Sindbis virus messenger RNA: the 5′-termini and methylated residues of 26 and 42 S RNA

Dubin, Donald T.; Stollar, Victor; HsuChen, Chuen-Chin; Timko, Kathleen D.; Guild, Gregory M.

doi:10.1016/0042-6822(77)90471-8

Cited by 75 publications

(34 citation statements)

References 48 publications

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“…In alphaviruses, such a domain may function to compete with cellular homologs or have separate RNA-associated activities mediated by a substrate-binding domain that is conserved in cellular and viral homologs also and that may target rRNA or viral RNA species (A. Gorbalenya, personal communication). Consistent with the absence of active 2Ј-O-MT, the SIN genome and 26S mRNA possess only type 0 cap structures (8). Finding a similar phenotype for nsP2 mutants that map outside of this domain suggests the 2Ј-O-MT-associated function is not responsible (solely) for the PI phenotype or that substitution at distant sites affects this C-domain sequence once it is folded.…”

Section: Discussionmentioning

confidence: 52%

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

Sawicki

Perri²,

Polo³

et al. 2006

J Virol

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In order to establish nonlytic persistent infections (PI) of BHK cells, replicons derived from Sindbis (SIN) and Semliki Forest (SFV) viruses have mutations in nsP2. Five different nsP2 PI replicons were compared to wild-type (wt) SIN, SFV, and wt nsPs SIN replicons. Replicon PI BHK21 cells had viral RNA synthesis rates that were less than 5% of those of the wt virus and ϳ10% or less of those of SIN wt replicon-infected cells, and, in contrast to wt virus and replicons containing wt nsP2, all showed a phenotype of continuous minus-strand synthesis and of unstable, mature replication/transcription complexes (RC؉) that are active in plus-strand synthesis. Minus-strand synthesis and incorporation of [ 3 H]uridine into replicative intermediates differed among PI replicons, depending on the location of the mutation in nsP2. Minus-strand synthesis by PI cells appeared normal; it was dependent on continuous P123 and P1234 polyprotein synthesis and ceased when protein synthesis was inhibited. The failure by the PI replicons to shut off minus-strand synthesis was not due to some defect in the PI cells but rather was due to the loss of some function in the mutated nsP2. This was demonstrated by showing that superinfection of PI cells with wt SFV triggered the shutdown of minus-strand synthesis, which we believe is a host response to infection with alphaviruses. Together, the results indicate alphavirus nsP2 functions to engage the host response to infection and activate a switch from the early-to-late phase. The loss of this function leads to continuous viral minus-strand synthesis and the production of unstable RC؉.The alphaviruses Sindbis (SIN) and Semliki Forest (SFV) are plus-stranded RNA viruses whose greater than 40S (11.7 kb) genomes encode four nonstructural proteins (nsP1-4), numbered according to their gene order (reviewed in reference 55), that are the essential components of the viral replicases and transcriptase. They are synthesized initially as polyproteins P1234 and P123, the former by readthrough of an opal termination codon between the nsP3 and nsP4 genes (54). The replicases involved in minus-strand (P123, P23) and genome (P23 or fully cleaved nsPs) synthesis contain uncleaved nsP2-containing polyproteins (32,33,52,59). The transcriptase makes a subgenomic 26S mRNA that encodes the viral structural proteins. Capping of viral genome and 26S plus strands employs methyltransferase and guanylyltransferase activities present within the nsP1 protein (2-4, 23, 35, 51, 58) and 5Ј-triphosphatase activity resident in the N domain of nsP2 (57). The nsP1 protein also is involved in the initiation of minusstrand synthesis (21, 50, 53, 60) and interacts with nsP4 (12, 53), which is the RNA-dependent RNA polymerase and also affects host cell-dependent replication (11,33). The N half to two-thirds of the nsP3 phosphoprotein is conserved among alphaviruses and provides essential functions for minus-strand and 26S mRNA syntheses (5,26,29,30,38,59); it also includes a conserved macrohistone 2A-like sequence predicted to b...

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Section: Discussionmentioning

confidence: 52%

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

Sawicki

Perri²,

Polo³

et al. 2006

J Virol

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“…The presence of small amounts of m 5 C in Sindbis viral mRNA, Adenovirus 2 mRNA, and in cellular mRNA from cultured hamster (BHK-21) cells was reported in early studies (Dubin et al 1975a(Dubin et al , 1975b(Dubin et al , 1977. Interestingly, m 5 C was not detected in mRNA from Novikoff hepatoma cells (Desrosiers et al 1974(Desrosiers et al , 1975, HeLa (Wei et al 1976(Wei et al , 1977, or mouse L cells .…”

Section: -Methylcytidine (M 5 C)mentioning

confidence: 75%

The biosynthesis and functional roles of methylated nucleosides in eukaryotic mRNA

