scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

scholarly journals Expression of heterologous genes in oncolytic adenoviruses using picornaviral 2A sequences that trigger ribosome skipping

2008 ◽  
Vol 89 (2) ◽  
pp. 389-396 ◽  
Author(s):  
Garth M. Funston ◽  
Susanna E. Kallioinen ◽  
Pablo de Felipe ◽  
Martin D. Ryan ◽  
Richard D. Iggo
Keyword(s):  
Fluorescent Protein ◽  
Burst Size ◽  
Foot And Mouth Disease ◽  
Human Adenovirus ◽  
Peptide Bond ◽  
Fusion Partner ◽  
Reading Frame ◽  
Mouth Disease Virus ◽  
Green Fluorescent ◽  
Protein Ix

Insertion of picornaviral 2A sequences into mRNAs causes ribosomes to skip formation of a peptide bond at the junction of the 2A and downstream sequences, leading to the production of two proteins from a single open reading frame. Adenoviral protein IX is a minor capsid protein that has been used to display foreign peptides on the surface of the capsid. We have used 2A sequences from the foot-and-mouth disease virus (FMDV) and porcine teschovirus 1 (PTV-1) to express protein IX (pIX) and green fluorescent protein (GFP) from pIX–2A–GFP fusion genes in an oncolytic virus derived from human adenovirus 5. GFP was efficiently expressed by constructs containing either 2A sequence. Peptide bond skipping was more efficient with the 58 aa FMDV sequence than with the 22 aa PTV-1 2A sequence, but the virus with the FMDV 2A sequence showed a reduction in plaque size, cytopathic effect, viral burst size and capsid stability. We conclude that ribosome skipping induced by 2A sequences is an effective strategy to express heterologous genes in adenoviruses; however, careful selection or optimization of the 2A sequence may be required if protein IX is used as the fusion partner.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

scholarly journals The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences

2001 ◽  
Vol 82 (5) ◽  
pp. 1027-1041 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Lorraine E. Hughes ◽  
Garry Luke ◽  
Heidi Mendoza ◽  
Edwin ten Dam ◽  
...  
Keyword(s):  
Disease Virus ◽  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Reading Frame ◽  
Naturally Occurring ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Green Fluorescent ◽  
Translation Systems

The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins ‘cleaving’ at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to β-glucuronidase (GUS) – forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (∼90%) were in the form of the ‘cleavage’ products GUS and [GFP2A]. Alternative models have been proposed to account for the ‘cleavage’ activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring ‘2A-like’ sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that ‘cleavage’ occurred in constructs in which all the candidate nucleophilic residues were substituted – with the exception of aspartate-12. This residue is not, however, conserved amongst all functional ‘2A-like’ sequences. ‘2A-like’ sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial α-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial α-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the Picornaviridae. Taken together, these data provide additional evidence that neither FMDV 2A nor ‘2A-like’ sequences are autoproteolytic elements.

scholarly journals Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

2007 ◽  
Vol 18 (8) ◽  
pp. 2893-2903 ◽  
Author(s):  
Sarah L. Barker ◽  
Linda Lee ◽  
B. Daniel Pierce ◽  
Lymarie Maldonado-Báez ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Late Stage ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Temperature Sensitive ◽  
Type I ◽  
Reading Frame ◽  
Rich Domain ◽  
C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

scholarly journals Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Benjamin Brennan ◽  
Veronica V. Rezelj ◽  
Richard M. Elliott
Keyword(s):  
Virus Infection ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transcription Termination ◽  
Severe Disease ◽  
Open Reading Frame ◽  
Coding Region ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

scholarly journals Mutation in yaaT Leads to Significant Inhibition of Phosphorelay during Sporulation in Bacillus subtilis

2002 ◽  
Vol 184 (20) ◽  
pp. 5545-5553 ◽  
Author(s):  
Shigeo Hosoya ◽  
Kei Asai ◽  
Naotake Ogasawara ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Response Regulator ◽  
Significant Inhibition ◽  
Open Reading Frame ◽  
Histidine Kinases ◽  
Reading Frame ◽  
Green Fluorescent ◽  
Sporulation Genes

