scholarly journals Insertion of Enhanced Green Fluorescent Protein in a Hinge Region of Vesicular Stomatitis Virus L Polymerase Protein Creates a Temperature-Sensitive Virus That Displays No Virion-Associated Polymerase Activity In Vitro

2009 ◽  
Vol 83 (23) ◽  
pp. 12241-12252 ◽  
Author(s):  
John B. Ruedas ◽  
Jacques Perrault
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Polymerase Activity ◽  
Temperature Sensitive ◽  
Viral Mrna ◽  
L Protein ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

ABSTRACT The RNA-dependent RNA polymerase of viruses belonging to the order Mononegavirales is part of a large multifunctional L protein that also catalyzes viral mRNA capping and cap methylation. The L protein of this diverse group of agents displays six blocks of conserved sequences. The precise relationship between these conserved regions and individual functions is largely unknown, except for “domain” VI that clearly encodes a viral mRNA cap methylase. The L protein of morbilliviruses (family Paramyxoviridae) was reported to tolerate insertion of the enhanced green fluorescent protein (EGFP) in a region just upstream of domain VI. Recombinant viruses with this insertion grow well in cell culture but are highly attenuated in animal hosts. We show here that the L protein of vesicular stomatitis virus (VSV), the prototype of the Rhabdoviridae family, also tolerates insertion of EGFP at a similar site. The modified protein (LEGFP) and the resultant recombinant virus both demonstrated a sharp temperature-sensitive phenotype for polymerase activity, with reduced activity at 37°C and no activity at 37.5°C. Neither translation nor methylation of mutant virus transcripts was affected at 37°C. Curiously, mutant virus grown at permissive temperature contained about threefold-less L protein than the wild-type virus did and displayed no virion-associated polymerase activity in vitro. These findings support the notion that a flexible “hinge” region separates the cap methylase domain of L proteins from upstream functions and open up a number of avenues for studies of L-protein function in the more-tractable VSV model system.

scholarly journals The Methyltransferase Region of Vesicular Stomatitis Virus L Polymerase Is a Target Site for Functional Intramolecular Insertion

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 989
Author(s):  
Emmanuel Heilmann ◽  
Janine Kimpel ◽  
Stephan Geley ◽  
Andreas Naschberger ◽  
Carles Urbiola ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Insertion Site ◽  
Polymerase Activity ◽  
Temperature Sensitive ◽  
Single Chain ◽  
Intracellular Location ◽  
L Protein ◽  
Vesicular Stomatitis

The L-protein of vesicular stomatitis virus (VSV) is a single-chain multi-domain RNA-dependent RNA polymerase. Previously reported attempts of intramolecular insertions of fluorescent proteins into the L-protein resulted in temperature-sensitive and highly attenuated polymerase activity. Here, we describe a novel insertion site that was selected based on in silico prediction. Of five preselected locations, insertion of the fluorescent protein mCherry in the VSV polymerase between amino acids 1620 and 1621 preserved polymerase function even after extended passaging and showed only mild attenuation compared to wildtype VSV polymerase. High magnification fluorescence imaging revealed a corpuscular cytosolic pattern for the L-protein. To confirm that the insertion site tolerates inclusion of proteins others than mCherry, we cloned mWasabi into the same position in L, generating a VSV-LmWasabi, which was also functional. We also generated a functional dual-color-dual-insertion VSV construct with intramolecularly labeled P and L-proteins. Together, our data present an approach to tag VSV polymerase intramolecularly without perturbing enzymatic activity. This L fusion protein might enable future tracing studies to monitor intracellular location of the VSV transcription and replication machinery in real-time life-imaging studies.

scholarly journals Formation of Guanosine(5′)Tetraphospho(5′)Adenosine Cap Structure by an Unconventional mRNA Capping Enzyme of Vesicular Stomatitis Virus

2008 ◽  
Vol 82 (15) ◽  
pp. 7729-7734 ◽  
Author(s):  
Tomoaki Ogino ◽  
Amiya K. Banerjee
Keyword(s):  
Vesicular Stomatitis Virus ◽  
In Vitro Transcription ◽  
Viral Mrna ◽  
Viral Mrnas ◽  
L Protein ◽  
Transcription System ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Vesicular Stomatitis

ABSTRACT The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV) elicits GTPase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities to produce a 5′-cap core structure, guanosine(5′)triphospho(5′)adenosine (GpppA), on viral mRNAs. Here, we report that the L protein produces an unusual cap structure, guanosine(5′)tetraphospho(5′)adenosine (GppppA), that is formed by the transfer of the 5′-monophosphorylated viral mRNA start sequence to GTP by the PRNTase activity before the removal of the γ-phosphate from GTP by GTPase. Interestingly, GppppA-capped and polyadenylated full-length mRNAs were also found to be synthesized by an in vitro transcription system with the native VSV RNP.

scholarly journals The p14 FAST Protein of Reptilian Reovirus Increases Vesicular Stomatitis Virus Neuropathogenesis

2008 ◽  
Vol 83 (2) ◽  
pp. 552-561 ◽  
Author(s):  
Christopher W. Brown ◽  
Kyle B. Stephenson ◽  
Stephen Hanson ◽  
Michael Kucharczyk ◽  
Roy Duncan ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Fluorescent Protein ◽  
Syncytium Formation ◽  
Gene Encoding ◽  
Fast Proteins ◽  
Bona Fide ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

