even in cells from Trex1-mutated AGS patients. Here, I summarise their findings and discuss them in context with the other AGS causative genes which encode subunits of the RNase H2 complex. I describe mechanisms by which the inappropriate intracellular accumulation of nucleic acid species might deleteriously impact upon normal cell cycle progression. Finally, using the example of Systemic Lupus Erythematosus (SLE), I also summarise the evidence suggesting that the failure to process intermediates of nucleic acid metabolism can result in the activation of uncontrolled autoimmunity.     

Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease

Trex1 is the major 3' DNA exonuclease in mammalian cells, and mutations in the human TREX1 gene can cause Aicardi-Goutières syndrome, characterized by perturbed immunity. Similarly, Trex1(-/-) mice have an autoinflammatory phenotype; however, the mechanism of Trex1-deficient disease is unknown. We report that Trex1, ordinarily associated with the endoplasmic reticulum (ER), relocalizes to the S phase nucleus after gamma irradiation or hydroxyurea treatment. Notably, Trex1-deficient cells show defective G1/S transition and chronic ATM-dependent checkpoint activation, even in the absence of exogenous stress, correlating with persistent single-stranded DNA molecules produced in S phase, which accumulate in the ER. Our data indicate that Trex1 acts on a single-stranded DNA polynucleotide species generated from processing of aberrant replication intermediates to attenuate DNA damage checkpoint signaling and prevent pathological immune activation.     

Unmasking immune sensing of retroviruses: Interplay between innate sensors and host effectors

Retroviruses can selectively trigger an array of innate immune responses through various PRR. The identification and the characterization of the molecular basis of retroviral DNA sensing by the DNA sensors IFI16 and cGAS has been one of the most exciting developments in viral immunology in recent years. DNA sensing by these cytosolic sensors not only leads to the initiation of the type I interferon (IFN) antiviral response and the induction of the inflammatory response, but also triggers cell death mechanisms including pyroptosis and apoptosis in retrovirus-infected cells, thereby providing important insights into the pathophysiology of chronic retroviral infection. Host restriction factors such as SAMHD1 and Trex1 play important roles in regulating innate immune sensing, and have led to the idea that innate immune defense and host restriction actually converge at different levels to determine the outcome of retroviral infection. In this review, we discuss the sensing of retroviruses by cytosolic DNA sensors, the relevance of host factors during retroviral infection, and the interplay between host factors and the innate antiviral response in different cell types, within the context of two human pathogenic retroviruses – human immunodeficiency virus (HIV-1) and human T cell-leukemia virus type I (HTLV-1).     

Trex2 enables spontaneous sister chromatid exchanges without facilitating DNA double-strand break repair

Trex2 is a 3' → 5' exonuclease that removes 3'-mismatched sequences in a biochemical assay; however, its biological function remains unclear. To address biology we previously generated trex2(null) mouse embryonic stem (ES) cells and expressed in these cells wild-type human TREX2 cDNA (Trex2(hTX2)) or cDNA with a single-amino-acid change in the catalytic domain (Trex2(H188A)) or in the DNA-binding domain (Trex2(R167A)). We found the trex2(null) and Trex2(H188A) cells exhibited spontaneous broken chromosomes and trex2(null) cells exhibited spontaneous chromosomal rearrangements. We also found ectopically expressed human TREX2 was active at the 3' ends of I-SceI-induced chromosomal double-strand breaks (DSBs). Therefore, we hypothesized Trex2 participates in DNA DSB repair by modifying 3' ends. This may be especially important for ends with damaged nucleotides. Here we present data that are unexpected and prompt a new model. We found Trex2-altered cells (null, H188A, and R167A) were not hypersensitive to camptothecin, a type-1 topoisomerase inhibitor that induces DSBs at replication forks. In addition, Trex2-altered cells were not hypersensitive to γ-radiation, an agent that causes DSBs throughout the cell cycle. This observation held true even in cells compromised for one of the two major DSB repair pathways: homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Trex2 deletion also enhanced repair of an I-SceI-induced DSB by both HDR and NHEJ without affecting pathway choice. Interestingly, however, trex2(null) cells exhibited reduced spontaneous sister chromatid exchanges (SCEs) but this was not due to a defect in HDR-mediated crossing over. Therefore, reduced spontaneous SCE could be a manifestation of the same defect that caused spontaneous broken chromosomes and spontaneous chromosomal rearrangements. These unexpected data suggest Trex2 does not enable DSB repair and prompt a new model that posits Trex2 suppresses the formation of broken chromosomes.     

Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts.     

SAMHD1抗艾滋病病毒作用研究进展

SAMHD1蛋白全称为不育-α-基序结构域(SAM域)和组氨酸/天冬氨酸残基双联体结构域(HD域)包涵蛋白1,是由真核生物SAMHD1基因编码的蛋白质。研究表明SAMHD1蛋白对机体固有免疫有调控作用,它能够明显上调抗病毒免疫应答,介导由干扰素引起的炎症反应,参与宿主对入侵病毒的防御体系。早期的研究主要集中在其基因突变引起的Aicardi-Goutières综合征(AGS),最新研究发现SAMHD1作为一种核酸水解酶,具有代谢耗竭髓样细胞和树突状细胞内dNTP池从而抑制I型艾滋病病毒(Human immunodeficiency virus,HIV-1)cDNA合成的功能。而HIV-2和猴免疫缺陷病毒(SIVsm/mac)辅助基因编码的Vpx蛋白则能拮抗SAMHD1对病毒复制的抑制作用。近年来,有关SAMHD1蛋白的功能及其抗病毒作用机制的研究进展迅速,本文主要对此加以综述。     

Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

Human APOBEC3A (A3A) is a single-domain cytidine deaminase that converts deoxycytidine residues to deoxyuridine in single-stranded DNA (ssDNA). It inhibits a wide range of viruses and endogenous retroelements such as LINE-1, but it can also edit genomic DNA, which may play a role in carcinogenesis. Here, we extend our recent findings on the NMR structure of A3A and report structural, biochemical and cell-based mutagenesis studies to further characterize A3A’s deaminase and nucleic acid binding activities. We find that A3A binds ssRNA, but the RNA and DNA binding interfaces differ and no deamination of ssRNA is detected. Surprisingly, with only one exception (G105A), alanine substitution mutants with changes in residues affected by specific ssDNA binding retain deaminase activity. Furthermore, A3A binds and deaminates ssDNA in a length-dependent manner. Using catalytically active and inactive A3A mutants, we show that the determinants of A3A deaminase activity and anti-LINE-1 activity are not the same. Finally, we demonstrate A3A’s potential to mutate genomic DNA during transient strand separation and show that this process could be counteracted by ssDNA binding proteins. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions.     

Excision of 3' termini by the Trex1 and TREX2 3'-->5' exonucleases. Characterization of the recombinant proteins

The excision of nucleotides from DNA 3' termini is an important step in DNA replication, repair, and recombination pathways to generate correctly base paired termini for subsequent processing. The mammalian TREX1 and TREX2 proteins contain potent 3'-->5' exonucleases capable of functioning in this capacity. To study the activities of these exonucleases we have developed strategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical characterization. The Trex1 and TREX2 proteins are homodimers that exhibit robust 3' excision activities with very similar preferred reaction conditions and preferences for specific DNA substrates. In a steady-state kinetic analysis, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2. The Michaelis constants derived from these data indicate similar apparent kcat values of 22 s(-1) for Trex1 and 16 s(-1) for TREX2 using single-stranded oligonucleotides. The apparent KM values of 19 nm for Trex1 and 190 nm for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2. An exonuclease competition assay was designed using heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2. The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for producing correctly base paired 3' termini.     

Autoimmunity caused by constitutive activation of cytoplasmic viral RNA sensors

RIG-I-like receptors (RLRs) are well-known viral sensors that trigger the antiviral interferon (IFN) response by recognizing the non-self signatures of viral RNAs. The proper induction of the IFN response is known to play a crucial role in protecting against viral infections, whereas aberrant activation can lead to autoimmune disorders. We herein provided an overview of the antiviral IFN response and autoimmunity, with a focus on recent studies describing autoimmunity caused by mutations in the cytoplasmic viral RNA sensor, melanoma differentiation-associated gene 5 (MDA5).     

APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.     

Human Endogenous Retrovirus Protein Activates Innate Immunity and Promotes Experimental Allergic Encephalomyelitis in Mice

Multiple sclerosis (MS) is a complex multifactorial disease of the central nervous system (CNS) for which animal models have mainly addressed downstream immunopathology but not potential inducers of autoimmunity. In the absence of a pathogen known to cause neuroinflammation in MS, Mycobacterial lysate is commonly used in the form of complete Freund''s adjuvant to induce autoimmunity to myelin proteins in Experimental Allergic Encephalomyelitis (EAE), an animal model for MS. The present study demonstrates that a protein from the human endogenous retrovirus HERV-W family (MSRV-Env) can be used instead of mycobacterial lysate to induce autoimmunity and EAE in mice injected with MOG, with typical anti-myelin response and CNS lesions normally seen in this model. MSRV-Env was shown to induce proinflammatory response in human macrophage cells through TLR4 activation pathway. The present results demonstrate a similar activation of murine dendritic cells and show the ability of MSRV-Env to trigger EAE in mice. In previous studies, MSRV-Env protein was reproducibly detected in MS brain lesions within microglia and perivascular macrophages. The present results are therefore likely to provide a model for MS, in which the upstream adjuvant triggering neuroinflammation is the one detected in MS active lesions. This model now allows pre-clinical studies with therapeutic agents targeting this endogenous retroviral protein in MS.     

DNA mismanagement leads to immune system oversight

Trex1, a major 3' DNA exonuclease in mammalian cells, has been thought to act primarily in DNA replication or repair. Surprisingly, the major phenotype resulting from Trex1 deficiency in humans and mice is a chronic inflammatory disease. In this issue, Yang et al. (2007) report that Trex1 deficiency causes chronic activation of the ATM-dependent DNA-damage checkpoint and accumulation of a discrete single-stranded DNA species in the cytoplasm, either of which could contribute to chronic inflammation.     

LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus.     

Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer‐related death worldwide. Activation of c‐MET increases tumour cell survival through the initiation of the DNA damage repair pathway. PARP is an essential key in the DNA damage repair pathway. The primary role of PARP is to detect and initiate an immediate cellular response to single‐strand DNA breaks. Tumours suppressor genes such as BRCA1/2 are closely associated with the DNA repair pathway. In BRCA1/2 mutations or deficiency status, cells are more likely to develop additional genetic alterations and chromosomal instability and can lead to cancer. In this study, we investigate the role of c‐MET and PARP inhibition in a gastric cancer model. We exploited functional in vitro and in vivo experiments to assess the antitumour potential of co‐inhibition of c‐MET (SU11274) and PARP (NU1025). This leads to a reduction of gastric cancer cells viability, especially after knockdown of BRCA1/2 through apoptosis and induction of γ‐Η2ΑΧ. Moreover, in AGS xenograft models, the combinatorial treatment of NU1025 plus SU11274 reduced tumour growth and triggers apoptosis. Collectively, our data may represent a new therapeutic approach for GC thought co‐inhibition of c‐MET and PARP, especially for patients with BRCA1/2 deficiency tumours.     

The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(ΔVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(ΔVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(ΔVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.     

NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway

Alagille syndrome (AGS) is caused by mutations in the gene for the Notch signaling pathway ligand Jagged1 (JAG1), which are found in 94% of patients. To identify the cause of disease in patients without JAG1 mutations, we screened 11 JAG1 mutation-negative probands with AGS for alterations in the gene for the Notch2 receptor (NOTCH2). We found NOTCH2 mutations segregating in two families and identified five affected individuals. Renal manifestations, a minor feature in AGS, were present in all the affected individuals. This demonstrates that AGS is a heterogeneous disorder and implicates NOTCH2 mutations in human disease.