scholarly journals Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

2009 ◽  
Vol 84 (3) ◽  
pp. 1477-1488 ◽  
Author(s):  
Natalya L. Teterina ◽  
Eric A. Levenson ◽  
Ellie Ehrenfeld
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Viral Rna ◽  
Progeny Virus ◽  
C Terminus ◽  
Membrane Reorganization ◽  
Infected Cells ◽  
Viral Polyprotein ◽  
Fluorescent Protein Tags

ABSTRACT The 2A proteins of the Picornaviridae enterovirus genus are small cysteine proteinases that catalyze essential cleavages in the viral polyprotein in cis and in several cellular proteins in trans. In addition, 2A has been implicated in the process of viral RNA replication, independent of its protease functions. We have generated viable polioviruses that encode 2A proteins containing fluorescent protein tag insertions at either of two sites in the 2A protein structure. Viruses containing an insertion of Discosoma sp. red fluorescent protein (DsRed) after residue 144 of 2A, near the C terminus, produced plaques only slightly smaller than wild-type (wt) virus. The polyprotein harboring the 2A-DsRed fusion protein was efficiently and accurately cleaved; fluorescent 2A proteinase retained protease activity in trans and supported translation and replication of viral RNA, both in vitro and in infected cells. Intracellular membrane reorganization to support viral RNA synthesis was indistinguishable from that induced by wt virus. Infected cells exhibited strong red fluorescence from expression of the 2A-DsRed fusion protein, and the progeny virus was stable for three to four passages, after which deletions within the DsRed coding sequence began to accumulate. Confocal microscopic imaging and analysis revealed a portion of 2A-DsRed in punctate foci concentrated in the perinuclear region that colocalized with replication protein 2C. The majority of 2A, however, was associated with an extensive structural matrix throughout the cytoplasm and was not released from infected cells permeabilized with digitonin.

scholarly journals Cloning and identification of regulatory gene UL76 of human cytomegalovirus

2000 ◽  
Vol 81 (10) ◽  
pp. 2407-2416 ◽  
Author(s):  
Shang-Kwei Wang ◽  
Chang-Yih Duh ◽  
Tai-Tsung Chang
Keyword(s):  
Fusion Protein ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Immediate Early ◽  
Free Form ◽  
Infected Cells ◽  
Green Fluorescent

The major immediate-early promoter/enhancer (MIEP, −1139 to +52) of human cytomegalovirus (HCMV) is regulated by cell type-specific transcriptional factors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that the regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD169) genomic sublibraries were constructed and in vitro transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression was exhibited by a number of sublibraries, from one of which a genomic clone was selected for augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stages of the reproductive cycle. Characterized by its highly basic composition (predicted pI 11·6), a free form of pUL76 tagged with green fluorescent protein was found to localize exclusively to the nucleus. In this report, pUL76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and the ratio of transfected effector DNA.

scholarly journals Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis

2012 ◽  
Vol 87 (3) ◽  
pp. 1454-1464 ◽  
Author(s):  
Adriaan H. de Wilde ◽  
Yanhua Li ◽  
Yvonne van der Meer ◽  
Grégoire Vuagniaux ◽  
Robert Lysek ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Fluorescent Protein ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Gfp Reporter ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Unit Reduction

ABSTRACTVirus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC50) of 5.2 μM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC50of 0.95 μM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC50that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC50similar to that of CsA. The addition of 4 μM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in anin vitroassay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Endotoxin Stimulates Liver Macrophages To Release Mediators That Inhibit an Early Step in Hepadnavirus Replication

2000 ◽  
Vol 74 (12) ◽  
pp. 5525-5533 ◽  
Author(s):  
Uta Klöcker ◽  
Ursula Schultz ◽  
Heinz Schaller ◽  
Ulrike Protzer
Keyword(s):  
Fluorescent Protein ◽  
Virus Production ◽  
Antiviral Effect ◽  
Progeny Virus ◽  
Dna Templates ◽  
Liver Macrophages ◽  
Hepatocyte Cultures ◽  
Proinflammatory Mediators ◽  
Duck Hepatitis

