Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA.
ABSTRACT: The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X dsRNA, of the L-BC dsRNA virus, and of the single-stranded replicon 20S RNA. We show that SKI2 encodes a 145-kDa protein with motifs characteristic of helicases and nucleolar proteins and is essential only in cells carrying M dsRNA. Unexpectedly, Ski2p does not repress M1 dsRNA copy number when M1 is supported by aN L-A cDNA clone; nonetheless, it did lower the levels of M1 dsRNA-encoded toxin produced. Since toxin secretion from cDNA clones of M1 is unaffected by Ski2p, these data suggest that Ski2p acts by specifically blocking translation of viral mRNAs, perhaps recognizing the absence of cap or poly(A). In support of this idea, we find that Ski2p represses production of beta-galactosidase from RNA polymerase I [no cap and no poly(A)] transcripts but not from RNA polymerase II (capped) transcripts.
Project description:The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p-like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at approximately 90 degrees C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 A using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N-terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix-hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex.
Project description:Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5'-terminal coding region followed by two internal A-rich sequences and a 3'-terminal region without coding capacity. Mlus positive strands carry cis-acting signals at their 5' and 3' termini for transcription and replication similar to those of killer viruses. The open reading frame (ORF) at the 5' portion codes for a putative preprotoxin with an N-terminal secretion signal, potential Kex2p/Kexlp processing sites, and N-glycosylation sites. No sequence homology was found either between the Mlus dsRNA and M1, M2, or M28 dsRNA or between Klus and the K1, K2, or K28 toxin. The Klus amino acid sequence, however, showed a significant degree of conservation with that of the product of the host chromosomally encoded ORF YFR020W of unknown function, thus suggesting an evolutionary relationship.
Project description:Ski2 is a cytoplasmic RNA helicase that functions together with the exosome in the turnover and quality control of mRNAs. Ski2 is conserved in eukaryotes and is related to the helicase Mtr4, a cofactor of the nuclear exosome involved in the processing and quality control of a variety of structured RNAs. We have determined the 2.4 Å resolution crystal structure of the 113 kDa helicase region of Saccharomyces cerevisiae Ski2. The structure shows that Ski2 has an overall architecture similar to that of Mtr4, with a core DExH region and an extended insertion domain. The insertion is not required for the formation of the Ski2-Ski3-Ski8 complex, but is instead an RNA-binding domain. While this is reminiscent of the Mtr4 insertion, there are specific structural and biochemical differences between the two helicases. The insertion of yeast Mtr4 consists of a ?-barrel domain that is flexibly attached to a helical stalk, contains a KOW signature motif, and binds in vitro-transcribed tRNA(i)(Met), but not single-stranded RNA. The ?-barrel domain of yeast Ski2 does not contain a KOW motif and is tightly packed against the helical stalk, forming a single structural unit maintained by a zinc-binding site. Biochemically, the Ski2 insertion has broad substrate specificity, binding both single-stranded and double-stranded RNAs. We speculate that the Ski2 and Mtr4 insertion domains have evolved with different properties tailored to the type of transcripts that are the substrates of the cytoplasmic and nuclear exosome.
Project description:A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5'-3' exoribonuclease of the ?-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein-protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally, Thermococcus barophilus whole-cell extract fractionation experiments provide evidences that an aRNase J/ASH-Ski2 complex might exist in vivo and hint at an association of aRNase J with the ribosome that is emphasised in absence of ASH-Ski2. Whilst aRNase J homologues are found among bacteria, the RNA exosome and the Ski2-like RNA helicase have eukaryotic homologues, underlining the mosaic aspect of archaeal RNA machines. Altogether, these results suggest a fundamental role of ?-CASP RNase/helicase complex in archaeal RNA metabolism.
Project description:A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.
Project description:A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.
Project description:A small double-stranded (ds) RNA element was isolated from a moderately hypovirulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. from eastern New Jersey. Virulence was somewhat lower in the dsRNA-containing strain than in a virulent dsRNA-free control strain, but colony morphology and sporulation levels were comparable. A library of cDNA clones was constructed, and overlapping clones representing the entire genome were sequenced. The 2728-bp dsRNA was considerably smaller than previously characterized C. parasitica dsRNAs, which are 12-13 kb and ancestrally related to the Potyviridae family of plant viruses. Sequence analysis revealed one large open reading frame, but only if mitochondrial codon usage (UGA = Trp) was invoked. Nuclease assays of purified mitochondria confirmed that the dsRNA was localized within mitochondria. Assuming mitochondrial translation, the deduced amino acid sequence had landmarks typical of RNA-dependent RNA polymerases. Alignments of the conserved regions indicate that this dsRNA is more closely related to yeast T and W dsRNAs and single-stranded RNA bacteriophages such as Q beta than to other hypovirulence-associated dsRNAs.
Project description:To determine transcriptomic changes in cellular targets induced by MCV-miR-M1. Briefly, HEK293 cells were transfected with either MCV-miR-M1-5p, MCV-miR-M1-3p or control mimic (Thermo Fisher Scientific) prior to RNA extraction and confirmation of MCV miRNA 5p and 3p expression via stem loop qRT-PCR. Total RNA libraries were prepared using TruSeq Stranded Total RNA Sample Prep Kit (Illumina, USA) and the TruSeq cDNA libraries were analysed via Illumina HiSeq2500 paired end 100bp.
Project description:Three small and distinct satellite double-stranded RNAs (dsRNAs) denoted s1, s1', and s2 were recently described for a Trichomonas vaginalis isolate harboring a dsRNA virus. Since characterization of these satellite dsRNAs might provide insight into the virus replication cycle and virus-host interactions, full-length cDNAs to s1 and s1' dsRNAs were synthesized and sequenced. s1 dsRNA has 688 bp, and s1' dsRNA has 616 bp. A 228-bp open reading frame that begins at nucleotide 37 was detected on a putative sense strand of s1. All satellite RNAs were found associated with RNA-dependent RNA polymerase (RDRP) activity that banded on CsCl gradients. Within carrier trichomonads, satellite RNAs synthesized single-stranded replicative intermediates. An in vitro assay was established to assess replication of satellite RNAs. Transcripts generated from s1 cDNA, for example, served as a template for the viral RDRP. These templates had a polarity similar to that of the replicative intermediate found in the satellite-harboring parasites. Importantly, the recognition of s1 RNA was shown to be specific, since unrelated RNAs did not serve as templates for RDRP under the same experimental conditions. The data indicate that the cDNA of s1 has a specific and essential sequence needed for recognition by the viral RDRP and for subsequent RNA synthesis. Both s1 and s1' have conserved domains, albeit of unproven function, but which may be required for replication.
Project description:Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.