scholarly journals Sendai Virus Y Proteins Are Initiated by a Ribosomal Shunt

1998 ◽  
Vol 18 (9) ◽  
pp. 5021-5031 ◽  
Author(s):  
Patrizia Latorre ◽  
Daniel Kolakofsky ◽  
Joseph Curran
Keyword(s):  
Sendai Virus ◽  
Donor Site ◽  
Initiation Site ◽  
Acceptor Site ◽  
Mrna Editing ◽  
Translational Initiation ◽  
Reading Frame ◽  
P Protein ◽  
Ca 50 ◽  
Essential Subunit

ABSTRACT The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C′, C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C′ to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5′ cap and then scan ca. 50 nucleotides (nt) of the 5′ untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

scholarly journals Two Nucleotides Immediately Upstream of the Essential A6G3 Slippery Sequence Modulate the Pattern of G Insertions during Sendai Virus mRNA Editing

1999 ◽  
Vol 73 (1) ◽  
pp. 343-351 ◽  
Author(s):  
Stéphane Hausmann ◽  
Dominique Garcin ◽  
Anne-Sophie Morel ◽  
Daniel Kolakofsky
Keyword(s):  
Sendai Virus ◽  
Cell System ◽  
Mrna Editing ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
P Gene ◽  
Minimum Number ◽  
Type 3 ◽  
Slippery Sequence ◽  
Bovine Parainfluenza Virus

ABSTRACT Editing of paramyxovirus P gene mRNAs occurs cotranscriptionally and functions to fuse an alternate downstream open reading frame to the N-terminal half of the P protein. G residues are inserted into a short G run contained within a larger purine run (A n G n ) in this process, by a mechanism whereby the transcribing polymerase stutters (i.e., reads the same template cytosine more than once). Although Sendai virus (SeV) and bovine parainfluenza virus type 3 (bPIV3) are closely related, the G insertions in their P mRNAs are distributed differently. SeV predominantly inserts a single G residue within the G run of the sequence 5′ AACAAAAAAGGG, whereas bPIV3 inserts one to six G’s at roughly equal frequency within the sequence 5′ AUUAAAAAAGGGG(differences are underlined). We have examined how thecis-acting editing sequence determines the number of G’s inserted, both in a transfected cell system using minigenome analogues and by generating recombinant viruses. We found that the presence of four rather than three G’s in the purine run did not affect the distribution of G insertions. However, when the underlined AC of the SeV sequence was replaced by the UU found in bPIV3, the editing phenotype from both the minigenome and the recombinant virus resembled that found in natural bPIV3 infections (i.e., a significant fraction of the mRNAs contained two to six G insertions). The two nucleotides located just upstream of the polypurine tract are thus key determinants of the editing phenotype of these viruses. Moreover, the minimum number of A residues that will promote SeV editing phenotype is six but can be reduced to five when the upstream AC is replaced by UU. A model for how the upstream dinucleotide controls the insertion phenotype is presented.

scholarly journals Versatility of the Accessory C Proteins of Sendai Virus: Contribution to Virus Assembly as an Additional Role

2000 ◽  
Vol 74 (12) ◽  
pp. 5619-5628 ◽  
Author(s):  
Mohammad K. Hasan ◽  
Atsushi Kato ◽  
Miki Muranaka ◽  
Ryoji Yamaguchi ◽  
Yuko Sakai ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Sendai Virus ◽  
Virus Assembly ◽  
Virus Multiplication ◽  
Interferon Α ◽  
Reading Frame ◽  
P Protein ◽  
The Matrix ◽  
Infected Cells ◽  
C Proteins

ABSTRACT The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C′, C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(−) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111–124, 1998). Here, we further characterized the 4C(−) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(−) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-α/β. SeV C proteins thus appear to be quite versatile.

scholarly journals Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles

1990 ◽  
Vol 10 (4) ◽  
pp. 1423-1431
Author(s):  
L L Searles ◽  
R S Ruth ◽  
A M Pret ◽  
R A Fridell ◽  
A J Ali
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Donor Site ◽  
Initiation Site ◽  
Splice Donor Site ◽  
Wild Type ◽  
Upstream Site ◽  
Reading Frame ◽  
Exon 1 ◽  
A Minor

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been examined. The v36f mutation is a B104/roo insertion in intron 4 near the splice donor site. A mutant carrying this alteration accumulates a very low level of mRNA that is apparently polyadenylated at a site within the B104/roo transposon. The v48a mutation, which deletes approximately 200 nt of DNA, fuses portions of exons 3 and 4 without disruption of the translational reading frame. A smaller transcript accumulates at a wild-type level, and thus an altered, nonfunctional polypeptide is likely to be synthesized in strains carrying this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Structure and transcription of the Drosophila melanogaster vermilion gene and several mutant alleles.

1990 ◽  
Vol 10 (4) ◽  
pp. 1423-1431 ◽  
Author(s):  
L L Searles ◽  
R S Ruth ◽  
A M Pret ◽  
R A Fridell ◽  
A J Ali
Keyword(s):  
Drosophila Melanogaster ◽  
Transcription Initiation ◽  
Donor Site ◽  
Initiation Site ◽  
Splice Donor Site ◽  
Wild Type ◽  
Upstream Site ◽  
Reading Frame ◽  
Exon 1 ◽  
A Minor

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been examined. The v36f mutation is a B104/roo insertion in intron 4 near the splice donor site. A mutant carrying this alteration accumulates a very low level of mRNA that is apparently polyadenylated at a site within the B104/roo transposon. The v48a mutation, which deletes approximately 200 nt of DNA, fuses portions of exons 3 and 4 without disruption of the translational reading frame. A smaller transcript accumulates at a wild-type level, and thus an altered, nonfunctional polypeptide is likely to be synthesized in strains carrying this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Analyses of the 5-prime Ends of Escherichia coli ORFs