ABSTRACT In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of σH, which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles

1999 ◽  
Vol 112 (15) ◽  
pp. 2473-2484 ◽  
Author(s):  
A. Biederbick ◽  
S. Rose ◽  
H.P. Elsasser
Keyword(s):  
Fluorescent Protein ◽  
Signal Sequence ◽  
In Vitro Transcription ◽  
Autophagic Vacuole ◽  
Reading Frame ◽  
Vacuole Membrane ◽  
Conserved Regions ◽  
Punctate Pattern ◽  
Green Fluorescent

Using antibodies against autophagic vacuole membrane proteins we identified a human cDNA with an open reading frame of 1848 bp, encoding a protein of 70 kDa, which we named lysosomal apyrase-like protein of 70 kDa (LALP70). Sequence analysis revealed that LALP70 belongs to the apyrase or GDA1/CD39 family and is almost identical to a human uridine diphosphatase, with the exception of nine extra amino acids in LALP70. Members of this family were originally described as ectoenzymes, with some intracellular exceptions. Transfected LALP70 fused to the green fluorescent protein localized in the cytoplasm with a punctate pattern in the perinuclear space. These structures colocalized with the autophagic marker monodansylcadaverine and the lysosomal protein lamp1. Hydrophobicity analysis of the encoded protein revealed a transmembrane region at the N and C termini. Most of the sequence is arranged between these transmembrane domains, and contains four apyrase conserved regions. In vitro transcription/translation in the presence of microsomes showed that no signal sequence is cleaved off and that the translation product is protected from trypsin treatment. Our data indicate that LALP70 is a type III lysosomal/autophagic vacuole membrane protein with the apyrase conserved regions facing the luminal space of the vacuoles.

CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLS IN VITRO

2008 ◽  
Vol 11 (01) ◽  
pp. 1-7 ◽  
Author(s):  
Xiangping Liu ◽  
Haibo Wang ◽  
Kun Yang ◽  
Aihua Sui ◽  
Yanming Wang ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Fluorescent Protein ◽  
Interleukin 10 ◽  
Eukaryotic Expression ◽  
Cell Culture Supernatant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synovial Cells ◽  
Reading Frame ◽  
Green Fluorescent

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

scholarly journals Identification of a Hydrophobic Domain of HA2 Essential to Morphogenesis of Helicoverpa armigera Nucleopolyhedrovirus

2008 ◽  
Vol 82 (8) ◽  
pp. 4072-4081 ◽  
Author(s):  
Qian Wang ◽  
Yun Wang ◽  
Changyong Liang ◽  
Jianhua Song ◽  
Xinwen Chen
Keyword(s):  
Helicoverpa Armigera ◽  
Fluorescent Protein ◽  
Hydrophobic Domain ◽  
C Terminus ◽  
Localization Analysis ◽  
Helicoverpa Armígera ◽  
Budded Virus ◽  
Green Fluorescent ◽  
Vitro Analysis

ABSTRACT The HA2 protein of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) is a WASP homology protein capable of nucleating branched actin filaments in the presence of the Arp2/3 complex in vitro. To determine the role of ha2 in the HearNPV life cycle, ha2 knockout and ha2 repair bacmids were constructed. Transfection and infection analysis demonstrated that the ha2 null bacmid was unable to produce infectious budded virus (BV), while the repair bacmid rescued the defect. In vitro analysis demonstrated that the WCA domain of HA2 accelerates Arp2/3-mediated actin assembly and is indispensable to the function of HA2. However, analysis of the repaired recombinant with a series of truncated ha2 mutants demonstrated that the WCA domain was essential but not enough to yield infectious virions, and a hydrophobic domain (H domain) consisting of amino acids (aa) 167 to 193 played a pivotal role in the production of BV. Subcellular localization analysis with enhanced green fluorescent protein fusions showed that the H domain functioned as a nuclear localization signal. In addition, deletion of the C terminus of the ha2 product, a phosphatidylinositol 4-kinase homolog, dramatically decreased the viral titer, while deletion of 128 aa from the N terminus did not affect HA2 function.

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