Telomeric RNAs as a novel player in telomeric integrity

Telomeres protect linear chromosome ends from being recognized and processed as double-strand breaks by DNA repair activities. This protective function of telomeres is essential for chromosome stability. Until recently, telomeres have been considered to be transcriptionally silent. This notion was overturned in a series of recent papers that describe the existence of telomeric repeat-containing RNAs (TERRAs) in vertebrates and yeast. Here, we summarize recent developments in this field of telomere research, in particular the possible mechanisms that control TERRA expression

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2pro activity by the NS3 protease domain. NS2pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2pro activity mimic structural changes occurring during NS3-mediated NS2pro activation. Introducing these activating NS2pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2pro activation by NS3

Complex signals in the genomic 3′ nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA

The genomes of positive-strand RNA viruses strongly resemble cellular mRNAs. However, besides operating as a messenger to generate the virus-encoded proteins, the viral RNA serves also as a template during replication. A central issue of the viral life cycle, the coordination of protein and RNA synthesis, is yet poorly understood. Examining bovine viral diarrhea virus (BVDV), we report here on the role of the variable 3′V portion of the viral 3′ nontranslated region (3′NTR). Genetic studies and structure probing revealed that 3′V represents a complex RNA motif that is composed of synergistically acting sequence and structure elements. Correct formation of the 3′V motif was shown to be an important determinant of the viral RNA replication process. Most interestingly, we found that a proper conformation of 3′V is required for accurate termination of translation at the stop-codon of the viral open reading frame and that efficient termination of translation is essential for efficient replication of the viral RNA. Within the viral 3′NTR, the complex 3′V motif constitutes also the binding site of recently characterized cellular host factors, the so-called NFAR proteins. Considering that the NFAR proteins associate also with the 5′NTR of the BVDV genome, we propose a model where the viral 3′NTR has a bipartite functional organization: The conserved 3′ portion (3′C) is part of the nascent replication complex; the variable 5′ portion (3′V) is involved in the coordination of the viral translation and replication. Our data suggest the accuracy of translation termination as a sophisticated device determining viral adaptation to the host

Dom zu Fritzlar, Nordquerhaus. Fassadeninstandsetzung. Fugenarbeiten Dokumentation der Restaurierungsarbeiten am Querhaus Nord 1991

Videofilm (19 min) und BrochuereAvailable from TIB Hannover: YG3(1) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely

Replication Studies Using Genotype 1a Subgenomic Hepatitis C Virus Replicons

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-α), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-α may be attributed directly to the type 1a polymerase

Polyprotein driven formation of two independent sets of complexes supporting hepatitis C virus genome replication

Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from ORF2 and a second duplicate NS polyprotein from ORF1. Using these constructs and other methods, we have assessed polyprotein requirements needed for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings; those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue; one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N) and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, a S1977P mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two inter-dependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.IMPORTANCE:Positive strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing sub-genomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behaviour of distinct NS3 helicase knock-out mutations hints that certain conformations of this enzyme might be particularly deleterious for replication