scholarly journals The 29-Nucleotide Deletion Present in Human but Not in Animal Severe Acute Respiratory Syndrome Coronaviruses Disrupts the Functional Expression of Open Reading Frame 8

2007 ◽  
Vol 81 (24) ◽  
pp. 13876-13888 ◽  
Author(s):  
Monique Oostra ◽  
Cornelis A. M. de Haan ◽  
Peter J. M. Rottier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Signal Sequence ◽  
Expression System ◽  
Functional Expression ◽  
Open Reading Frame ◽  
T7 Promoter ◽  
Reading Frame ◽  
Genomic Changes ◽  
Nucleotide Deletion ◽  
Infected Cells

ABSTRACT One of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (SARS-CoV) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. This occurred in open reading frame 8 (ORF8), one of the accessory genes unique to the SARS-CoV. The function of ORF8 and the significance of the deletion are unknown. The intact ORF8 present in animal and some early human isolates encodes a 122-amino-acid polypeptide (8ab+), which we expressed in cells using the vaccinia virus T7 expression system. It was found to contain a cleavable signal sequence, which directs the precursor to the endoplasmic reticulum (ER) and mediates its translocation into the lumen. The cleaved protein became N-glycosylated, assembled into disulfide-linked homomultimeric complexes, and remained stably in the ER. The 29-nucleotide deletion splits ORF8 into two ORFs, 8a and 8b, encoding 39- and 84-residue polypeptides. The 8a polypeptide is likely to remain in the cytoplasm, as it is too small for its signal sequence to function and will therefore be directly released from the ribosome. However, we could not confirm this experimentally due to the lack of proper antibodies. ORF8b appeared not to be expressed in SARS-CoV-infected cells or when expressed from mRNA's mimicking mRNA8. This was due to the context of the internal AUG initiation codon, as we demonstrated after placing the ORF8b immediately behind the T7 promoter. A soluble, unmodified and monomeric 8b protein was now expressed in the cytoplasm, which was highly unstable and rapidly degraded. Clearly, the 29-nucleotide deletion disrupts the proper expression of the SARS-CoV ORF8, the implications of which are discussed.

scholarly journals In-Frame 12-Nucleotide Deletion within Open Reading Frame 3a in a SARS-CoV-2 Strain Isolated from a Patient Hospitalized with COVID-19

2021 ◽  
Vol 10 (8) ◽  
Author(s):  
John A. Lednicky ◽  
Kartikeya Cherabuddi ◽  
Massimiliano S. Tagliamonte ◽  
Maha A. Elbadry ◽  
Kuttichantran Subramaniam ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Nucleotide Deletion

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain UF-8, with an in-frame 12-nucleotide deletion within open reading frame 3a (ORF3a), was isolated from a 78-year-old COVID-19 patient in March 2020.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure

1996 ◽  
Vol 16 (1) ◽  
pp. 73-80 ◽  
Author(s):  
D A Rubin ◽  
J H Youson ◽  
L E Marra ◽  
R M Dores
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Russian Sturgeon ◽  
Reading Frame ◽  
Terminal Codon

ABSTRACT A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3′ untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3′ untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes — gar and bowfin; one chondrostean fish — the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

scholarly journals Nipah Virus Edits Its P Gene at High Frequency To Express the V and W Proteins

2009 ◽  
Vol 83 (8) ◽  
pp. 3982-3987 ◽  
Author(s):  
Sachin Kulkarni ◽  
Valentina Volchkova ◽  
Christopher F. Basler ◽  
Peter Palese ◽  
Viktor E. Volchkov ◽  
...  
Keyword(s):  
High Frequency ◽  
Nipah Virus ◽  
Open Reading Frame ◽  
Mrna Editing ◽  
Reading Frame ◽  
Specific Antibodies ◽  
P Gene ◽  
Gene Transcripts ◽  
Infected Cells ◽  
Over Time

ABSTRACT Nipah virus (NiV) is predicted to encode four proteins from its P gene (P, V, W, and C) via mRNA editing and an alternate open reading frame. By use of specific antibodies, the expression of the V, W, and C proteins in NiV-infected cells has now been confirmed. Analysis of the P-gene transcripts shows a ratio of P:V:W mRNA of 1:1:1, but this differs over time, with greater proportions of V and W transcripts observed as the infection progresses. Eighty-two percent of transcripts are edited, with up to 11 G insertions observed. This exceptionally high editing frequency ensures expression of the V and W proteins.

scholarly journals Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis bywprA Gene Disruption

