Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5'-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

HIV-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors (PRRs). We hypothesized that, if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors, then manipulation of specific interactions between HIV-1 capsid (CA) and host factors that putatively regulate these processes should trigger PRRs and stimulate type 1 interferon secretion. Here we show that HIV-1 CA mutants N74D and P90A, which are impaired for interaction with cofactors Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA) respectively(1-2), cannot replicate in primary human monocyte derived macrophages (MDM) because they trigger innate sensors leading to nuclear translocation of NFκB and IRF3, the production of soluble type-1 interferon (IFN) and induction of an antiviral state. Depletion of CPSF6 with shRNA expression allows wild type virus to trigger innate sensors and interferon production. In each case, suppressed replication is rescued by IFN-receptor blockade demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration suggesting that a viral reverse transcription product comprises the HIV-1 pathogen associated molecular pattern (PAMP). Finally, we show that we can pharmacologically induce wild type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to utilize CPSF6 and cyclophilins to cloak its replication allowing evasion of innate immune sensors and induction of a cell autonomous innate immune response in primary human macrophages (Extended Data Fig 1).

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

BackgroundXenotropic murine leukaemia viruses (MLV-X) are endogenous gammaretroviruses that infect cells from many species, including humans. Xenotropic murine leukaemia virus-related virus (XMRV) is a retrovirus that has been the subject of intense debate since its detection in samples from humans with prostate cancer (PC) and chronic fatigue syndrome (CFS). Controversy has arisen from the failure of some studies to detect XMRV in PC or CFS patients and from inconsistent detection of XMRV in healthy controls.ResultsHere we demonstrate that Taqman PCR primers previously described as XMRV-specific can amplify common murine endogenous viral sequences from mouse suggesting that mouse DNA can contaminate patient samples and confound specific XMRV detection. To consider the provenance of XMRV we sequenced XMRV from the cell line 22Rv1, which is infected with an MLV-X that is indistinguishable from patient derived XMRV. Bayesian phylogenies clearly show that XMRV sequences reportedly derived from unlinked patients form a monophyletic clade with interspersed 22Rv1 clones (posterior probability >0.99). The cell line-derived sequences are ancestral to the patient-derived sequences (posterior probability >0.99). Furthermore, pol sequences apparently amplified from PC patient material (VP29 and VP184) are recombinants of XMRV and Moloney MLV (MoMLV) a virus with an envelope that lacks tropism for human cells. Considering the diversity of XMRV we show that the mean pairwise genetic distance among env and pol 22Rv1-derived sequences exceeds that of patient-associated sequences (Wilcoxon rank sum test: p = 0.005 and p < 0.001 for pol and env, respectively). Thus XMRV sequences acquire diversity in a cell line but not in patient samples. These observations are difficult to reconcile with the hypothesis that published XMRV sequences are related by a process of infectious transmission.ConclusionsWe provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice. We propose that XMRV might not be a genuine human pathogen.

TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved.

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive “signaling waves” revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.

DNA methylation is vital for proper chromatin structure and function in mammalian cells. Genetic removal of the enzymes that catalyze DNA methylation results in defective imprinting, transposon silencing, X chromosome dosage compensation, and genome stability. This epigenetic modification is interpreted by methyl-DNA binding domain (MBD) proteins. MBD proteins respond to methylated DNA by recruiting histone deacetylases (HDAC) and other transcription repression factors to the chromatin. The MBD2 protein is dispensable for animal viability, but it is implicated in the genesis of colon tumors. Here we report that the MBD2 protein is controlled by arginine methylation. We identify the protein arginine methyltransferase enzymes that catalyze this modification and show that arginine methylation inhibits the function of MBD2. Arginine methylation of MBD2 reduces MBD2-methyl-DNA complex formation, reduces MBD2-HDAC repression complex formation, and impairs the transcription repression function of MBD2 in cells. Our report provides a molecular description of a potential regulatory mechanism for an MBD protein family member. It is the first to demonstrate that protein arginine methyltransferases participate in the DNA methylation system of chromatin control.The information contained within the DNA sequences of many organisms is augmented by epigenetic modifications of DNA and proteins bound to it. Methylation of cytosines in the context of CG dinucleotides is the predominant epigenetic modification of vertebrate genomes (27,43). The majority of CG sites appears to be methylated in nonembryonic cells; only CG-rich segments located in gene control regions are generally unmethylated (7,71).Methylation is catalyzed postsynthetically by DNA methyltransferase (DNMT) enzymes (15, 27). DNMT1 is the major maintenance methyltransferase, and it ensures that newly synthesized DNA retains the methylation pattern of the template strand; DNMT3a and DNMT3b are de novo methyltransferases, setting up the methyl-CG landscape of the genome early in development. DNMT3L has no intrinsic enzyme activity, but it is essential for genome methylation, serving as a cofactor for DNMT3a and DNMT3b. DNMT2 has no detectable DNA methylation activity and was recently reclassified as a tRNA methyltransferase (28).DNA methylation is vital for proper chromatin structure and function: genetic inactivation of each DNMT reveals its roles in X chromosome dosage compensation (3, 68), transposon silencing (12, 79), imprinting (13,30,37,42,53), and chromosome stability (20). These physiological phenomena have in common chromatin silencing.At the molecular level, the methyl-CG mark can be attractive or repulsive to DNA binding factors that affect chromatin activity (43). The most thoroughly characterized set of factors that are attracted to methyl-CG is the methyl-DNA binding domain (MBD) protein family (31,52,59,64). These proteins share a highly conserved MBD, which most of the five family members use to recognize methylated DNA (Fig. 1). Outside this region, the...

c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. We demonstrate here that Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role.c-Abl is a ubiquitously expressed non-receptor tyrosine kinase. This protein has Src homology domains (SH1, SH2, and SH3) 1 at the N terminus and a DNA binding domain, an actin binding domain, three nucleus localization signals, and a proline-rich motif at the C terminus (reviewed in Refs. 1 and 2). The C terminus is essential for c-Abl function and is a unique feature not found in other Src family members. c-Abl is localized in both the nucleus and the cytoplasm. Movement between these two compartments may play an important role in regulating c-Abl function (3, 4). c-Abl is activated by DNA damage, oxidative stress, cell adhesion to extracellular matrix, growth factors, and Src family kinases (reviewed in Refs. 1 and 2). However, the physiological function of c-Abl is not well understood. Mice lacking c-Abl showed perinatal death, runtedness, reduced fertility, lymphopenia, and osteoporosis (5-7). Arg (Abl-related protein), a c-Abl homologue, has a similar structure (8). Arg(Ϫ/Ϫ) mice develop normally, whereas embryos deficient in both Abl and Arg suffer from defects in neurulation and die before 11 days postcoitum (9). c-Abl plays an important role in apoptosis (1). Ectopic expression of c-Abl in fibroblasts causes apoptosis in a p53-independent manner (10, 11). Epithelial cells...