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 A system of six yeast chromosomal genes (SK12, -3, -4, -6, -7, and -8) lowers the copy numbers of the L-A, M, X, and L-BC dsRNA viruses and of the ssRNA replicon 20S RNA (3,17,44,54,65 (70), and the ski mutations also suppress mkt mutations (54).The SK13 and SK18 genes have been cloned and characterized. The SK13 product is a 163-kDa nuclear protein (52) with several copies of an amino acid repeat pattern, the TPR repeat, of unknown function (62). The SK18 protein has two copies of a different repeat amino acid sequence pattern first identified in ,B-transducin (45). Deletion mutations of either SK13 or SK18 had no effect on cell growth unless an M replicon was present. In that case, the cells were cold sensitive for growth as discussed above. For this reason, we view the SKI system as a dedicated antiviral system. However, the mechanism by which the SKI proteins interfere with the propagation of the RNA replicons is completely unknown.We report here that Ski2p resembles helicases and nucleolar proteins and present evidence that Ski2p acts by blocking the translation of viral mRNA. MATERIALS AND METHODSStrains and media. The yeast strains used in this study are shown in Table 1. Escherichia coli DHSa was used to propagate plasmid DNA, strain MV1190 and M13 helper phage K07 were used for the isolation of single-stranded plasmid DNA for DNA sequencing, and strain CJ236 was used for the production of uracil-containing single-stranded plasmid DNA for site-directed mutagenesis (40). Yeast strains were grown on YPAD, SD, H-His, H-Trp, H-Ura, or 4.7MB medium (70). E. coli strains were grown on LB, TB, or M9 medium (43).The strains in which M or X is supported by an L-A cDNA clone were constructed as follows: a strain derived from strain 3221 harboring L-A and either M1 or X (produced by cytoduction into strain 3221) was transformed with the L-A 4331

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

“…A centromeric SKI2 plasmid (pAJ39) was constructed by moving a SKI2-containing XbaI/Sst I fragment from pAJ63 (p2-18/8-4) (Johnson & Kolodner, 1995) into XbaI/Sst I-digested pRS315+ Because epitope tagging Ski2p at the amino or carboxyl terminal ends yielded a nonfunctional protein (Widner & Wickner, 1993), we created an internally triple-c-myc-tagged Ski2p by inserting the epitope-containing 123-bp BamHI fragment from pKB241 (S+ Kron and G+ Fink) into BamHI-digested pRDK331, thus replacing a 27-bp BamHI fragment of the SKI2 gene+ SKI2cmyc was moved from the resultant plasmid (pAJ159) to pRS315 on an Sst I fragment, creating pAJ160+ Both SKI2cmyc-bearing plasmids complemented the lethality of xrn1⌬ ski2-1-14 (data not shown)+ The SKI8 gene was amplified by PCR from wild-type genomic DNA using primers containing Sal I and HindIII sites and then digested and ligated into Sal I/HindIII-digested pEG(KT), creating a galactose inducible GST-SKI8 fusion (pAJ261)+ A galactose-inducible wild-type SKI8 construct (pAJ267) was made by moving SKI8 on a Sal I/HindIII fragment from pAJ261 into XhoI/HindIII-digested pRDK249 (Johnson & Kolodner, 1991)+ The SKI8 gene was also moved on a XhoI/HindIII fragment from pAJ261 into XhoI/HindIII-digested pRS416, placing SKI8 under the control of the CYC1 promoter (pAJ581)+…”

“…The superkiller (SKI ) genes in yeast are encoded in the nuclear genome+ They were initially identified from mutations that caused overexpression of a killer toxin encoded by the endogenous double-stranded RNA, denoted as M, a satellite of the double-stranded RNA virus L-A (Toh-e et al+, 1978;Ridley et al+, 1984;reviewed in Wickner, 1996areviewed in Wickner, , 1996b)+ Subsequent work demonstrated that SKI2, SKI3, SKI6, SKI7, and SKI8 are necessary to repress translation of poly(A) minus RNAs (Widner & Wickner, 1993;Masison et al+, 1995;Benard et al+, 1998Benard et al+, , 1999, whereas SKI1 is XRN1 (Johnson & Kolodner, 1995) and encodes a 59-exoribonuclease (Larimer et al+, 1992)+ In strains lacking both M and L-A, double mutants of xrn1⌬ and either ski2 or ski3 mutations are not viable (Johnson & Kolodner, 1995) or are temperature sensitive (Benard et al+, 1999), depending on strain background+ These observations indicate that the SKI genes are necessary for a general cellular function in addition to an antiviral activity (Johnson & Kolodner, 1995)+ ski8 mutations also are synthetic lethal with xrn1⌬ (Jacobs Anderson & Parker, 1998; J+T+ Brown & A+W+ Johnson, unpubl+ observation)+ More recently, SKI2, SKI3, SKI6, and SKI8 have been shown to be required for a 39-mRNA degradation pathway (Jacobs Anderson & Parker, 1998)+ Ski6p/Rrp41p itself is a 39-exoribonuclease and is an essential component of the exosome (Mitchell et al+, 1997), a large complex of 39-exoribonucleases that is involved in multiple RNA processing events in ribosome biogenesis (Mitchell et al+, 1996(Mitchell et al+, , 1997Zanchin & Goldfarb, 1999)+…”

