Viral Mutagenesis as a Means for Generating Novel Proteins

Davis, J. Nathan; Pol, Anthony N. van den

doi:10.1128/jvi.01747-09

Cited by 10 publications

(11 citation statements)

References 25 publications

(31 reference statements)

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“…1B) was constructed as follows. The plasmid pVSV1XN-dsRed (28,34) was digested using XhoI and NotI to remove the 0.75-kb insert coding for dsRed in the first position, replacing it with a 1.5-kb insert containing two genes, one encoding a green fluorescent protein (GFP) derived from Aequorea victoria (avGFP) and the other humanized Renilla GFP (rrGFP) derived from Renilla reniformis. This 1,503-bp insert was commercially synthesized (Genscript, Piscataway, NJ) using the sequence shown in Fig.…”

Section: Vsv-12=gfp Plasmid Construction Virus Recovery and Verificmentioning

confidence: 99%

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Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

Pol

Davis

2013

J Virol

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Vesicular stomatitis virus (VSV) has shown considerable promise both as an immunization vector and as an oncolytic virus. In both applications, an important concern is the safety profile of the virus. To generate a highly attenuated virus, we added two reporter genes to the 3= end of the VSV genome, thereby shifting the NPMGL genes from positions 1 to 5 to positions 3 to 7. The resulting virus (VSV-12=GFP) was highly attenuated, generating smaller plaques than four other attenuated VSVs. In one-step growth curves, VSV-12=GFP displayed the slowest growth kinetics. The mechanism of attenuation appears to be due to reduced expression of VSV genes downstream of the reporter genes, as suggested by a 10.4-fold reduction in L-protein RNA transcript. Although attenuated, VSV-12=GFP was highly effective at generating an immune response, indicated by a high-titer antibody response against the green fluorescent protein (GFP) expressed by the virus. Although VSV-12=GFP was more attenuated than other VSVs on both normal and cancer cells, it nonetheless showed a greater level of infection of human cancer cells (glioma and melanoma) than of normal cells, and this effect was magnified in glioma by interferon application, indicating selective oncolysis. Intravenous VSV-12=GFP selectively infected human gliomas implanted into SCID mice subcutaneously or intracranially. All postnatal day 16 mice given intranasal VSV-12=GFP survived, whereas only 10% of those given VSV-G/GFP survived, indicating reduced neurotoxicity. Intratumoral injection of tumors with VSV-12=GFP dramatically suppressed tumor growth and enhanced survival. Together these data suggest this recombinant virus merits further study for its oncolytic and vaccine potential. V esicular stomatitis virus (VSV) is an enveloped nonsegmented negative-strand RNA virus of the Rhabdoviridae family with a simply organized genome of 11.2 kb that encodes just five genes (N, P, M, G, and L) (1, 2). The ability to recover fully replicationcompetent VSV from suitably engineered plasmid DNA (3, 4) has enabled the generation of modified recombinant versions of VSV (rVSV), some of which are currently under active investigation for their therapeutic potential as replicating or nonreplicating vaccine vectors (5-8) and as oncolytic agents for the treatment of a number of different types of human cancer (9-12).In nature, VSV is a pathogen of livestock, such as horses, cattle, and swine, with infection of humans being relatively rare and resulting typically in subclinical or mild flu-like symptoms (13,14). Although encephalitis is not a characteristic of natural VSV infection (13), experimental infection of brain cells has been found in animal models (15)(16)(17)(18). We have previously shown that the use of recombinant attenuated VSV and peripheral immunization reduced or blocked the ability of VSV to infect central nervous system (CNS) cells (12,19). Further refinement of recombinant VSVs for therapeutic application, particularly within the brain, may benefit from additional viral attenuati...

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Section: Vsv-12=gfp Plasmid Construction Virus Recovery and Verificmentioning

confidence: 99%

“…Given the possibility of mutations in the VSV genome (34,58), one concern is the stability of VSV-12=GFP over time. To study the phenotype of VSV-12=GFP, we injected tumors in vivo with the virus and harvested virus-infected tumors 24 h later for comparison in a plaque size assay.…”

Section: Vsv-12=gfp Targets Brain Tumors In Vivomentioning

confidence: 99%

Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

Pol

Davis

2013

J Virol

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“…In eukaryotic systems, different approaches must be taken to generate large mutant libraries. A recombinant vesicular stomatitis virus was recently reported to generate randomly mutated fluorescent proteins amenable to screening in mammalian cells 23 . The mutation rate and the size of the library were controlled by regulating the number of infected cells and the number of rounds of viral replication.…”

Section: Directed Evolution Of Fluorescent Proteinsmentioning

confidence: 99%

Directed molecular evolution to design advanced red fluorescent proteins

2011

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Fluorescent proteins have become indispensable imaging tools for biomedical research. continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

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“…However, phage-assisted continuous evolution is also limited to evolving interactions within E. coli. Similar approaches using retroviruses and mammalian cell culture (12,13) do not sample as large a diversity of mutants.…”

Section: Introductionmentioning

confidence: 99%

Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM

Finney-Manchester

Maheshri

2013

Nucleic Acids Research

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A major hurdle to evolutionary engineering approaches for multigenic phenotypes is the ability to simultaneously modify multiple genes rapidly and selectively. Here, we describe a method for in vivo-targeted mutagenesis in yeast, targeting glycosylases to embedded arrays for mutagenesis (TaGTEAM). By fusing the yeast 3-methyladenine DNA glycosylase MAG1 to a tetR DNA-binding domain, we are able to elevate mutation rates >800 fold in a specific ∼20-kb region of the genome or on a plasmid that contains an array of tetO sites. A wide spectrum of transitions, transversions and single base deletions are observed. We provide evidence that TaGTEAM generated point mutations occur through error-prone homologous recombination (HR) and depend on resectioning and the error-prone polymerase Pol ζ. We show that HR is error-prone in this context because of DNA damage checkpoint activation and base pair lesions and use this knowledge to shift the primary mutagenic outcome of targeted endonuclease breaks from HR-independent rearrangements to HR-dependent point mutations. The ability to switch repair in this way opens up the possibility of using targeted endonucleases in diverse organisms for in vivo-targeted mutagenesis.

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Viral Mutagenesis as a Means for Generating Novel Proteins

Cited by 10 publications

References 25 publications

Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

Highly Attenuated Recombinant Vesicular Stomatitis Virus VSV-12′GFP Displays Immunogenic and Oncolytic Activity

Directed molecular evolution to design advanced red fluorescent proteins

Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM

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