Bokar

2005

Fine-Tuning of RNA Functions by Modification and Editing

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Modified nucleosides are present in mRNA of all eukaryotes, albeit at much lower levels than in other RNA moieties such as rRNA, tRNA, and snRNA. Modification by methylation occurs on the terminal guanosine of the cap (N 7 -methylguanosine), and the first two encoded nucleosides (2'-O-methylnuculeosides) in most higher eukaryotes. Additional modifications of cap nucleosides occur in special cases where the cap is derived by trans-splicing in nematodes and kinetoplastids. Modification by methylation also occurs at internal adenosine residues in many species (N 6 -methyladenosine). Modification by deamination occurs at specific adenosine residues (forming inosine) and cytidine residues (forming uridine) in very specific cases leading to post-transcriptional editing. Numerous studies have shown the importance of the cap N 7 -methylguanosine in translation, splicing, transport, and mRNA stability. The role of the 2'-O-methylnucleosides is not as well understood, but there is evidence that these modifications play some role in translation efficiency. The role of internal N 6 -methyladenosine residues is least known, and is the focus of this review. The formation of N 6 -methyladenosine is catalyzed by a complex enzyme containing a subunit (MT-A70) that co-localizes with nuclear speckles and appears to be widely expressed in all higher eukaryotes. Loss of this enzyme leads to a sporulation defect in yeast and to apoptosis in mammalian cells, although the exact mechanism by which the effects occur remains obscure.

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“…The parental 42 S RNA is copied to complementary minus-strands, which in turn are used as templates for the synthesis of new 42 S RNA plus-strands and subgenomic 26 S mRNAs. During plus-strand synthesis, the 5Ј ends of the 42 S and 26 S RNAs become modified with covalently attached m 7 GpppA (the cap0 structure) (3)(4)(5). Capping of the RNAs is believed to be obligatory also for the replication of Alphavirus, since a point mutation specifically destroying the guanylyltransferase activity of Nsp1 is lethal for the virus (6).…”

Section: Semliki Forest Virus (Sfv)mentioning

confidence: 99%

Identification of a Novel Function of the AlphavirusCapping Apparatus

Vasiljeva

Merits

Auvinen

et al. 2000

Journal of Biological Chemistry

175

162

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Semliki Forest virus (SFV)1 is member of the Alphavirus genus of the Togaviridae family. SFV has a positive-stranded 42 S RNA genome of 11.5 kilobases. RNA replication of SFV takes place in the cytoplasm and is catalyzed by the viral RNA-dependent RNA polymerase, which contains the virusspecific proteins Nsp1-4. These are cleavage products of a large (2342 aa) nonstructural polyprotein P1234. In the RNA polymerase complex all Nsps are in close association with each other (1, 2). The parental 42 S RNA is copied to complementary minus-strands, which in turn are used as templates for the synthesis of new 42 S RNA plus-strands and subgenomic 26 S mRNAs. During plus-strand synthesis, the 5Ј ends of the 42 S and 26 S RNAs become modified with covalently attached m 7 GpppA (the cap0 structure) (3-5). Capping of the RNAs is believed to be obligatory also for the replication of Alphavirus, since a point mutation specifically destroying the guanylyltransferase activity of Nsp1 is lethal for the virus (6).The functions of Alphavirus Nsps in replication have been studied using various genetic and biochemical approaches (1, 2). Nsp4 is the catalytic component of the RNA polymerase (7,8), whereas the functions of the phosphoprotein Nsp3 (9) are poorly defined (10, 11). Nsp2 has several distinct functions. It has nucleoside triphosphatase (NTPase) activity at its aminoterminal half (12), which is vital for the virus replication (13). Nsp2 has RNA helicase activity, which utilizes NTP hydrolysis as the energy source (14). The carboxyl-terminal part of the protein is a papain-like protease responsible for the autocatalytic cleavages of the nonstructural polyprotein (2, 15, 16). The carboxyl-terminal part has a nuclear localization sequence, which is responsible for sequestering of about half of the molecules to the nucleus during infection (17, 18). Furthermore, Nsp2 regulates transcription of the subgenomic 26 S RNA (Ref.19 and references therein). Here we show that Nsp2 has yet an additional activity required for capping of the virus mRNAs.Capping of cellular mRNAs occurs in the nucleus and comprises four different reactions. RNA 5Ј-triphosphatase removes the ␥-phosphate from the 5Ј end of the nascent RNA molecule (pppRNA 3 ppRNA). Guanylyltransferase reacts with a GTP molecule to form a covalent complex with GMP, which is then transferred from guanylyltransferase to the 5Ј end of RNA to form G(5Ј)ppp(5Ј)NpRNA. Methylation by guanine-7N-methyltransferase yields an RNA molecule with the cap0 structure (m 7 GpppNpRNA). Further methylation by nucleoside-2Ј-Omethyltransferase of the riboses of the penultimate and the adjacent nucleotides yields cap1 and cap2 structures, respectively (20,21).Unlike cellular mRNAs, the capping of Alphavirus RNAs takes place in the cytoplasm and is carried out by reactions that differ from the nuclear reactions as follows. (i) Nsp1 catalyzes transfer of the methyl group from S-adenosylmethionine to GTP to yield m 7 GTP (methyltransferase reaction), and (ii) a covalent guanylate complex Nsp1-m 7 GMP i...

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Sindbis virus messenger RNA: the 5′-termini and methylated residues of 26 and 42 S RNA

Cited by 75 publications

References 48 publications

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

Role for nsP2 Proteins in the Cessation of Alphavirus Minus-Strand Synthesis by Host Cells

The biosynthesis and functional roles of methylated nucleosides in eukaryotic mRNA

Identification of a Novel Function of the AlphavirusCapping Apparatus

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