ABSTRACT Hepadnaviruses are known to be sensitive to various extracellular mediators. Therefore, bacterial endotoxin, which induces the secretion of proinflammatory mediators in the liver, was studied for its effect on hepadnavirus infection in vitro using the duck hepatitis B virus (DHBV) model. In initial experiments, endotoxin was shown to inhibit DHBV replication in primary duck hepatocyte cultures prepared by standard collagenase perfusion. As a primary endotoxin target, hepatic nonparenchymal cells (NPC) contaminating primary hepatocyte cultures, and among these probably macrophages (Kupffer cells), were identified to secrete polypeptide mediators into the cell culture medium. When added during DHBV infection, these mediators elicited the principal antiviral effect in a dose-dependent fashion. On the molecular level, they inhibited accumulation of viral proteins as well as amplification of the nuclear extrachromosomal DHBV DNA templates. In hepatocytes with an established DHBV infection, DHBV protein and progeny virus production was inhibited while the levels of established nuclear DHBV DNA templates and viral transcripts remained unaffected. Finally, in hepatocytes infected with a replication-deficient recombinant DHBV-green fluorescent protein (GFP) virus, the endotoxin-induced mediators markedly reduced GFP expression from chimeric DHBV-GFP transcripts, indicating that the major effect is at a level of translation of viral RNAs. Taken together, the data obtained demonstrate that antiviral mediators, and among these the cytokines alpha interferon (IFN-α) and IFN-γ, are released from hepatic NPC, most probably liver macrophages, upon endotoxin stimulation; furthermore, these mediators act at a posttranscriptional step of hepadnavirus replication.

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 Conventional Kinesin Mediates Microtubule-Microtubule Interactions In Vivo

2006 ◽  
Vol 17 (2) ◽  
pp. 907-916 ◽  
Author(s):  
Anne Straube ◽  
Gerd Hause ◽  
Gero Fink ◽  
Gero Steinberg
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Activity ◽  
C Terminus ◽  
Cross Bridges ◽  
Conventional Kinesin ◽  
Motor Head

Conventional kinesin is a ubiquitous organelle transporter that moves cargo toward the plus-ends of microtubules. In addition, several in vitro studies indicated a role of conventional kinesin in cross-bridging and sliding microtubules, but in vivo evidence for such a role is missing. In this study, we show that conventional kinesin mediates microtubule-microtubule interactions in the model fungus Ustilago maydis. Live cell imaging and ultrastructural analysis of various mutants in Kin1 revealed that this kinesin-1 motor is required for efficient microtubule bundling and participates in microtubule bending in vivo. High levels of Kin1 led to increased microtubule bending, whereas a rigor-mutation in the motor head suppressed all microtubule motility and promoted strong microtubule bundling, indicating that kinesin can form cross-bridges between microtubules in living cells. This effect required a conserved region in the C terminus of Kin1, which was shown to bind microtubules in vitro. In addition, a fusion protein of yellow fluorescent protein and the Kin1tail localized to microtubule bundles, further supporting the idea that a conserved microtubule binding activity in the tail of conventional kinesins mediates microtubule-microtubule interactions in vivo.

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.

scholarly journals Preferential Translation of Vesicular Stomatitis Virus mRNAs Is Conferred by Transcription from the Viral Genome

2006 ◽  
Vol 80 (23) ◽  
pp. 11733-11742 ◽  
Author(s):  
Zackary W. Whitlow ◽  
John H. Connor ◽  
Douglas S. Lyles
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Host Protein ◽  
Translation Efficiency ◽  
Viral Mrnas ◽  
Translation Inhibition ◽  
Infected Cells ◽  
Vesicular Stomatitis

ABSTRACT Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

Sign in / Sign up

Export Citation Format

Share Document