Meztli Arguello

mTORC1 Controls Mitochondrial Activity and Biogenesis through 4E-BP-Dependent Translational Regulation

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

Chemical targeting of the innate antiviral response by histone deacetylase inhibitors renders refractory cancers sensitive to viral oncolysis

Intratumoral innate immunity can play a significant role in blocking the effective therapeutic spread of a number of oncolytic viruses (OVs). Histone deacetylase inhibitors (HDIs) are known to influence epigenetic modifications of chromatin and can blunt the cellular antiviral response. We reasoned that pretreatment of tumors with HDIs could enhance the replication and spread of OVs within malignancies. Here, we show that HDIs markedly enhance the spread of vesicular stomatitis virus (VSV) in a variety of cancer cells in vitro, in primary tumor tissue explants and in multiple animal models. This increased oncolytic activity correlated with a dampening of cellular IFN responses and augmentation of virus-induced apoptosis. These results illustrate the general utility of HDIs as chemical switches to regulate cellular innate antiviral responses and to provide controlled growth of therapeutic viruses within malignancies. HDIs could have a profoundly positive impact on the clinical implementation of OV therapeutics.

A functional C-terminal TRAF3-binding site in MAVS participates in positive and negative regulation of the IFN antiviral response

Recognition of viral RNA structures by the cytosolic sensor retinoic acid-inducible gene-I (RIG-I) results in the activation of signaling cascades that culminate with the generation of the type I interferon (IFN) antiviral response. Onset of antiviral and inflammatory responses to viral pathogens necessitates the regulated spatiotemporal recruitment of signaling adapters, kinases and transcriptional proteins to the mitochondrial antiviral signaling protein (MAVS). We previously demonstrated that the serine/threonine kinase IKKε is recruited to the C-terminal region of MAVS following Sendai or vesicular stomatitis virus (VSV) infection, mediated by Lys63-linked polyubiquitination of MAVS at Lys500, resulting in inhibition of downstream IFN signaling (Paz et al, Mol Cell Biol, 2009). In this study, we demonstrate that C-terminus of MAVS harbors a novel TRAF3-binding site in the aa450-468 region of MAVS. A consensus TRAF-interacting motif (TIM), 455-PEENEY-460, within this site is required for TRAF3 binding and activation of IFN antiviral response genes, whereas mutation of the TIM eliminates TRAF3 binding and the downstream IFN response. Reconstitution of MAVS−/− mouse embryo fibroblasts with a construct expressing a TIM-mutated version of MAVS failed to restore the antiviral response or block VSV replication, whereas wild-type MAVS reconstituted antiviral inhibition of VSV replication. Furthermore, recruitment of IKKε to an adjacent C-terminal site (aa 468–540) in MAVS via Lys500 ubiquitination decreased TRAF3 binding and protein stability, thus contributing to IKKε-mediated shutdown of the IFN response. This study demonstrates that MAVS harbors a functional C-terminal TRAF3-binding site that participates in positive and negative regulation of the IFN antiviral response.

Ubiquitin-regulated recruitment of IkappaB kinase epsilon to the MAVS interferon signaling adapter.

ABSTRACT Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-κB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IκB kinase ε (IKKε) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKε to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKε to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-κB activation. Thus, recruitment of IKKε to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-κB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKε and TBK-1 in the RIG-I/Mda5 pathway.

Disruption of the B-cell specific transcriptional program in HHV-8 associated primary effusion lymphoma cell lines.

Primary effusion lymphoma (PEL) is a lymphoproliferative disease of B-cell origin that is associated with HHV-8 infection. PEL cells harbor a non-B, non-T phenotype and lack significant surface immunoglobulin (Ig) expression, a characteristic that has not been fully explained. In the present study, we demonstrate that PEL cells constitutively express interferon regulatory factor (IRF)-4, a transcription factor that regulates the activity of the immunoglobulin light-chain enhancer elements λB and κE3′ through binding to a composite Ets-IRF site. IRF-4 activity requires its physical interaction with PU.1, an Ets family member involved in the activation of genes essential for B-cell development. However, in PEL-derived B-cell lines, PU.1 expression was completely abrogated; expression of the B cell specific transcription factor Oct-2, which is known to regulate PU.1 expression, was also abolished. Moreover, the B-cell-specific coactivator of octamer factors, BOB-1/OcaB, was expressed at very decreased levels in PEL cells. Ectopic expression of Oct-2 was able to fully restore PU.1 promoter activity in the PEL cell line BCBL-1, while PU.1 expression also reconstituted the activity of the λB Ets-IRF site. In addition, protein levels of BSAP/Pax-5 and IRF-8/ICSBP were undetectable in PEL cells. The pattern of transcription factor ablation observed in PEL was found to be comparable to that observed in classical Hodgkins disease-derived cell lines, which also lack B-cell-specific surface markers. These observations indicate that disruption of the B-cell-specific transcriptional program is likely to contribute to the incomplete B-cell phenotype characteristic of PEL cells.

Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5′ triphosphate (5′ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5′pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5′pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5′pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5′pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5′pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

Polo-like kinase 1 (PLK1) regulates interferon (IFN) induction by MAVS.

The mitochondria-bound adapter MAVS participates in IFN induction by recruitment of downstream partners such as members of the TRAF family, leading to activation of NF-κB, and the IRF3 pathways. A yeast two-hybrid search for MAVS-interacting proteins yielded the Polo-box domain (PBD) of the mitotic Polo-like kinase PLK1. We showed that PBD associates with two different domains of MAVS in both dependent and independent phosphorylation events. The phosphodependent association requires the phosphopeptide binding ability of PBD. It takes place downstream of the proline-rich domain of MAVS, within an STP motif, characteristic of the binding of PLK1 to its targets, where the central Thr234 residue is phosphorylated. Its phosphoindependent association takes place at the C terminus of MAVS. PLK1 strongly inhibits the ability of MAVS to activate the IRF3 and NF-κB pathways and to induce IFN. Reciprocally, depletion of PLK1 can increase IFN induction in response to RIG-I/SeV or RIG-I/poly(I)-poly(C) treatments. This inhibition is dependent on the phosphoindependent association of PBD at the C terminus of MAVS where it disrupts the association of MAVS with its downstream partner TRAF3. IFN induction was strongly inhibited in cells arrested in G2/M by nocodazole, which provokes increased expression of endogenous PLK1. Interestingly, depletion of PLK1 from these nocodazole-treated cells could restore, at least partially, IFN induction. Altogether, these data demonstrate a new function for PLK1 as a regulator of IFN induction and provide the basis for the development of inhibitors preventing the PLK1/MAVS association to sustain innate immunity.

Vesicular Stomatitis Virus Oncolysis of T Lymphocytes Requires Cell Cycle Entry and Translation Initiation

ABSTRACT Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4+ T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G0. Activation of primary CD4+ T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH2-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G1 arrest, but not by aphidicolin or Taxol, which blocks at the G11S or G21M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G0/G1 transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G0 phase to the G1 phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.

The 4E-BP–eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes

Lymphocyte reliance on 4E-BP2 and eIF4E for growth and proliferation underlies their exquisite sensitivity to the drug rapamycin. 4E-BP, the key to lymphocyte sensitivity In most cells, the mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) regulates cell growth through the ribosomal S6 kinases (S6Ks) and cell proliferation through translation-regulating proteins of the eIF4E-binding protein (4E-BP) family, respectively. Although mTORC1 is present in all cells, the mTORC1 inhibitor rapamycin is an effective immunosuppressant that blocks lymphocyte proliferation (see the Focus by Abraham). But, why are lymphocytes so exquisitely sensitive? So et al. found that lymphocytes did not depend on S6K signaling to promote growth or proliferation in response to antigen receptor stimulation. Instead, these cells relied on 4E-BP proteins for both processes. Unlike in nonlymphoid cells, the 4E-BP2 isoform was more abundant than the 4E-BP1 isoform in lymphocytes, and its phosphorylation by mTORC1 was more sensitive to rapamycin. These data suggest that the exquisite sensitivity of lymphocytes to rapamycin may be due to their complete reliance on 4E-BP2 for both growth and proliferation. Rapamycin has been used as a clinical immunosuppressant for many years; however, the molecular basis for its selective effects on lymphocytes remains unclear. We investigated the role of two canonical effectors of the mammalian target of rapamycin (mTOR): ribosomal S6 kinases (S6Ks) and eukaryotic initiation factor 4E (eIF4E)–binding proteins (4E-BPs). S6Ks are thought to regulate cell growth (increase in cell size), and 4E-BPs are thought to control proliferation (increase in cell number), with mTORC1 signaling serving to integrate these processes. However, we found that the 4E-BP–eIF4E signaling axis controlled both the growth and proliferation of lymphocytes, processes for which the S6Ks were dispensable. Furthermore, rapamycin disrupted eIF4E function selectively in lymphocytes, which was due to the increased abundance of 4E-BP2 relative to that of 4E-BP1 in these cells and the greater sensitivity of 4E-BP2 to rapamycin. Together, our findings suggest that the 4E-BP–eIF4E axis is uniquely rapamycin-sensitive in lymphocytes and that this axis promotes clonal expansion of these cells by coordinating growth and proliferation.

The eukaryotic translation initiation factor eIF4E is a direct transcriptional target of NF-κB and is aberrantly regulated in acute myeloid leukemia

The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.