scholarly journals Efficient Homologous RNA Recombination and Requirement for an Open Reading Frame during Replication of Equine Arteritis Virus Defective Interfering RNAs

2000 ◽  
Vol 74 (19) ◽  
pp. 9062-9070 ◽  
Author(s):  
Richard Molenkamp ◽  
Sophie Greve ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Virus ◽  
Rna Replication ◽  
Proximal Region ◽  
Full Length ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Replicase Gene ◽  
Rna Recombination ◽  
Reading Frame ◽  
A Genome

ABSTRACT Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156–3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3′- and 5′-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5′-proximal region was found to be approximately 100-fold lower than that in the 3′-proximal part of the genome.

scholarly journals Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

Genetics ◽  
1992 ◽  
Vol 132 (1) ◽  
pp. 125-133 ◽  
Author(s):  
N L Glass ◽  
L Lee
Keyword(s):  
Neurospora Crassa ◽  
Mating Type ◽  
Single Copy ◽  
Open Reading Frame ◽  
Sexual Cycle ◽  
Reading Frame ◽  
Heterokaryon Incompatibility ◽  
Functional Regions ◽  
A Genome ◽  
Ascospore Formation

Abstract In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.

scholarly journals Yeast Lsm1p-7p/Pat1p Deadenylation-Dependent mRNA-Decapping Factors Are Required for Brome Mosaic Virus Genomic RNA Translation

2003 ◽  
Vol 23 (12) ◽  
pp. 4094-4106 ◽  
Author(s):  
Amine O. Noueiry ◽  
Juana Diez ◽  
Shaun P. Falk ◽  
Jianbo Chen ◽  
Paul Ahlquist
Keyword(s):  
Mosaic Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Open Reading Frame ◽  
Viral Rna ◽  
Genomic Rna ◽  
Reading Frame ◽  
Mrna Decapping ◽  
Rna Translation ◽  
Positive Strand Rna

ABSTRACT Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5′ and 3′ noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.

scholarly journals A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus (Geobacillus) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

2004 ◽  
Vol 70 (12) ◽  
pp. 7140-7147 ◽  
Author(s):  
Jaishree Vellore ◽  
Samuel E. Moretz ◽  
Bert C. Lampson
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Protein Product ◽  
Thermophilic Bacterium ◽  
Full Length ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Group Ii

ABSTRACT The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75°C.

scholarly journals Isolation and Characterization of an Arterivirus Defective Interfering RNA Genome

2000 ◽  
Vol 74 (7) ◽  
pp. 3156-3165 ◽  
Author(s):  
Richard Molenkamp ◽  
Babette C. D. Rozier ◽  
Sophie Greve ◽  
Willy J. M. Spaan ◽  
Eric J. Snijder
Keyword(s):  
Rna Virus ◽  
Equine Arteritis Virus ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Family ◽  
Defective Interfering Rna ◽  
Rna Genome ◽  
Interfering Rna ◽  
Discontinuous Transcription

ABSTRACT Equine arteritis virus (EAV), the type member of the family Arteriviridae, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.

scholarly journals Proteolytic Processing of the Open Reading Frame 1b-Encoded Part of Arterivirus Replicase Is Mediated by nsp4 Serine Protease and Is Essential for Virus Replication

1999 ◽  
Vol 73 (3) ◽  
pp. 2027-2037 ◽  
Author(s):  
Leonie C. van Dinten ◽  
Sietske Rensen ◽  
Alexander E. Gorbalenya ◽  
Eric J. Snijder
Keyword(s):  
Serine Protease ◽  
Proteolytic Processing ◽  
Equine Arteritis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Nonstructural Proteins ◽  
Site Mutation ◽  
Reading Frame ◽  
Infected Cells ◽  
Unusual Phenotype

ABSTRACT The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625–6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864–4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837→Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.

scholarly journals Uncoupled Expression of p33 and p92 Permits Amplification of Tomato Bushy Stunt Virus RNAs

1998 ◽  
Vol 72 (7) ◽  
pp. 5845-5851 ◽  
Author(s):  
Sara K. Oster ◽  
Baodong Wu ◽  
K. Andrew White
Keyword(s):  
Viral Genome ◽  
Stop Codon ◽  
Rna Virus ◽  
Rna Replication ◽  
Tomato Bushy Stunt Virus ◽  
Viral Rna ◽  
Rna Amplification ◽  
Viral Rnas ◽  
Reading Frame ◽  
Translational Enhancer

ABSTRACT Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5′-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal ∼6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3′-located segment, region 3.5, was tested. However, when 5′-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5′ cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.

scholarly journals Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

1991 ◽  
Vol 277 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R Dumas ◽  
M Lebrun ◽  
R Douce
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Single Gene ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Open Reading Frame ◽  
Amino Acid Residues ◽  
Protein Precursor ◽  
Reading Frame ◽  
Full Length Cdna

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the ‘fingerprint’ region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

scholarly journals Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

2003 ◽  
Vol 77 (6) ◽  
pp. 3569-3577 ◽  
Author(s):  
Pamela J. Glass ◽  
Carl Q. Zeng ◽  
Mary K. Estes
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Capsid Protein ◽  
Full Length ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Two Dimensional Gel Electrophoresis ◽  
Reading Frame ◽  
Orf3 Protein ◽  
Orf2 Protein

ABSTRACT Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF31-179, ORF31-152, and ORF31-107) no longer produced the 35K form. An N-terminal truncation mutant (ORF351-212) and a site-directed mutant (ORF3T201V) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3FL), ORF31-179, ORF31-152, and ORF351-212 interacted with the ORF2 protein, while an ORF31-107 protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

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