2021 ◽  
Author(s):  
James F Curran ◽  
Michael Ward
Keyword(s):  
Escherichia Coli ◽  
Nearest Neighbor ◽  
Initiation Site ◽  
Translational Efficiency ◽  
Translational Initiation ◽  
Reading Frame ◽  
Internal Sequence ◽  
Prime Ends ◽  
Selection For ◽  
Reading Frames

Sequence biases at 5-prime ends of coding sequences differ from those of the remainder of ORFs, reflecting differences in function. Internal sequence biases promote translational efficiency by several mechanisms including correlating codon usage and tRNA concentration. However, the early region may also facilitate translational initiation, establishment of the reading frame, and polypeptide processing. Here we examine the beginnings of the ORFs of an Escherichia coli K12 reference genome. The results extend previous observations of A-richness to include an overabundance of the AAA triplet in all reading frames, consistent with the hypothesis that the beginnings of ORFs contribute to initiation site accessibility. Results are also consistent with the idea that the first two amino acids are under selection because they facilitate solvation of the amino-terminus at the end of the ribosomal exit channel. Moreover, serine is highly overrepresented as the second amino acid, possibly because it can facilitate removal of the terminal formylmethionine. Non-AUG initiation codons are known to be less efficient than AUG at directing initiation, presumably because of relatively weak base pairing to the initiator-tRNA. But non-UAG initiation codons are not followed by unusual 3-prime nearest neighbor codons. Moreover, the four NUG initiation codons do not differ in their propensity to frameshift in an assay known to be sensitive to base pair strength. Altogether, these data suggest that the 5-prime ends of ORFs are under selection for several functions, and that initiation codon identity may not critical beyond its role in initiation.

scholarly journals The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 1143-1151
Author(s):  
Gil Shalev ◽  
Avraham A Levy
Keyword(s):  
Transposable Element ◽  
Donor Site ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Blue Staining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Reading Frame ◽  
Strand Breaks ◽  
Ectopic Recombination

The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repair pathway. To this end, we developed an assay whereby β-Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonallelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5′ end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5′ end of the GUS open reading frame and had a deletion at the 3′ end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac-induced ectopic recombination. The occurrence of ectopic recombination following double-strand breaks is a potentially important factor in plant genome evolution.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

scholarly journals Identification and characterization of a novel paramyxovirus, porcine parainfluenza virus 1, from deceased pigs

2013 ◽  
Vol 94 (10) ◽  
pp. 2184-2190 ◽  
Author(s):  
Susanna K. P. Lau ◽  
Patrick C. Y. Woo ◽  
Ying Wu ◽  
Annette Y. P. Wong ◽  
Beatrice H. L. Wong ◽  
...  
Keyword(s):  
Rectal Swab ◽  
Sendai Virus ◽  
Parainfluenza Virus ◽  
Mrna Editing ◽  
Viral Loads ◽  
Reverse Transcription Pcr ◽  
Phosphoprotein Gene ◽  
Human Parainfluenza Virus ◽  
Identification And Characterization

We describe the discovery and characterization of a novel paramyxovirus, porcine parainfluenza virus 1 (PPIV-1), from swine. The virus was detected in 12 (3.1 %) of 386 nasopharyngeal and two (0.7 %) of 303 rectal swab samples from 386 deceased pigs by reverse transcription-PCR, with viral loads of up to 106 copies ml−1. Complete genome sequencing and phylogenetic analysis showed that PPIV-1 represented a novel paramyxovirus within the genus Respirovirus, being most closely related to human parainfluenza virus 1 (HPIV-1) and Sendai virus (SeV). In contrast to HPIV-1, PPIV-1 possessed a mRNA editing function in the phosphoprotein gene. Moreover, PPIV-1 was unique among respiroviruses in having two G residues instead of three to five G residues following the A6 run at the editing site. Nevertheless, PPIV-1, HPIV-1 and SeV share common genomic features and may belong to a separate group within the genus Respirovirus. The presence of PPIV-1 in mainly respiratory samples suggests a possible association with respiratory disease, similar to HPIV-1 and SeV.

scholarly journals Role of a Cytotoxic-T-Lymphocyte Epitope-Defined, Alternative gag Open Reading Frame in the Pathogenesis of a Murine Retrovirus-Induced Immunodeficiency Syndrome

2005 ◽  
Vol 79 (7) ◽  
pp. 4308-4315 ◽  
Author(s):  
Arti Gaur ◽  
William R. Green
Keyword(s):  
T Lymphocyte ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Protein S ◽  
Initiation Site ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Ctl Epitope ◽  
Immunodeficiency Syndrome

ABSTRACT LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60 gag ). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

scholarly journals Untangling the sequence of events during the S2→ S3transition in photosystem II and implications for the water oxidation mechanism

2020 ◽  
Vol 117 (23) ◽  
pp. 12624-12635 ◽  
Author(s):  
Mohamed Ibrahim ◽  
Thomas Fransson ◽  
Ruchira Chatterjee ◽  
Mun Hon Cheah ◽  
Rana Hussein ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Water Oxidation ◽  
Donor Site ◽  
Oxidation Mechanism ◽  
Oxygenic Photosynthesis ◽  
Acceptor Site ◽  
Oxygen Evolving Complex ◽  
Ps Ii ◽  
Sequence Of Events ◽  
Large Channel

In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2→ S3transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2→ S3transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QAand QB, are observed. At the donor site, tyrosine YZand His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2→ S3transition mirrors the appearance of OXelectron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

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