Search results for "RNA-Dependent RNA Polymerase"

Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.

2000

AbstractPosttranscriptional gene silencing (PTGS) in plants results from the degradation of mRNAs and shows phenomenological similarities with quelling in fungi and RNAi in animals. Here, we report the isolation of sgs2 and sgs3 Arabidopsis mutants impaired in PTGS. We establish a mechanistic link between PTGS, quelling, and RNAi since the Arabidopsis SGS2 protein is similar to an RNA-dependent RNA polymerase like N. crassa QDE-1, controlling quelling, and C. elegans EGO-1, controlling RNAi. In contrast, SGS3 shows no significant similarity with any known or putative protein, thus defining a specific step of PTGS in plants. Both sgs2 and sgs3 mutants show enhanced susceptibility to virus, d…

Engineering of a DNA Polymerase for Direct m6A Sequencing

2017

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N6-methyladenosine (m6A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m6A-containing RNA prior to sequencing, since m6A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m6A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for…

Sentinel hospital-based surveillance for norovirus infection in children with gastroenteritis between 2015 and 2016 in Italy

2018

Noroviruses are one of the leading causes of gastro-enteric diseases worldwide in all age groups. Novel epidemic noroviruses with GII.P16 polymerase and GII.2 or GII.4 capsid type have emerged worldwide in late 2015 and in 2016. We performed a molecular epidemiological study of the noroviruses circulating in Italy to investigate the emergence of new norovirus strains. Sentinel hospital-based surveillance, in three different Italian regions, revealed increased prevalence of norovirus infection in children (<15 years) in 2016 (14.4% versus 9.8% in 2015) and the emergence of GII.P16 strains in late 2016, which accounted for 23.0% of norovirus infections. The majority of the strains with a GII.…

2015

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m(1)A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m(1)A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored…

Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein.

2003

The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular co-localization of endogenous a…

Inhibitors acting on nucleic acid synthesis in an oncogenic RNA virus.

1971

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell1–4. Several specific inhibitors of viral DNA polymerases have been found5–7 and Spiegelman8 has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of…

A comparative analysis to study editing of small noncoding BC200- and Alu transcripts in brain of prion-inoculated rhesus monkeys (M. Mulatta).

2012

Small retroelements (short interspersed elements, abbreviated SINEs) are abundant in vertebrate genomes. Using RNA isolated from rhesus monkey cerebellum and buffy coat, reverse-transcription polymerase chain reaction (RT PCR) was applied to clone cDNA of BC200 and Alu RNAs. Transcripts containing Alu-SINE sequences may be subjected to extensive RNA editing by ADAR (adenosine deaminases that act on RNA) deamination. Abundance of Alu transcripts was determined with real-time RT PCR and was significantly higher than BC200 (brain cytoplasmic) in cerebellum. BC200 transcripts were absent from buffy coat cells. Availability of the rhesus genome sequence allowed the BC200 transcripts to be mapped…

A frame shift mutation in a hot spot region of the nuclear autoantigen La (SS-B).

1996

A hot spot region was identified in the exon 7 of the nuclear autoantigen La (SS-B). Two La cDNAs were identified which contained a frame shift mutation in the hot spot region. One La cDNA was isolated from a cDNA library made from peripheral blood lymphocytes of an autoimmune patient with primary Sjogren's Syndrome, the other La cDNA was isolated from a human liver cDNA library. The patient's La cDNA had a deletion and the liver La cDNA had an insert of an (A)-residue at the same position. Inserts of 4, 16 and 24 more or less homogeneous (A)-residues were found at the same site in the three La retropseudogenes. The hot spot region located in one of the major autoepitope regions of the La a…

Cloning and expression of a cDNA copy of the viral K28 killer toxin gene in yeast

1995

The killer toxin K28, secreted by certain killer strains of the yeast Saccharomyces cerevisiae is genetically encoded by a 1.9 kb double-stranded RNA, M-dsRNA (M28), that is present within the cell as a cytoplasmically inherited virus-like particle (VLP). For stable maintenance and replication, M28-VLPs depend on a second dsRNA virus (LA), which has been shown to encode the major capsid protein (cap) and a capsid-polymerase fusion protein (cap-pol) that provides the toxin-coding M-satellites with their transcription and replicase functions. K28 toxin-coding M28-VLPs were isolated, purified and used in vitro for the synthesis of the single-stranded M28 transcript, which was shown to be of pl…

Selective Stimulation of Hepatitis C Virus and Pestivirus NS5B RNA Polymerase Activity by GTP

1999

NS5B of the hepatitis C virus is an RNA template-dependent RNA polymerase and therefore the key player of the viral replicase complex. Using a highly purified enzyme expressed with recombinant baculoviruses in insect cells, we demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP. Enhancement of RNA synthesis was found with various heteropolymeric RNA templates, with poly(C)-oligo(G)12 but not with poly(A)-oligo(U)12. Several amino acid substitutions in polymerase motifs B, C, and D previously shown to be crucial for RdRp activity were tested for GTP stimulation of RNA synthesis. Most of these mutations, …