2000 ◽  
Vol 66 (2) ◽  
pp. 476-480 ◽  
Author(s):  
Sang Jun Lee ◽  
Dong Min Kim ◽  
Kwang Hee Bae ◽  
Si Myung Byun ◽  
Jae Hoon Chung
Keyword(s):  
Bacillus Subtilis ◽  
Gene Disruption ◽  
Therapeutic Potential ◽  
Signal Sequence ◽  
Open Reading Frame ◽  
Expression Plasmid ◽  
Reading Frame ◽  
Extracellular Milieu ◽  
Foreign Proteins

ABSTRACT Staphylokinase (SAK), a polypeptide secreted byStaphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Open Reading Frame 57 Encodes a Posttranscriptional Regulator with Multiple Distinct Activities

2000 ◽  
Vol 74 (8) ◽  
pp. 3586-3597 ◽  
Author(s):  
Jessica R. Kirshner ◽  
David M. Lukac ◽  
Jean Chang ◽  
Don Ganem
Keyword(s):  
Posttranscriptional Regulation ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Open Reading Frame ◽  
Luciferase Reporter ◽  
Reading Frame ◽  
Infected Cells ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Coding Potential

ABSTRACT Open reading frame (ORF) 57 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a homolog of known posttranscriptional regulators that are essential for replication in other herpesviruses. Here, we examined the expression of this gene and the function(s) of its product. KSHV ORF 57 is expressed very early in infection from a 1.6-kb spliced RNA bearing several in-frame initiation codons. Its product is a nuclear protein that, in transient assays, has little effect on the expression of luciferase reporter genes driven by a variety of KSHV and heterologous promoters. However, ORF 57 protein enhances the accumulation of several viral transcripts, in a manner suggesting posttranscriptional regulation. These transcripts include not only known cytoplasmic mRNAs (e.g., ORF 59) but also a nuclear RNA (nut-1) that lacks coding potential. Finally, ORF 57 protein can also modulate the effects of the ORF 50 gene product, a classical transactivator known to be required for lytic induction. The expression from some (e.g., nut-1) but not all (e.g., tk) ORF 50-responsive promoters can be synergistically enhanced by coexpression of ORF 50 and ORF 57. This effect is not due to upregulation of ORF 50 expression but rather to a posttranslational enhancement of the transcriptional activity of ORF 50. These data indicate that ORF 57 is a powerful pleiotropic effector that can act on several posttranscriptional levels to modulate the expression of viral genes in infected cells.

scholarly journals Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2265-2271 ◽  
Author(s):  
Xiao Tao Lu ◽  
Amy C. Sims ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Reading Frame ◽  
Amino Terminal ◽  
Infected Cells ◽  
Carboxy Terminal ◽  
A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

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 Bioinformatics Analysis Identifies a Small ORF in the Genome of Fish Nidoviruses of Genus Oncotshavirus Predicted to Encode a Novel Integral Protein

2021 ◽  
Vol 12 (4) ◽  
pp. 753-764
Author(s):  
Frederick Kibenge ◽  
Ashley McKibbon ◽  
Molly Kibenge ◽  
Yingwei Wang
Keyword(s):  
Bioinformatics Analysis ◽  
Signal Sequence ◽  
Open Reading Frame ◽  
Type I ◽  
Structural Protein ◽  
Genome Sequence Analysis ◽  
Reading Frame ◽  
The Third ◽  
E Protein ◽  
Small Open Reading Frame

Genome sequence analysis of Atlantic salmon bafinivirus (ASBV) revealed a small open reading frame (ORF) predicted to encode a Type I membrane protein with an N-terminal cleaved signal sequence (110 aa), likely an envelope (E) protein. Bioinformatic analyses showed that the predicted protein is strikingly similar to the coronavirus E protein in structure. This is the first report to identify a putative E protein ORF in the genome of members of the Oncotshavirus genus (subfamily Piscavirinae, family Tobaniviridae, order Nidovirales) and, if expressed would be the third family (after Coronaviridae and Arteriviridae) within the order to have the E protein as a major structural protein.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide

2019 ◽  
Vol 0 (0) ◽  
Author(s):  
Amir Hossein Momen ◽  
Naser Harzandi ◽  
Azam Haddadi ◽  
Bijan Bambai
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Peptide Yy ◽  
Signal Sequence ◽  
Expression System ◽  
Promoter Sequence ◽  
Scale Production ◽  
Signal Sequences ◽  
T7 Promoter ◽  
E Coli

Abstract Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

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