Moloney leukemia virus type 10 inhibits reverse transcription and retrotransposition of intracisternal a particles.

Moloney leukemia virus type 10 protein (MOV10) is an RNA helicase that is induced by type I interferon. It inhibits HIV replication at several steps of its replicative cycle. Of interest, MOV10 is a component of mRNA processing (P) bodies, which inhibit retrotransposition (RTP) of intracisternal A particles (IAP). In this report, we studied the effects of MOV10 on IAP RTP and its dependence on P bodies. Indeed, MOV10 inhibited IAP RTP. It decreased significantly not only the products of reverse transcriptase but also its endogenous activity. MOV10 also associated with IAP RNA. Furthermore, although it was found in IAP virus-like particles, it did not affect their incorporation of IAP RNA, primer tRNAPhe (phenylalanine tRNA), or IAP Gag. Concerning P bodies, the exogenously expressed MOV10 had no effect on their size and number, and the inhibition of IAP RTP persisted despite the depletion of their RCK subunit. Thus, by interfering with reverse transcription, MOV10 inhibits IAP RTP, and this inhibition is independent of P bodies.

P bodies inhibit retrotransposition of endogenous intracisternal a particles.

mRNA-processing bodies (P bodies) are cytoplasmic foci that contain translationally repressed mRNA. Since they are important for the retrotransposition of Ty elements and brome mosaic virus in yeast cells, we assessed the role of P bodies in the movement of endogenous intracisternal A particles (IAPs) in mammalian cells. In contrast to the case for these other systems, their disruption via knockdown of RCK or eukaryotic initiation factor E transporter (eIF4E-T) increased IAP retrotransposition as well as levels of IAP transcripts, Gag proteins, and reverse transcription products. This increase was not mediated by impairing the microRNA pathway. Rather, the removal of P bodies shifted IAP mRNA from nonpolysomal to polysomal fractions. Although IAP mRNA localized to P bodies, Gag was targeted to the endoplasmic reticulum (ER), from which IAP buds. Thus, by sequestering IAP mRNA away from Gag, P bodies inhibit rather than promote IAP retrotransposition.

Frataxin deficiency induces Schwann cell inflammation and death.

Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.

Cell functions impaired by frataxin deficiency are restored by drug-mediated iron relocation.

Various human disorders are associated with misdistribution of iron within or across cells. Friedreich ataxia (FRDA), a deficiency in the mitochondrial iron-chaperone frataxin, results in defective use of iron and its misdistribution between mitochondria and cytosol. We assessed the possibility of functionally correcting the cellular properties affected by frataxin deficiency with a siderophore capable of relocating iron and facilitating its metabolic use. Adding the chelator deferiprone at clinical concentrations to inducibly frataxin-deficient HEK-293 cells resulted in chelation of mitochondrial labile iron involved in oxidative stress and in reactivation of iron-depleted aconitase. These led to (1) restoration of impaired mitochondrial membrane and redox potentials, (2) increased adenosine triphosphate production and oxygen consumption, and (3) attenuation of mitochondrial DNA damage and reversal of hypersensitivity to staurosporine-induced apoptosis. Permeant chelators of higher affinity than deferiprone were not as efficient in restoring affected functions. Thus, although iron chelation might protect cells from iron toxicity, rendering the chelated iron bioavailable might underlie the capacity of deferiprone to restore cell functions affected by frataxin deficiency, as also observed in FRDA patients. The siderophore-like properties of deferiprone provide a rational basis for treating diseases of iron misdistribution, such as FRDA, anemia of chronic disease, and X-linked sideroblastic anemia with ataxia.

Frataxin knockdown causes loss of cytoplasmic iron-sulfur cluster functions, redox alterations and induction of heme transcripts.

Frataxin protein deficiency causes the neurodegenerative disease Friedreich ataxia. We used inducible siRNA to order the consequences of frataxin deficiency that we and others have previously observed. The earliest consequence of frataxin deficiency was a defect in cytoplasmic iron-sulfur proteins. In the second phase, protein oxidative damage increased, and CuZnSOD was induced, as was the unfolded protein response (UPR), long before any decline in mitochondrial aconitase activity. In the third phase, mitochondrial aconitase activity declined. And in the fourth phase, coincident with the decrease in heme-containing cytochrome c protein, a transcriptional induction of the heme-dependent transcripts ALAS1 and MAOA occurred. These observations suggest that the earliest consequences of frataxin deficiency occur in ISC proteins of the cytoplasm, resulting in oxidative damage and stress and activation of the unfolded protein response which has been associated with neurological disease, and that later consequences involve mitochondrial iron-sulfur cluster deficiency, heme deficiency, and then increased heme biosynthesis.