“…The relationship between 39-degradation and repression of translation of poly(A) minus mRNA by the SKI genes is unclear+ The SKI gene products may act directly in a 39-mRNA degradation pathway with translation effects being secondary (Jacobs Anderson & Parker, 1998)+ Ski2p is a 146-kDa putative RNA helicase of the DEVH family (Widner & Wickner, 1993) and shares some similarity with Suv3p, a yeast mitochondrial RNA helicase (Stepien et al+, 1992)+ Suv3p is a component of the mitochondrial degradosome that also contains the 39-exoribonuclease Dss1p/Msu1p (Dziembowski et al+, 1998) and is required for intron degradation in mitochondria (Margossian et al+, 1996)+ Similarly, the Escherichia coli degradosome, a complex containing RNase E, polynucleotide phosphorylase, and the DEADbox RNA helicase RhlB, is required for 39 degradation of mRNA (Py et al+, 1996;Coburn & Mackie, 1999;reviewed in Carpousis et al+, 1999)+ In vitro analysis of the E. coli degradosome indicates that RhlB is required for degradation past secondary structures (Py et al+, 1996)+ Because among the SKI genes only SKI6 is known to encode an exonuclease activity, Ski2p, Ski3p and Ski8p may act to recruit the exosome to mRNA or act on the mRNP to allow access by the exosome (Jacobs Anderson & Parker, 1998)+ Alternatively, it has been suggested that the SKI gene products act in some way on the ribosome to confer specificity for translating poly(A) mRNA (Wickner, 1996a(Wickner, , 1996bBenard et al+, 1998Benard et al+, , 1999)+ This interpretation is supported by the finding that a ski6-2 mutant displays a defect in 60S ribosomal subunit biogenesis (Benard et al+, 1998) and poly(A) minus mRNA is translated at levels 10-to 34-fold higher when electroporated into ski2, ski3, ski6, or ski8 mutants as compared to wildtype (Masison et al+, 1995;Benard et al+, 1998)+ Furthermore, the human homolog Ski2w is localized primarily to the nucleolus with a smaller amount in the cytoplasm in apparent association with 40S ribosomal subunits (Qu et al+, 1998), suggesting a direct role in ribosome assembly or function+…”

Domain interactions within the Ski2/3/8 complex and between the Ski complex and Ski7p

“…Defects in the SKI genes lead to increased translation of poly(A) minus mRNAs (Benard et al 1998(Benard et al , 1999. Recently, our lab has shown that the effect of ski mutants to enhance translation of poly(A) minus mRNA (Widner and Wickner 1993) can be phenocopied for a given mRNA by the inclusion of RNA elements in cis within the 3 0 -UTR of the transcript that inhibit 3 0 -decay (Brown and Johnson 2001). Thus, the effect of ski mutations on poly(A) minus mRNA is likely due to defects in 3 0 -decay rather than a direct effect on the translation machinery itself (Widner and Wickner 1993;Benard et al 1998Benard et al , 1999.…”

Monitoring Sentinel of Diarrhea: A Rotavirus in the Site of Hospital Complex Pediatrique of Kingasani II: 2009-2014

“…Clinicians who are members of the site identify the patients and complete an investigation form for each case admitted with severe acute diarrhea. This form is completed at the site laboratory with all test results and then returned to the clinicians for patient follow-up until discharge, then sent to the manager for compilation and re-sent to the expanded immunization program managers (EPI), the National Institute for Biological Research (INRB), and the WHO were the analysis is carried out at each level [14][15][16][17][18][19].…”