ABSTRACT The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins may serve as virulence factors by promoting virus dissemination and increased or altered cytopathology. To directly test this hypothesis, the gene encoding the p14 FAST protein of reptilian reovirus was inserted into the genome of a heterologous virus that does not naturally form syncytia, vesicular stomatitis virus (VSV). Expression of the p14 FAST protein by the VSV/FAST recombinant gave the virus a highly fusogenic phenotype in cell culture. The growth of this recombinant fusogenic VSV strain was unaltered in vitro but was significantly enhanced in vivo. The VSV/FAST recombinant consistently generated higher titers of virus in the brains of BALB/c mice after intranasal or intravenous infection compared to the parental VSV/green fluorescent protein (GFP) strain that expresses GFP in place of p14. The VSV/FAST recombinant also resulted in an increased incidence of hind-limb paralysis, it infected a larger volume of brain tissue, and it induced more extensive neuropathology, thus leading to a lower maximum tolerable dose than that for the VSV/GFP parental virus. In contrast, an interferon-inducing mutant of VSV expressing p14 was still attenuated, indicating that this interferon-inducing phenotype is dominant to the fusogenic properties conveyed by the FAST protein. Based on this evidence, we conclude that the reovirus p14 FAST protein can function as a bona fide virulence factor.

scholarly journals A Natural Botanical Product, Resveratrol, Effectively Suppresses Vesicular Stomatitis Virus Infection In Vitro

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1231
Author(s):  
Shih-Chao Lin ◽  
Xiang Zhang ◽  
Caitlin W. Lehman ◽  
Han-Chi Pan ◽  
Ya Wen ◽  
...  
Keyword(s):  
Viral Replication ◽  
Vesicular Stomatitis Virus ◽  
Active Sites ◽  
Fluorescent Protein ◽  
Economic Loss ◽  
Potential Effect ◽  
Therapeutic Development ◽  
Vesicular Stomatitis ◽  
Green Fluorescent

Numerous natural phytochemicals such as resveratrol are acknowledged as potent botanical agents in regulating immune responses. However, it is less understood whether such immunomodulatory phytochemicals are appropriate for use as direct treatments in veterinary viral diseases. In the present study, we investigated the efficacy of resveratrol in suppressing vesicular stomatitis virus (VSV) infection. Outbreaks of VSV can cause massive economic loss in poultry and livestock husbandry farming, and VSV treatment is in need of therapeutic development. We utilized a recombinant VSV that expresses green fluorescent protein (GFP) to measure viral replication in cells treated with resveratrol. Our findings revealed that resveratrol treatment affords a protective effect, shown by increased viability and reduced viral replication, as indicated by a reduction in fluorescent signals. Additionally, we found that resveratrol inhibition of VSV infection occurs via suppression of the caspase cascade. Structural analysis also indicated that resveratrol potentially interacts with the active sites of caspase-3 and -7, facilitating antiviral activity. The potential effect of resveratrol on reducing VSV infection in vitro suggests that resveratrol should be further investigated as a potential veterinary therapeutic or prophylactic agent.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

Lumenal targeted GFP, used as a marker of soluble cargo, visualises rapid ERGIC to Golgi traffic by a tubulo-vesicular network

2000 ◽  
Vol 113 (18) ◽  
pp. 3151-3159 ◽  
Author(s):  
R. Blum ◽  
D.J. Stephens ◽  
I. Schulz
Keyword(s):  
Fluorescent Protein ◽  
Time Lapse ◽  
Temperature Sensitive ◽  
Long Distance ◽  
Anterograde Transport ◽  
Network Transport ◽  
Vesicular Stomatitis ◽  
Green Fluorescent ◽  
Time Lapse Imaging ◽  
Tubular Membranes

The mechanism by which soluble proteins without sorting motifs are transported to the cell surface is not clear. Here we show that soluble green fluorescent protein (GFP) targeted to the lumen of the endoplasmic reticulum but lacking any known retrieval, retention or targeting motifs, was accumulated in the lumen of the ERGIC if cells were kept at reduced temperature. Upon activation of anterograde transport by rewarming of cells, lumenal GFP stained a microtubule-dependent, pre-Golgi tubulo-vesicular network that served as transport structure between peripheral ERGIC-elements and the perinuclear Golgi complex. Individual examples of these tubular elements up to 20 microm in length were observed. Time lapse imaging indicated rapid anterograde flow of soluble lumenal GFP through this network. Transport tubules, stained by lumenal GFP, segregated rapidly from COPI-positive membranes after transport activation. A transmembrane cargo marker, the temperature sensitive glycoprotein of the vesicular stomatitis virus, ts-045 G, is also not present in tubules which contained the soluble cargo marker lum-GFP. These results suggest a role for pre-Golgi vesicular tubular membranes in long distance anterograde transport of soluble cargo. http://www.biologists.com/JCS/movies/jcs1334.html

432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 373
Author(s):  
M. Reichenbach ◽  
F. A. Habermann ◽  
H. D. Reichenbach ◽  
T. Guengoer ◽  
F. Weber ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Phosphoglycerate Kinase ◽  
Healthy Male ◽  
Transgenic Founder ◽  
Green Fluorescent

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.

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